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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.

Title: COMPARISON OF HUMAN PAPILLOMAVIRUS (HPV) DETECTION IN URINE AND CERVICAL SWAB SAMPLES USING THE HPV GENOARRAY DIAGNOSTIC ASSAY Review round: 2 Reviewer: 2

Basic reporting: No Comments Experimental design: No Comments Validity of the findings: An additional method of comparison might further validate the findings of the article. Additional comments: While the authors have not provided any further experimental evidence to address the comments / concerns raised previously, they have addressed them in the discussion and via other textual changes.

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.

Title: HISTAMINE-2 RECEPTOR ANTAGONIST FAMOTIDINE MODULATES CARDIAC STEM CELL CHARACTERISTICS IN HYPERTENSIVE HEART DISEASE Review round: 1 Reviewer: 1

Basic reporting: No comment Experimental design: Results seem like rather preliminary. 1. Provide representative images for figures 1a, 2a and 2c. 2. There is no mechanism explaining salutary effects of famotidine on CSCs. It would help if authors can provide a molecular basis for the observed effects. 3. Do CSCs express histamine receptor? 4. Authors note that histamine receptor is present primarily on mast cells, known to express c-kit. Can the authors provide results showing that their CSC population is negative for mast cells population tryptase. 5. What happens to wild type CSC proliferation and growth kinetics after treatment with famotidine in vitro. Validity of the findings: The results are inconclusive and preliminary. More rigorous analysis required. Additional comments: No additional comments

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.

Title: HISTAMINE-2 RECEPTOR ANTAGONIST FAMOTIDINE MODULATES CARDIAC STEM CELL CHARACTERISTICS IN HYPERTENSIVE HEART DISEASE Review round: 1 Reviewer: 2

Basic reporting: The manuscript is clear but unfortunately includes no raw data. All results are presented as graphs. The authors should provide representative images for all three groups for Figures 1A, 2A, 2B and 2C. Furthermore, flow plots should be presented to justify the claim that the cells were 92+/-3% ckit+ and negative for CD45 and CD31. The introduction is brief but adequate. The discussion should include more examples of comparision between this work and what is known in the field. Experimental design: The experimental design is sound but the details of the methods are somewhat brief. For example, were the explants plated on coated flasks, what medium was used, which antibodies were used for flow, what were the cells stained with in the colony forming assay, how was the cell number obtained for the proliferation assay? Minor comment - what is 'Age associated variation in fig 1D? Validity of the findings: It is hard to assess the data when no raw images have been presented. If all the results are confirmed by raw data then the conclusions are sound. Additional comments: This is an interesting piece of work but is marred by the lack of raw data and the brief discussion.

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.

Title: HISTAMINE-2 RECEPTOR ANTAGONIST FAMOTIDINE MODULATES CARDIAC STEM CELL CHARACTERISTICS IN HYPERTENSIVE HEART DISEASE Review round: 2 Reviewer: 1

Basic reporting: The manuscript is clear and significantly improved in the revised version. Experimental design: The overall hypothesis is novel and interesting and addition of new methodology and results have improved the manuscript. Validity of the findings: The findings are novel and well supported by the data. Additional comments: The authors have addressed all the concerns raised.

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.

Title: HISTAMINE-2 RECEPTOR ANTAGONIST FAMOTIDINE MODULATES CARDIAC STEM CELL CHARACTERISTICS IN HYPERTENSIVE HEART DISEASE Review round: 2 Reviewer: 2

Basic reporting: In this revision the authors now include improved discussion of their results and representative images to support their data. However, the images in figure 5 to show b-Gal staining and the Western blot in Fig 5E are not very convincing. Experimental design: The methods are now improved with sufficient detail in most cases, although the quantities of FGF and insulin-selenium-transferrin are not given in 2.2 Validity of the findings: The data appears robust, although better images should be provided for fig 5 if possible. Additional comments: The manuscript is much improved but requires some editing for English.

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.

Title: INFLUENCE OF EXERCISE DURATION ON CARDIORESPIRATORY RESPONSES, ENERGY COST AND TISSUE OXYGENATION WITHIN A 6 HOUR TREADMILL RUN Review round: 1 Reviewer: 1

Basic reporting: Abstract: Please insert the correct VO2max units throughout the manuscript: "mL•min•kg-1" change to "mL•kg-1•min-1" Since you did not directly measure beta-oxidation. I would change to a reduced RER. I would suggest the authors estimate fat oxidation with the use of RER, and Oxygen Utilization. If you observed an increase in VO2 at the 10 km/h running speed, this would indicate a reduced running economy, despite no change in the energy cost of running. Introduction: Line 52. "It has been suggested increases".... Change to "It has been suggested that increases..." Did you record years of training experience? Or average weekly training time? These are often more informative then VO2max values. Was nutrition monitored or recorded? I know it was not controlled. But nutrition could have had a large effect on the results. Line 156-157. RER is VCO2:VO2. You have it reversed? Please correct. I am not clear how you obtained J•kg•m-1 . Can you please show me how you got these units? It is unclear why the two-way RM ANOVA and three-way RM ANOVA's were used. These were not clear from the aim of the study. Experimental design: I completely understand the small sample size, but was a sample size calculation done prior to the study? Validity of the findings: Was the metabolic system calibrated before and after? What is the reliability of the metabolic system in the laboratory used? Additional comments: The study is very unique, with a 6 hours of treadmill running. The authors point out on several occasions that no downhill running was included as this may alter the findings. However, what is the external validity of such findings (ie. are there any ultramarathon courses with no downhill sections)? I also feel that is important to point out that running economy was reduced despite no change in the energy cost of running. Lastly, I suggest the authors calculate fat oxidation instead of assuming a reduction in RER is equivalent to an increase in beta-oxidation.

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.

Title: INFLUENCE OF EXERCISE DURATION ON CARDIORESPIRATORY RESPONSES, ENERGY COST AND TISSUE OXYGENATION WITHIN A 6 HOUR TREADMILL RUN Review round: 1 Reviewer: 2

Basic reporting: no comment Experimental design: no comment Validity of the findings: Speculation is welcome, but should be identified as such. Additional comments: General comments: The current paper is relatively well-written and the topic related to endurance exercise and including many laboratory-running measurements is quite interesting. However, the paper requires some important clarifications, especially during the methods and discussion parts. To improve the global reading of the present manuscript, it would be judicious to use more abreviations throughout the text, such as 10-CR or HILL from the beginning to the end of proposed statements. The authors are requested to improve the quality of their work by taking account into the specific comments described below. INTRODUCTION: . L46: suggestion: Maximal oxygen uptake (VO2max), the fractional rate of VO2max (%VO2MAX) and the energy cost of running (Cr), i.e. the submaximal metabolic demand (VO2) per unit of distance covered are… . L48: Insert “may be” instead of “are” affected . L52: Insert “that” after suggested. . L53-56 : The reviewer is in agreement with the statements used by the authors but the sentence needs to be re-written by presenting firstly the effects of exercise duration until 8-h on VO2 and secondly, the effects of ultramarathon on the same variable. . L56-61 : Could you reformulate these sentences please. METHODS : Could you extend or precise the weekly running mileage for your experimental group. Does your population cycle during their training sessions ? L111: Replace maximum VO2 by VO2max. L132 : Why did you select a +10% slope ? L133 : For HILL, is the speed corresponding to 70%VO2max or the highest possible speed as you reported in the manuscript ? clarify the confusion please. L160 : It would have also been interesting to convert J.kg-1.m-1 in J.m-1 shunting the body mass which may decrease with exercise duration and exert an additional influence on final CrJ values. What ‘s your position about this choice of unit. L176 : What was the average time to attach and remove the NIRS devices during the four modules ? Could you provide more details about the position of the NIRS device on the prefrontal cortex. RESULTS : well presented. DISCUSSION : in the paragraph 17, I suggest that you also present the results including oxygenation and metabolic responses, not only PCI and RPE. L245-246 ; precise the two exercise intensities. L250- : Could you reformulate this sentence (i.e. repeated terms) and by including RER values to characterize the beta-oxidation. L253 : Could you reformulate « at intensities < 80% of maximum ». L257 : Beta-oxidation is not directly measured in the current work but extrapolated from RER values. It should be more appropriate to use the RER decrease firstly before inferring on beta-oxidation. L263 : replace « mild » by small. In the paragraph 19, the authors discussed their results focusing on the lack of CrJ alteration with exercise duration. Some hypotheses are proposed but the training status of the experimental group has not discussed in this part. The high training background and thereby, muscular adaptations in ultramarathon runners might constitute an additional hypothesis to explain the lack of CrJ alteration with exercise duration, either on level ground or HILL condition. Could you explore this type of hypothesis. You observed an absence of CrJ alteration on level ground, but at a moderate intensity (10 km/h) or at a self-selected speed or both. Could you precise in the discussion part what « level ground » means to clarify the reading. Furthermore, for the self-selected speed-based period of measurement, you observed a decrease in running speed across the modules and this decrease in speed may explain why CrJ did not change across the time in the case where VO2 continuously increase. You have not discussed the ratio between change in VO2 and change in speed to explain CrJ responses across the time. L283-286 : could you reformulate this sentence. L290-295 : The authors hypothesized that it is likely that the exercise duration during the HILL and 10-CR modules was too short to induce any significant changes in TSI responses throughout muscular sites. A link with a 24-h outdoor exercise using biopsies methods has been used, but it would have been better to create a link with studies investigating the evolution of NIRS parameters with exercise duration. Furthermore, it would be interesting to present limitations of NIRS devices in dynamic exercices in an attempt to explain the lack of TSI changes with exercise duration. L312 : I am a bit confused when the authors reported that the 6TR did affect the energy cost of running. Conclusions : insert future research directions at the end of conclusion part. Title table 1. Heart rate is mentioned twice. Change heart rate relative to individual maximum. What is the signification of the statistical symbol used ? Which difference ? Could you apply these modifications to table 2 and table 3. Figure 1. Could you insert the abreviations 10-CR and HILL, moderate and heavy exercises in the figure content In the results section, you wrote that TSI responses were significantly higher in PFC compared to VL and GL, but when observed the figure 3 and especially B and C, the reviewer has the impression that no difference exist between sites. Given that this impression is purely visual, could you confirm the statistical inference of your results. I suggest that you also change the statistical representation, not really clear. .

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.

Title: INFLUENCE OF EXERCISE DURATION ON CARDIORESPIRATORY RESPONSES, ENERGY COST AND TISSUE OXYGENATION WITHIN A 6 HOUR TREADMILL RUN Review round: 1 Reviewer: 3

Basic reporting: -This is a nicely written manuscript that is clear in its rationale, methods, analyses, and conclusions. No major concerns. - No notable grammatical issues. - Data is nicely presented. Experimental design: - The design is appropriate for the research question. - Hypotheses are appropriate and rationalized. - Methods are nicely described - no concerns. Validity of the findings: - The findings are interesting and worth publishing. I'd suggest linking the data with work showing the effects of prior heavy exercise on moderate intensity exercise (references noted below). - Statistics are nicely described. Additional comments: - Please define "reverse cardiovascular drift". - Would suggest inclusion of some priming literature on the amplitude of VO2 -- as these studies provide some physiologic insight into the mechanisms of how prior heavy exercise influences subsequent moderate intensity exercise (DiMenna et al. J Appl Physiol 2009 107(6):1743-56; Wilkerson et al. J Appl Physiol. 2004 97(4):1227-36; Gurd et al. J Appl Physiol. 2005 98(4):1371-8). These seminal papers highlight important physiology about priming effects on VO2. -

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.

Title: INFLUENCE OF EXERCISE DURATION ON CARDIORESPIRATORY RESPONSES, ENERGY COST AND TISSUE OXYGENATION WITHIN A 6 HOUR TREADMILL RUN Review round: 2 Reviewer: 1

Basic reporting: no comment Experimental design: clear Validity of the findings: clear Additional comments: Responses and/or modifications from the authors are considerably improved the manuscript in terms of methodological quality, results and discussion presentation. I recommend the publication of the current work in the Running scope, when applying the minor specific comments below. L50: ….that increases in Cr correlate positively with…. L54: remove “s” : afetr a 65-km trail ultramarathon L54: suggestion : Similarly, following a 65 km mountain ultramarathon…. L132: I suggest authors to report why the +10% slope was selected in this protocol, on the basis of feedback comments. Paragraph number 7: I suggest authors to remove “complete” (repeated twice) in the following sentence…. to complete the condition and/or (HERE) the 6TR,....

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.

Title: INFLUENCE OF EXERCISE DURATION ON CARDIORESPIRATORY RESPONSES, ENERGY COST AND TISSUE OXYGENATION WITHIN A 6 HOUR TREADMILL RUN Review round: 2 Reviewer: 2

Basic reporting: The authors have addressed my comments. Experimental design: The authors have addressed my comments. Validity of the findings: The authors have addressed my comments. Additional comments: The authors have addressed my comments.

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.

Title: INDEPENDENT EVOLUTION OF TETRALOOP IN ENTEROVIRUS ORIL REPLICATIVE ELEMENT AND ITS PUTATIVE BINDING PARTNERS IN VIRUS PROTEIN 3C Review round: 1 Reviewer: 1

Basic reporting: The language used by Prostova et al. in their paper is clear and unambiguous, yet I would like to recommend a further checking to improve wording/phrasing in some sections to improve the overall readability. Some specific notes on this regard: Line 43: I suggest adding long at the end of the sentence “… with plus strand genome about 7500 nt long” Line 60: I suggest changing "leafs" to "leaves" Line 69: I suggest changing "extremal" to "extreme" Line 107: I suggest changing "formed" to "created" Lines 119-122: I suggest expressing the total sequences per organism in the clearer format organism (N total) Line 144: I suggest changing "conservative" to "conserved" Line 299: please remove the comma Line 398: I would say that data "suggests" rather than "demonstrates" Overall, the submission is ‘self-contained’ with a clear aim and clear results, which can be used for future experimental validation of the hypothesis made by the authors in the paper. The authors made a good job in properly referencing and giving relevant background to their work. Their paper conforms to the journal guidelines, figures are clear and the raw data is appropriately given as supplementary material. To improve their manuscript, I would like to suggest to the authors to expand the Conclusions section to include more details on the final message of their paper. Experimental design: The work successfully qualifies for the Aims and Scope of the journal, conveying new findings on a relevant biological system using widely known bioinformatics techniques. The research question is well defined in the first sections of the paper, with several mentions of the significance of their study from various biological points of view. However, I wish the authors would put more emphasis on how their study contributes to fill the knowledge gap they are trying to address. The methodology used and described in the paper is sound enough to support their conclusions, though I would suggest to the authors to give more details on some methodological approaches that can be relevant to make their work reproducible. For instance in line 94 the authors report they used Clustal for sequence alignments, but without giving more details on the parameters that have been used (and if they differ between RNA and protein analysis). Another example is in lines 113-114 where the Vienna RNA Websuit has been used for secondary structure fold. I suggest adding some more details about the algorithm and parameters that have been used. Validity of the findings: The data presented by the authors are clear and robust, which support their conclusions. The speculative part is somewhat limited and identified as such, without falling in a common pitfall in this type of study of over interpretation of raw bioinformatics data to extract stretched biological meanings. A note I wish to make to the authors is that starting from line 312 in the Discussion they highlight how their data suggest a recognition structure-based rather than sequence-based, which to me is worth to be mentioned again towards the Conclusions part, being a significant result for general macromolecular recognition rules. Additional comments: All things considered, the work by Prostova et al. is in my opinion suitable to be published in PeerJ and can be further improved with some minor revisions/improvements I mentioned in the previous sections

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.

Title: INDEPENDENT EVOLUTION OF TETRALOOP IN ENTEROVIRUS ORIL REPLICATIVE ELEMENT AND ITS PUTATIVE BINDING PARTNERS IN VIRUS PROTEIN 3C Review round: 1 Reviewer: 2

Basic reporting: Overall the paper is clear and there are no widespread severe language issues present. However, there are some small grammatical and wording issues. I have listed a few example below. 1. On line 69 the phrase 'extermal stability' should be 'extreme stability'. 2. On line 215: 'the conservative motif KFRDI' should be 'the conserved motif KFRDI'. 3. On line 228: 'tripeptide (position 155) was invariantly Gly.' should be 'tripeptide (position 155) was always Gly.' This is not a comprehensive list of all such issues. Overall the paper could use some small editing for these minor wording issues. The introduction and background provide a clear summary of the subject and enough context to understand the question being asked. In addition, the paper is well structured and organized. The paper as a whole is an easy read. In addition the figures are generally good. Figure 1 is very helpful for understanding the biological context of the research and Figure 2 helps in understanding the finer details of the loop in question. However, there are some issues with Figure 3. They are: 1. Part A of the figure may best be presented as a simple table. This would give more complete information to the reader as it would be easier to see the exact counts of type of loop. 2. Part B needs some modifications. The arrows in B that indicate the important positions in the protein are very helpful it would be ideal if the number in the sequence logo was consistent with the stated numbers. That is the position labeled with 'proteolytic Glu 71' should be number 71 instead of 2. Finally, it would be helpful to label the arrow in the lower right as Lys153 from Rhinovirus A as it is not clear that that motif is from Rhiovirus A. Overall, the paper is well presented but some edits are necessary. Experimental design: The authors aim is to find a correlation between sequence conservation of domain d loop regions in Enteroviruses and the interacting amino acid sequences from protein 3C. They used a simple straightforward method to find any correlation. The methods section explains much of the techniques used. However there are some issues with their methods: 1. They do not justify the inclusion of otherwise excluded loop sequences in their analysis. For example, on line 171 the authors mention adding 3 unique tetra loop sequences back into the data set after filtration. I suspect this is to maintain diversity of loop sequences from the species. However, there is it is not clear to me why this must be done. The reasons for this decision must be clearly justified in the text. In addition, I would be interested to know if their conclusions change when these sequences are included. 2. They use a 99% percent cutoff and claim that a 95% cutoff results in a 'dramatic loss of unique tetraloop variants', providing a number of sequences lost would help justify the selection of 99% cutoff. In summary, I believe their methods are correct but some details require explanation. Validity of the findings: The authors conclusions about lack of a relationship between the sequence conservation in the domain d loop and the interacting protein are well supported by the data. As they state there is no clear pattern of loop sequence to protein sequence conservation. In addition, their observation that the protein sequence appears to be more diverse than the RNA sequence is both interesting and supported. The authors may wish to emphasize this contrast more. However, the authors may be underestimating the degeneracy of the sequence to structure relationship. In Petrov et. al (doi:10.1261/rna.039438.113) it was found that some loops may differing sequences as well as number of nucleotides but form the same overall 3D structure. Observations such as this strength the their conclusions and the authors may wish to cite such work. In addition, there has been some work studying the structure RNA/protein interactions. The authors may want to review this literature to suggest a how the diverse protein and loop sequence interact. If their suggestion that the overall 3D structure is maintained then the interaction should also be maintained. This may be outside the scope of the current work and I understand if the authors wish to leave such work for another publication. Additional comments: No additional comments

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.

Title: INDEPENDENT EVOLUTION OF TETRALOOP IN ENTEROVIRUS ORIL REPLICATIVE ELEMENT AND ITS PUTATIVE BINDING PARTNERS IN VIRUS PROTEIN 3C Review round: 1 Reviewer: 3

Basic reporting: The study is well-conducted and detailed. I have only few minor comments that might clarify the manuscript. 1) Table 2: It would be more clear if proportions of loop sequences instead (or in addition) to occurrence was indicated in the heatmap (i.e. percentage of a given loop sequence out of sequences observed in a given virus species). In addition, it would be interesting to see if there are statistically significant differences in the proportions of a given loop sequence between virus species. For example, loop sequence CUCG seems to be prevalent in EV-A species, but not in EV-B and EV-C species. Table S1 contains more information than Table 2 (regarding dataset bias - i.e. predominance of EV-A71 and PV), therefore I would prefer showing Table S1 in the main article. In Table S1, it would be more clear if two more columns; 'non-A71 EV-A' and 'non-polio EV-C' would be included. This would help the reader to understand the structure of the data (i.e. how much of the diversity in 'all' column is explained by EV-A71 and PV). 2) Fig 3: Only five species are shown - should others be included? e.g. rhinovirus B (with triloops) would be informational. 3) Supplementary Tables: Should protein 3C tripeptide sequences be also given for EV-B, EV-D, EV-E, EV-F and Rhinovirus B? Experimental design: no comment Validity of the findings: 1) Page 13 line 164-165: "Interestingly, the diversity of tetraloops among EV71 serotype was similar to the diversity of tetraloops in the whole Enterovirus A species (Table S1)." This statement needs clarification. According to Table S1 it seems that some tetraloops (such as UUCG, UAAG and UGAG) are found only in EV-A71 type but not in the other EV-A types.On the other hand, tetraloop UGCG seems to be highly prevalent among other EV-A types (N=112), but rare among EV-A71 (N=2), whereas CUCG seems to be prevalent among EV-A71 (N=127) but rare among the other types (N=5). Additional comments: Generally, the study is well-conducted and detailed. The conclusions are supported by the data. I have only few minor comments that might clarify the manuscript.

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.

Title: BENCHMARK DATASETS FOR PHYLOGENOMIC PIPELINE VALIDATION, APPLICATIONS FOR FOODBORNE PATHOGEN SURVEILLANCE Review round: 1 Reviewer: 1

Basic reporting: There's no question about basic reporting in this manuscript. Professional English is used throughout, sufficient background is provided, and the structure is professional and the manuscript self-contained Experimental design: Manuscript adheres to Aims and Scope. The research question is well defined and relevant, and the investigation has been performed to high technical and ethical standards. Validity of the findings: The findings are valid and the manuscript adheres to PeerJ's standards Additional comments: I think it’ll be helpful if the authors add information as to how they determined that the bacterial isolates are epidemiologically linked and in what direction. One key issue is establishing a phylogenetic relationship among isolates that mirrors the transmission network. I understand this is straightforward for the simulated data but it’s not clear to me how this was done for the real biological data. I mention this because the equivalency of phylogenies to transmission trees has been somewhat controversial (e.g., PMID: 24037268; PMID: 24675511; PMID: 26230489; PMID: 24916411). Consequently, methods has been develop to directly address this issue, e.g., PMID: 28545083 One minor thing that's hanging in my head is why providing example datasets in xlsx if the script takes tsv’s? The conversion is trivial but keeping everything tidy in open formats has its advantages.

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.

Title: BENCHMARK DATASETS FOR PHYLOGENOMIC PIPELINE VALIDATION, APPLICATIONS FOR FOODBORNE PATHOGEN SURVEILLANCE Review round: 1 Reviewer: 2

Basic reporting: Overall, the manuscript was well written. It is unclear why so many references are applied to the reference of Lyve-SET. I would just cite the manuscript that describes the pipeline, unless you are citing the specifics of diverse projects where Lyve-SET has been used. The NASP pipeline was mentioned, but wasn’t cited. L95: Peptoclostridium is not really used and is immediately followed by Clostridium in your text. GenBank currently uses “Clostrioides”. Experimental design: This manuscript describes a method to download and compare pipelines for WGS analyses. However, after downloading the repository, I saw no script referred to as “Gen-FS Gopher” or “GG”. There appears to be no comprehensive, documented workflow on how to download the 5 test datasets. The scripts provided to convert between “xlsx” and “tsv” required extra dependencies and I could not get them to work. Saving them as tsv files seemed to work and I was able to download the datasets, although I received an error. If these are benchmarking datasets, which SNPs should consistently be called? Simply obtaining the same tree doesn’t mean that any given pipeline is calling the correct SNPs. Why not release the SNPs that were used to infer the provided tree? As it reads now, it is unclear if the true utility of this paper is in benchmarking, or providing a tool to interface with the SRA to grab sequences and trees. Validity of the findings: Having a standard benchmarking scheme would be very helpful to standardize analyses across computational pipelines. Additional work on the repository associated with testing your scripts on multiple platforms and adding additional documentation would help users not familiar with these types of pipelines. Additional text on how these datasets can be used to benchmark pipelines would also help users assess new pipelines, using metrics that can be directly compared. Additional comments: This manuscript could be improved by working on the documentation, making sure that script names in the manuscript match those in the repository, and by providing specific metrics that users could use to assess new SNP pipelines. Providing standardized datasets to the community would be very helpful.

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.

Title: BENCHMARK DATASETS FOR PHYLOGENOMIC PIPELINE VALIDATION, APPLICATIONS FOR FOODBORNE PATHOGEN SURVEILLANCE Review round: 1 Reviewer: 3

Basic reporting: This article is generally clear and well written and the impact of the work is communicated clearly. Data and programs were accessible as indicated in the text. I found that the text was not clearly organized into Introduction/Materials and Methods, Results and Discussion. For example, the last paragraph of the introduction seems to be materials and methods. Some of the information on lines 102-112 (e.g. selection of datasets, data simulation) should be integrated into the materials and methods. Similarly, description of data simulation (lines 148-160) and the description of the development of the GG script also seem to fit within the scope of materials and methods. Authors should consider modifying organization and perhaps combine results/discussion. Given that the topic of this paper is somewhat different than a traditional scientific paper (announcement of the availability of data/tools rather than an evaluation of performance), perhaps traditional subheadings are not appropriate. Some minor comments: The Gen-FS gopher script is called “downloadDataset” on the github site which may lead to confusion. The pages preceding the tables are confusing. Eg:Table 2 “ Reviews and evaluates data submissions in food and color additive petitions and premarket notifications (GRAS and Food Contact Surfaces notifications) to determine the safety of the use of a product in foods within the context of applicab” Line 27: ortholog determination (no hyphen) Line 28: single-nucleotide polymorphisms Line 60/71: publically (should be publicly as in line 37?) Line 73: update number of genomes if possible Experimental design: The design of the study is clearly described. I would be interested in results of analysis of this dataset with different phylogenetic pipelines that are currently in use among Gen FS partners to demonstrate how data generated from this dataset would be interpreted. Given that reference genomes are provided, could SNP/SNVs locations be identified? It would also be appropriate to provide a figure showing phylogenetic trees. Validity of the findings: no comment Additional comments: This manuscript describes the development of benchmark NGS datasets for priority foodborne pathogens that can be used to evaluate the performance of phylogenomic pipelines, and addresses the development of standardized formats for submission of similar benchmark datasets in the future. This work represents an important contribution that will be particularly useful for regulators in determining the reliability of new phylogenomic pipelines applied to the surveillance of foodborne pathogens. The tool for rapid retrieval of validation data will be useful in providing easy access to this data.

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.

Title: HEARING ASSESSMENT DURING DEEP BRAIN STIMULATION OF THE CENTRAL NUCLEUS OF THE INFERIOR COLLICULUS AND DENTATE CEREBELLAR NUCLEUS IN RAT Review round: 1 Reviewer: 1

Basic reporting: Entirely adequate. Experimental design: Methods organization could be improved. The section describing DBS was buried in the ABR method section. It should have been a separate and more complete section, as it was in the Smit et al., (2016) research report. The low n (5 / group) of the present study sets it up for failure to find significant effects. This appears particularly true since non-parametric statistical tests, which do not take advantage of the repeated-measures design, were used. The authors should explain their choice of statistical tests and consider the issue of adequate statistical power to determine significance. Although it wasn’t well explained (but should have been), apparently DBS was delivered throughout the epoch devoted to ABR acquisition. Hence there was a need to remove stimulus artifacts, as shown, but not thoroughly explained, in Figure 2. Apparently the DBS, both high-frequency and low, were delivered without controlling phase relationship to the ABR acquisition. That experimental choice should be justified. DBS periods were either 10 msec or 100 msec. ABR inter-stimulus intervals were 45 msec, while the ABR recording periods were 50 msec. Rather than let these events wander in and out of phase, a more tightly controlled protocol could have been used. For example, if the ABR-eliciting sound pulses were out of phase with DBS, then the DBS stimulus artifact would not have to be filtered. This would eliminate the loss of data produced by EP filtering. Validity of the findings: Perhaps the most important results appear in Figure 4. In my opinion it would have been more informative to show thresholds, or ABR signal levels (such as RMS), separately for waves I through V. This, because it is surprising that DBS in the IC would not affect ABR wave V amplitudes, since the IC is a major source of wave V potentials. It wasn’t clear to me how the wave V/I amplitude ratios, reported in Table 1, addressed the issue of amplitude effects. Were all of the Table 1 data collected with sound pulses at 90 dB (SPL)? This was implied in Methods, but not clear. If yes, what about the dynamic range of wave V (and other wave components)? Furthermore, if both wave I and wave V diminished, the V/I ratio would not change. On the topic of threshold: Threshold is just one point of the dynamic range of evoked potentials. In this context Schaette (Schaette, 2014) and others have introduce the concept of “hidden hearing loss.” A more sensitive measure of loss would be to quantify the evoked response strength over the entire range of stimulus levels, for each test frequency and/or each ABR wave (I – V). Root mean square (RMS) is often used for that purpose. Another advantage of the dynamic-range approach is that thresholds do not have to be determined by experimenter judgment. One level of DBS (100 µA) was tested. Since this level was shown in the preceding tinnitus study (Smit, et al., 2016) to be effective in tinnitus attenuation, perhaps this is adequate to show that the tinnitus attenuation did not derive from a hearing threshold shift.. However, the value of the present study would have been improved if a range of DBS levels had been tested… analogous to testing a drug at more than one dose. Additional comments: The primary value of the present research might be the addition of the DCBN component. Note the published experiment (Smit et al., 2016) showed that DBS in the IC did not affect PPI. On this basis one would have to conclude that IC DBS did not interfere with central auditory processing. This would be true because PPI depends not only upon a functioning auditory brainstem (and ear), but also to some extent, a functioning auditory forebrain (Du et al., 2011). The point is that the prior published finding diminishes the knowledge added by the present work. Nevertheless, demonstrating an effect, or lack of effect, on the ABR has value. References Du, Y., Wu, X., Li, L. 2011. Differentially organized top-down modulation of prepulse inhibition of startle. J Neurosci 31, 13644-53. Schaette, R. 2014. Tinnitus in men, mice (as well as other rodents), and machines. Hear Res 311, 63-71.

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.

Title: HEARING ASSESSMENT DURING DEEP BRAIN STIMULATION OF THE CENTRAL NUCLEUS OF THE INFERIOR COLLICULUS AND DENTATE CEREBELLAR NUCLEUS IN RAT Review round: 1 Reviewer: 2

Basic reporting: The article is written in clear English and is well structured. The hypotheses are clearly explained, reasonable and supported by sufficient amount of existing work in the literature. The structure of the result reflects what is expected from the hypotheses tested by the authors. Experimental design: In this article, the authors test the potential of deep brain stimulation to treat tinnitus in two brain targets. Both targets are theoretically relevant and untested, moreover, they chose to implant the electrodes in the area, which is involved directly in auditory input processing and another area, which is not involved, but remains relevant based on lesion studies. The scientific is well formulated and directly tested. The investigation was performed rigorously and the authors have considerable experience with deep brain stimulation experimental design. This particularly visible in the method description section, in which is all relevant experimental details are well explained to allow for data reproduction. Validity of the findings: Although the data are mostly not significant, the authors performed strict scientific observation of their data collection. The data set is robust and is discussed adequately in the discussion. Statistical tools have been used appropriately and the data set has been rigorously and thoroughly explored. Additional comments: The article is well written and the authors discussed objectively their findings. The use of the ABR is welcome and provide an objective assessment of the efficiency of their method. Although the data set does not suggest the chosen targets to be relevant for treating tinnitus, this work is scientifically executed and deserve its place in te litterature, as it is very informative theoretically and technically.

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.

Title: HEARING ASSESSMENT DURING DEEP BRAIN STIMULATION OF THE CENTRAL NUCLEUS OF THE INFERIOR COLLICULUS AND DENTATE CEREBELLAR NUCLEUS IN RAT Review round: 2 Reviewer: 1

Basic reporting: Acceptable. Experimental design: Acceptable. Validity of the findings: Acceptable. Additional comments: This revised manuscript has been substantially improved. My questions have been satisfactorily answered. The descriptive data, e.g., Fig 4, certainly appear equivalent between treatment conditions (i.e., brain stimulation) and groups (recording site). This, of course, supports the conclusions. Suggested minor improvements. The authors discuss the importance of recording electrode placement for ABR acquisition. Equally, or perhaps more important is consistency of sound presentation. Sound presentation features should be more precisely explained, how was the speaker presenting sound to the right ear (lines 146 – 147) arranged and standardized between recording sessions? Our experience shows that more ABR between subject variance can be attributed to sound stimulus variation than to electrode placement. Line 176. Use “data” as a plural noun. Line 276. Substitute “why” for “that.” Although not that important for the present findings, the discussion paragraph considering why HF-DBS might disrupt tinnitus but not exteroceptive hearing (lines 291 -298) could be improved. In this paragraph HF-DBS is initially considered as having broad volume impact. Later in the paragraph it is hypothesized that HF-DBS might disrupt tinnitus because of a focal effect, leaving parallel pathways carrying external sound information unaffected. Overall this appears contradictory. A related consideration (maybe too distant to be included) is that most researchers now consider the pathophysiological underpinning of tinnitus to be broadly distributed in the CNS.

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.

Title: VOOMDDA: DISCOVERY OF DIAGNOSTIC BIOMARKERS AND CLASSIFICATION OF RNA-SEQ DATA Review round: 1 Reviewer: 1

Basic reporting: In summary, this study was well planned, efficiently organized, and competently reported. The manuscipt presentation and literacy standard are generally high. Experimental design: Some of the real RNA-sequencing datasets were download from public database. They may produce from different laboratories. Measurements are affected by laboratory conditions, reagent lots and personnel differences. Did the author takes the batch effect into account? Will this affect your predicted results? Validity of the findings: In evaluation process paragraph (line 428-429), the data were randomly split into two parts as training (70%) and test (30%) set. We know that test errors may differ for different train/test splits.(line 436).How dose the user determine the appropriate split ratio? Additional comments: No additional comments

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.

Title: VOOMDDA: DISCOVERY OF DIAGNOSTIC BIOMARKERS AND CLASSIFICATION OF RNA-SEQ DATA Review round: 1 Reviewer: 2

Basic reporting: The manuscript by Zararsiz et al. introduced a method which combined voom and nearest shrunken centroids/diagonal discriminant classifiers together to perform classification of RNA-seq data. The methods are interesting, particularly for its possible application in the discovery of diagnostic biomarkers based on RNA-Seq data. Nevertheless, there are several weaknesses that should be improved. My major concerns and specific comments are listed below. 1) The manuscript was not very well written, containing few typos and formatting/grammar errors. The equations lists in the manuscript seem to be correct but not easy to follow, detailed mathematical annotations are encouraged to be included. Experimental design: 2) The purposed method VoomDLDA/VoomDQDA or VoomNSC didn't show superior performance compared to conventional approaches, in both simulation (Fig3) and real data. It will thus limit the application in practice. 3) There is large paragraph descriping data normalization in the Materials & Methods section. However, the final results showed that expression data from raw read counts, median ratio normalization or TMM normalization are basically identical (Fig3 & Fig4), any explanation for that? Validity of the findings: 4) For the purpose of discovering diagnostic biomarkers, it would be more relevent to train the model to prioritize the known biomarkers. Unfortunately, I'm not ware the author performed analysis of the kind in the manuscript. 5) The author split the data into training and test sets with 70% and 30%, is it part of cross validation, please clarify. Additional comments: No additional comments

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.

Title: VOOMDDA: DISCOVERY OF DIAGNOSTIC BIOMARKERS AND CLASSIFICATION OF RNA-SEQ DATA Review round: 1 Reviewer: 3

Basic reporting: no comment Experimental design: no comment Validity of the findings: no comment Additional comments: The paper introduces a novel data normalization, processing, classification, and feature selection method for RNA-Seq count data, by combining Voom normal linear modeling method with NSC (nearest shrunken centroids) and other classification methods. The presentation and experimentation is comprehensive. The authors utilize both simulated and real datasets and compare their results with some of the commonly used classification methods. I commend the authors for making their source code available. The method is also made available via a web service. I do not have any major concerns. I have listed the following points which would improve the publication: * In the abstract, keep background info to a minimum (RNAseq advantages not necessary to list in abstract) and highligh the contributions more. * The authors call their method voomNSC in the abstract, but voomDDA in the web link and the title. Use one name for the method, consistently. What does voomDDA stand for? If voomDDA is the umbrella term, the abstract should explain that rather than just voomNSC. * Expand the Voom acronym in the abstract ("variance modeling at the observational level") * In intro, authors introduce Voom as a good recently developed method and list its advantages. But a motivation for extending Voom is missing. What does Voom not handle that this paper is addressing? * SVM seems to perform well, but the main criticism of the authors is its inability to produce sparse models. SVM is frequently applied in combination with a feature selection strategy (e.g., forward selection or backward elimination). Performing SVM without feature selection is an unfair comparison. * In Equation 1: what is X.i ? * In Tables, mark the winners in bold. * Include other classifiers (e.g., SVM) also in the webtool. * Network analysis and GO analysis may not be appropriate for the selected classifier genes. Sparse set of genes that are good for classification would mask other coregulated and functionally relevant genes that are necessary for meaningful downstream analysis. * The web address in pdf is hyperlinked to "http://www.biosoft.hacettepe.edu.tr/voomDDA/.%20". The extra .%20 causes problems. * Heatmap, Network, and Go features seem to require the user having to run VoomDDA analysis first. Disable Heatmap/Network/Go until VoomDDA is executed, or automatically execute it. * Proofread the paper for syntax and writing errors. Some errors are: * counts which [are] obtained from less advancements: awkward phrasing we benefit from the delta rule: replace "benefit" lead to obtain[ing] thenumber, usingdeseq, parameterthat, thenormal, miRNAswith, andlung, isapplied

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.

Title: VOOMDDA: DISCOVERY OF DIAGNOSTIC BIOMARKERS AND CLASSIFICATION OF RNA-SEQ DATA Review round: 2 Reviewer: 1

Basic reporting: no comment Experimental design: no comment Validity of the findings: no comment Additional comments: All my concerns were well answered.

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.

Title: VOOMDDA: DISCOVERY OF DIAGNOSTIC BIOMARKERS AND CLASSIFICATION OF RNA-SEQ DATA Review round: 2 Reviewer: 2

Basic reporting: The authors have largely addressed my comments. The revised manuscript is better and I am happy with the content of this manuscript. Experimental design: As above. Validity of the findings: As above. Additional comments: As above.

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.

Title: ANTI-APOPTOTIC PROPERTIES OF CARBON MONOXIDE IN PORCINE OOCYTE DURING IN VITRO AGING Review round: 1 Reviewer: 1

Basic reporting: The work is well written and presented and the data are clear. Insufficient citations of the literature. The authors should be more comprehensive of the literature as it relates to reviews cited (e.g. Motterlini) as well as some of the seminal papers showing the effects of HO-1/CO to mediate apoptosis (Brouard, Zuckerbraun, et al.). The results are relevant to the hypotheses. Experimental design: The experimental design is reasonable, but not terribly innovative given the available tools by which to study the effects of HO-1/CO on apoptosis. The authors use an outdated CORM (CO Releasing Molecule) and are missing important controls that include the metal core and the inactive compound. The use of a phamacologics to block HO-1 should be complemented with siRNA validation given the off-target effects of Zn-PP. The use of immunohistochemistry should be validated with PCR or Western blot data to show that there is true changes versus artifact of the staining and specificity of the antibodies. From a scientific standpoint, none of the data is terribly novel given the enormous literature base. Adding a cell target for CO would strengthen the work and increase the enthusiasm that this will be an advance for the field. Validity of the findings: Data is not terribly robust with the majority of findings dependent on immunostaining. Validation with qPCR or western blot is necessary. Given that both HO-1 and HO-2 are changing almost identically draws question as to whether the antibodies are specific. No controls are provided. HO-2 is typically constitutively expressed and if there is induction, this could be an important contribution. Conclusions are reasonable, but again based on limited data sets that confirm a large literature base. Adding more detail on cell targets would be a stronger advance for the field. Additional comments: No additional comments

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.

Title: ANTI-APOPTOTIC PROPERTIES OF CARBON MONOXIDE IN PORCINE OOCYTE DURING IN VITRO AGING Review round: 1 Reviewer: 2

Basic reporting: This work studied the role of CO as an antiapoptotic molecule in porcine oocytes. The paper is easy to read and the basic ideas are clear. The english and references are appropiated. As metioned the structure is fine, but the some figures are dificult to understand and some results needs to be more robust. Specific comments: Page 7, line 38, please change "animal species" for "mammals" Page 9, line 97, please change "carbon monoxide" for "CO" Page 9, line 126, why above do you use ethanol and acetic acid and then changed to paraformaldehyde to fix the cells ? In figure 2, change HO-1 for HO-2 Figures 4 and 5 are difficult to understand, can you show your results in a clearer way ? In all the figures, please change the A, B, C nomenclature for more traditional *,**,*** for statitical significance. Why in the text you add the mean values without error or standar deviation ? Is the entire HO-1 localized in the nucleus or is a fragment of it, which could be recognized by the antibody ? I have some concerns in relation with the HO inhibitor used in this work. In my undestanding (and as you commented in the discusion secction) the Zn-PP IX is a HO inhibitor but also inhibits other enzymes and receptors such as for example: interleukin-1 receptor, GCS, and all the NOS isoformos. Therefore, the results obtained could be subtantially improved if iRNA agaist HO is used. Additionally, is suggested to use another CO donors with different structures in order to compare your results (e.g. CORM-A1). This because CORM-2 as well known produces CO, but also produce several other molecules, are you confident that any of these additional molecules are not doing anything in your model ? Experimental design: The experimental design is fine, just minor comments, see above. The only concern is that the pharmacology can be improved (see comment above) Validity of the findings: The results are novel, but they are not so robust, they need more work in terms of pharmacology and use of iRNA. Conclusions are well stated and not to speculatives. Additional comments: No additional comments

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.

Title: ANTI-APOPTOTIC PROPERTIES OF CARBON MONOXIDE IN PORCINE OOCYTE DURING IN VITRO AGING Review round: 2 Reviewer: 1

Basic reporting: The responses of the authors were reasonable. unfortunately, the data in figures 3 and 10 are not complete. The authors need to show side by side the changes in HO-1 and caspase 3 cleavage. In figure 11, why is the dose response curve showing increased apoptosis with higher amounts of CORM at 24h? Experimental design: reasonable except for the comments above about including comparators. Validity of the findings: In light of the newly added data, it is difficult to see how the conclusions are being made and now need additional data. Additional comments: Overall, the responses are adequate, but the data are somewhat incomplete (see above).

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.

Title: ANTI-APOPTOTIC PROPERTIES OF CARBON MONOXIDE IN PORCINE OOCYTE DURING IN VITRO AGING Review round: 2 Reviewer: 2

Basic reporting: The manuscript have been improved. The authors asnwered to all my main concerns. However, still remains some point to be improved. In the introduction between lines 72 and 88 is given the same (more or less) information compared to the paragraph between lines 89-97. Please fuse them in only one paragraph. Line 238, change "they were" by we In Results. Figure 2 must be the new figure 1.Therefore the sentece between lines 199 and 205 should be "The objective of the experiment was to prove the presence of HO-1 and HO-2 proteins in meiotically mature porcine oocytes (MII) and oocytes exposed to in vitro aging for the period of 24, 48 and 72 hours. HO isoforms are present in porcine oocyte (FIG 1, ex figure 2), and their expression gradually increases during in vitro aging. In case of HO-1, the signal was predominant in the nucleus/perichromosomal area (Fig 2). On the contrary, in HO-2 the signal was primarily observed mainly in the oocyte cytoplasm (Fig. 3)." In Discussion: if Zn-PP IX can inhibits the NOS...why you do not try in inhibit the NOS with L-name or other and see if the effect is similar or complementary to the CO-induced effect. Experimental design: NA Validity of the findings: NA Additional comments: NA

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.

Title: EFFECTS OF ASTRAGALUS POLYSACCHARIDE ON THE ADHESION-RELATED IMMUNE RESPONSE OF ENDOTHELIAL CELLS STIMULATED WITH CSFV IN VITRO Review round: 1 Reviewer: 1

Basic reporting: It is a solid, well-written article on the effects of Astragalus polysaccharide on porcine endothelial cells. Figures are good, literature adequate. Experimental design: The article and data are original enough to be published in the journal. The experimental design is well prepared, experiments are good and results are sound. The only problem is the lack of definition of the Astralagus polysaccharide. As it is crucial for the investigation, the respective information has to be added. Validity of the findings: Data are find, sound and interesting. Conclusion is fully based on results. List of references is adequate and relevant. Additional comments: An overall solid paper with potential clinical and/or industrial interests has one problem - the lack of definition of the Astralagus polysaccharide. As it is crucial for the investigation, the respective information has to be added. It is not possible to publish biological study without attempting the fully describe the material used in the study.

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.

Title: EFFECTS OF ASTRAGALUS POLYSACCHARIDE ON THE ADHESION-RELATED IMMUNE RESPONSE OF ENDOTHELIAL CELLS STIMULATED WITH CSFV IN VITRO Review round: 1 Reviewer: 2

Basic reporting: 1. The introductions need more details about the relationships between ECs cells, hemorrhagic diseases, and the factors tested in this studies. 2. In the Discussion section, please refer to related figures in text. The authors did that in the Results section, but they are also needed in the Discussion section. Experimental design: 1. The authors need to indicate the catalog numbers or concentrations of the antibodies used in this study. 2. In Line 84, did the authors add serum and antibiotics to the medium? 3. In Lines 93-95, please write the amounts of HPR-labeled ABC and DAB were added into each well. 4. In Line 240-243, it looked like P-selection expression occurred later than E-selection expression in Figures 2A and 2B. Since the WPBs were released quickly, the authors cannot observe that kind of difference through qRT-PCR, which is used to study the changes at transcriptional level. The authors need to explain more clearly about their proposed mechanisms. 5. In Lines 366-367, what does “puce staining” mean? 6. In Lines 369-370, the authors need to refer the different bars to the culture conditions. Also do this in figure legends 3 and 4. Also, add “either” before “medium alone”, and replace “and CSFV plus APS” by “or CSFV plus APS”. Validity of the findings: 1. My only major concern is the claims about APS’s functions. In this work, the authors studied the gene expression profiles related to cell adhesion and immune response, but did not do functional assays to confirm APS’s protective role during CSFV infection nor use APS to treat hemorrhagic infection. 2. In the abstract, the authors concluded that APS is a potential adjuvant for CSFV vaccine. However, in the last paragraph of Introduction, the authors claimed that the adjuvant part was studied in their previous work, while the aim of this study was to identify a method for hemorrhagic treatment. Please make the conclusion clear and consistent throughout this manuscript. Additional comments: The paper studied the immunomodulatory effects of Astragalus polysaccharide, the key active component extracted from a traditional Chinese medicinal herb, on endothelial cells’s immune response to classical swine fever virus. The authors stimulated the ECs with CSFV in the presence of APS and investigated the gene expression profiles of key factors related to cell adhesion, cell growth and immune response. They observed that the addition of APS could downregulate the expression of selectins, tissue factor, TLR-4, interferon and TGF beta, thus indicating APS’s potential as an adjuvant for CSFV vaccine. The authors used qPT-PCR and ELISA to quantify gene expression, which is commonly used method in this area; the statistical analysis part is good. 1. In Lines 369-370, the authors need to refer the different bars to the culture conditions. Also do this in figure legends 3 and 4. Also, add “either” before “medium alone”, and replace “and CSFV plus APS” by “or CSFV plus APS”. 2. In Line 371, RT-PCR is real-time PCR here, the “real-time” can be deleted. 3. In Lines 371 and 377, it is “SYBR Green” not “SYBgreen”.

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.

Title: EFFECTS OF ASTRAGALUS POLYSACCHARIDE ON THE ADHESION-RELATED IMMUNE RESPONSE OF ENDOTHELIAL CELLS STIMULATED WITH CSFV IN VITRO Review round: 1 Reviewer: 3

Basic reporting: In the study entitled ‘Effects of astragalus polysaccharide on the adhesion-related Immune response of endothelial cells stimulated with CSFV in vitro’, the authors have studied the effects of Astragalus polysaccharide on porcine endothelial cells. They have quantified the mRNA expression levels of E and P-selectins ,TNF, VGF and cytokines after CSFV infection and the effect of APS either by pretreatment or addition along with CSFV. The have also studied the levels of cytokines in these cultures by ELISA. Overall, the experiments are well formulated but the manuscript lacks a detailed discussion of the results and interpretation of differences observed at different time points after stimulation. The authors should discuss their observations in the light of what has already been published in the literature in case of CSFV and other viral infection. APS has been observed to regulate the levels of immunomodulating compounds in many different cell types before (Zhou L, Sci Rep, 2017; Xue H, Sci Rep, 2017; Wu S, Sci Rep, 207 etc), whether these effects on Endothelial cells are specific for CSFV or true for most viral infection should be stated. Also, APS has been shown in previous studies to have an effect on many different cell types including myocardial functions, neuronal cells, macrophages etc, therefore, in general, its effects on various different cell types have been studied and its use as an adjuvant in vaccines against viral infections may be generic and not specific for CSFV and endothelial cells. Experimental design: Line 42-46) -The basis of the study that author’s state in introduction is that vascular endothelial cells play a role in recruitment of immune cells and influence the outcome of immune response, but not sufficient literature is cited to support this. Many different cell types are involved in innate immune defense in viral infections, and the authors have cited their own study on the effect of Astralagus polyscachharide on CSFV infection in peripheral blood mononuclear cells, published in Plos One in 2012. But they fail to convince in the introduction and discussion that ECs are infact important cells involved in primary line of immune defense against CSFV infection. Other such as Borca M, 2008 (Virus Res), Dong X, 2013 (Virology J), Glaude , 2008 (J virol) have studied CSFV infection in different cell types and should be cited. (Line 51-70)- The background on Astragalus polysaccharide is hugely similar to the previous study the authors have published on the role of APS in PBMCs (Zhuge ZY, 2012, Plos One) and should be re written. Validity of the findings: No comments Additional comments: No additional comments

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.

Title: EFFECTS OF ASTRAGALUS POLYSACCHARIDE ON THE ADHESION-RELATED IMMUNE RESPONSE OF ENDOTHELIAL CELLS STIMULATED WITH CSFV IN VITRO Review round: 2 Reviewer: 1

Basic reporting: Generally, my review of the new version remains the same: An overall solid paper with potential clinical and/or industrial interests has one problem - the lack of definition of the Astralagus polysaccharide. As it is crucial for the investigation, the respective information has to be added. It is not possible to publish biological study without attempting the fully describe the material used in the study. The authors revised or explained all requests from the second reviewer., however, in case of my request, nothing really changed. Line 73 does not explain anything. Additional references support the claims, we there is still a need to explain the material, i.e., composition, purity etc. Experimental design: No comments Validity of the findings: No comments Additional comments: From the reviewer;s pioint of view, nothing really changed. Line 73 does not explain anything. Additional references support the claims, we there is still a need to explain the material, i.e., composition, purity etc.

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.

Title: EFFECTS OF ASTRAGALUS POLYSACCHARIDE ON THE ADHESION-RELATED IMMUNE RESPONSE OF ENDOTHELIAL CELLS STIMULATED WITH CSFV IN VITRO Review round: 2 Reviewer: 2

Basic reporting: No comment Experimental design: No comment Validity of the findings: No comment Additional comments: The revised version addresses all my concerns and it is well-written. I recommend to accept the manuscript for the publication on PeerJ.

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.

Title: EFFECTS OF INVASION HISTORY ON PHYSIOLOGICAL RESPONSES TO IMMUNE SYSTEM ACTIVATION IN INVASIVE AUSTRALIAN CANE TOADS Review round: 1 Reviewer: 1

Basic reporting: Overall, I thought the article was well written and set up very well with previous research. I only have a couple of comments: Ln 70: "These responses are predicted (and have been 70 shown) to be down-regulated in invasive populations (Lee & Klasing 2004; Lee et al. 2005)." Some examples of types of immune responses and what taxa would be helpful here. Ln 71: What does standing immune responses mean? Does this mean constitutive? Experimental design: Ln 194: I assume the authors mean 30 ul of Luminol. Validity of the findings: Ln 115: Add the direction of the relationship. In the table, the effect of RBCs is significant. Is this due to the autoflorensce of the RBCs? Just a brief sentence explaining this may be helpful for those not familiar with the technique. Low sample size, but it is acknowledged by authors and conclusions are framed appropriately. Additional comments: No additional comments

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.

Title: EFFECTS OF INVASION HISTORY ON PHYSIOLOGICAL RESPONSES TO IMMUNE SYSTEM ACTIVATION IN INVASIVE AUSTRALIAN CANE TOADS Review round: 1 Reviewer: 2

Basic reporting: The manuscript is clear and very well written. Title and abstract are both informative. Introduction supports the study and adequately refers to pertinent literature. However, the logic flow of the introduction should be improved to explain more effectively the main hypothesis of the study (see specific comments below). The structure of the manuscript conforms to PeerJ standards. Figures and tables are all nice and properly labelled. Raw data are provided. Experimental design: The research is within the scope of the journal and the research questions are quite clearly stated (but see specific comments). The experimental protocol has been carefully selected not only to address the specific questions of the study but also to propose a novel effective procedure to investigate immune pathways. Since a previous work (Brown et al. 2015) which investigated the same aspect (immune response) in the same two populations of cane toad did not account for inter-individual variation, the study also represents a further step in our comprehension of invaders’ eco-immunology. The research protocol is rigorous and numerous details are provided to replicate the study. Validity of the findings: Although the main prediction of the study (that is higher less costly immune response in toads from the invasion front) is not confirmed by the results, the findings of the manuscript are still valid with several suggested explanations to support the null outcome. Also, limitations of the study are clearly recognized (e.g. it did not “account for differences in environmental effects prior to collection”, L393-394) and future more advanced studies are proposed. Data is controlled and statistics used is adequate. The sample size is small and this could partially obscure the results but the limitation is well recognized in the manuscript. Conclusions are limited to supporting results and speculations are recognized as such. However, I feel that the second part of the discussion is a little bit too long (one or two paragraphs should be shortened or removed) and speculative. Only most valid speculations should be incorporated (see specific comments). Additional comments: General comments In this study, immune response and glucocorticoid response are experimentally induced in two Australian invasive populations of cane toads (one from the core and one from the periphery of the invasion) by injecting an antigen lipopolysaccharide. The study is very interesting and well supported by the exceptional literature regarding this invasive species. Although the results are not fully conclusive (no difference detected between the populations), the robustness of the protocol and the novelty of an immune response measurement in vivo make its publication recommended. Although I do not have any major revision to recommend, I have added some comments below that I hope could improve the readability of the manuscript, especially in the introduction section. Specific comments L38 - add “wild-caught” before “toads”? L55-75 - The first part of the introduction, although logic and well structured, is a little bit misleading. The authors state correctly that a down-regulation of the costly systemic inflammatory response is expected in invasive populations (or in the populations at the invasion front); however eco-immunology theory predicts also that less costly immune pathways should be up-regulated by invasive populations or species (see for example “humoural” response in Lee and Klasing 2004). The authors, later in the text, hypothesize exactly this up-regulation (also in accordance to a similar study conducted by Brown et al. 2015 that compared the same two invasive populations). Therefore, they should clarify here (and not later, at L89-91) that two divergent eco-evolutionary scenarios (downregulation of costly immune pathways and upregulation of less costly immune pathways) are expected in invasive (or invasion front) populations. This should be helpful, especially for a general reader, to comprehend the aim of the study, its assumptions and also the main prediction reported at L129-132. L73 – “in human neutrophils” Could you find more references, not necessarily in humans, that support this? L76-77 “To Australia from Hawaii” - The cane toad did not arrive in Australia directly from the native range (South America) but rather it was moved from one area to another (e.g. Hawaii) until it established in Australia. It means that during its invasion history, it has been exposed to many different groups and sources of pathogens. Could have this promoted a strong less costly response of the immune system that become canalized (less plastic) in the invasive Australian populations? Since you are not comparing a native pop with an invasive population but two invasive populations, this should be somewhere stated in the manuscript, maybe in the discussion. L90-91 I would suggest “less costly” instead of “not costly”. Any immune response should have a cost, although minimal. L 104 “because this antigen is no longer part of a live organism, mutation and replication are not possible”. Any reference for this? L107-108 “We applied this experimental method to clarify immune function in different populations of invasive cane toads.” This sentence is too generic, please rephrase. L139-140 Could you please add the geographic coordinates for the two sites of capture (Cairns and Oombulgurri)? Also, a map of Australia with the cane toad invasion, although not essential, could help the reader to visualize introduction point and current invasion front. L290-291 “Contrary to our predictions, responses to LPS exposure did not differ significantly between toads from the invasion front population versus the range core population”. Your prediction was more specific than that because you were expecting an up-regulation; please rephrase. L291-293 “Our predictions were based upon the energetic costs associated with mounting immune responses while undergoing enemy release.” This sentence is too generic, please rephrase. Also, as many different types of immune responses can occur and some are not costly, I would suggest being more specific across the whole manuscript for example distinguishing between systemic inflammatory responses and humoral responses (as stated in Lee and Klasing 2004). L310-319 This could be summarized in only two sentences. L 330 “More mobile toads”. Please clarify this for a general reader. Do they move more frequently? Do they move longer? L 370-376 Since Brown et al. 2015 did not detect any sex effect, this paragraph could be removed because it does not add anything essential to the interpretation of your results. L574 Please fix reference (space issue) In the tables. Significant results in bold? Also, p-value is not defined anywhere in the manuscript.

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.

Title: QUALITATIVE STUDY OF HEALTHCARE PROVIDERS’ CURRENT PRACTICE PATTERNS AND BARRIERS TO SUCCESSFUL REHYDRATION FOR PEDIATRIC DIARRHEAL ILLNESSES IN KENYA Review round: 1 Reviewer: 1

Basic reporting: Clear English: Overall, the language used was clear, unambiguous, and reflects current professional standards. Some minor grammatical errors. Specifically, place a comma after using "despite" when followed by a contrasting clause (ex. lines 52 & 95). Additionally, some of language included may benefit from minor revision (examples below). Line 110: Use of the phrase “in our experience” seems very anecdotal. Though there may be very little, if any, research regarding the use of NGs, consider including literature that looks at which rehydration practices are most used in Kenyan healthcare settings. While your experiences are more than valid, offering a more concrete reference, if available, would strengthen your point. Line 113: “There is no literature” is a very strong statement. While I am sure your review of the available literature was exhaustive, you may want to avoid such an absolute. Consider using “based on an exhaustive (or extensive) review of the literature…” or “No literature pertaining to x was identified”. References: Most of your references are 6 or more years old. One is almost 30 years old. While many seem relevant to the current study, consider including literature published in the last 5 years. Line 92: You use a CDC reference from 2011. I would encourage you to use a more current reference. There are mortality estimates--those cited in CDC reports--that have been published in the last 3 years. Line 94: To my knowledge, cholera does not drive diarrheal rates among infants and young children in Kenya. Consider referencing the pathogen(s) that drive diarrheal rates among this population in this setting. The Global Enteric Multicenter Study has a number of published papers that could provide some additional information. Figures and tables: Table 1 could use some additional development. Consider including more demographic/background information in table 1, if available. Self-contained: Overall, the paper was strong and well-written. However, there are several areas of the paper that could benefit from additional consideration. Specifically, the methods and results sections (discussed below). Experimental design: Original research: The research is original and fits well within the scope of the journal. Research question: The research question is clear and fills a important knowledge gap in the literature. Rigorous investigation: Based on the information included, the research meets high technical and ethical standards. However, I do have one question regarding consent. Line 126: Since your study population was limited to pediatric care providers, all of whom are likely literate, why did you only collect verbal consent? Verbal consent is generally reserved for studies exploring socially sensitive/taboo topics or for populations where literacy is limited. Were there barriers to collecting written consent? Methods: When writing up a methods section, I generally work to answer the who, when, what, where, and how of the study. While simplistic, it serves to ensure that at least the basic components of the methods section are articulated. While you clearly address the where, who, and what - where (western Kenya), who (44 healthcare providers at various facilities around MTRH and AMPATH), what (a case-scenerio and semi-structured interviews), you do not provide any information as to “when” the study was conducted, “how” participants and health facilitates were selected for inclusion or “how” much time each participant contributed to the study. Further, I do not see any reference to your sampling technique. Though based on the description offered, it seems as convenience sampling was used. This should be made explicit. Noting the sampling techniques used, even when describing a study with a small sample, provides the reader with a better frame to interpret the results. Also, please describe the number of healthcare facilities in the region and the number you planned to include in the study. Line 148-156: The description of the study population needs to be improved. As noted, be sure to include a more developed description of sampling techniques used. As it stands, it is unclear if you selected either the research sites or the individual participants based on convenience, purposive, snowball, and/or random sampling. Though you have a small sample, this information in necessary for framing the results presented. Also, would community health workers receive training regarding NGs and/or would they typically be expected to administer them or be familiar with the process. If this is not included in their training, their responses may skew the results. Lastly, how many healthcare facilitates sit within a certain radius of MTRH and AMPATH and how many of these healthcare facilities were represented in data collection? Validity of the findings: Once other comments are considered, you may want to update the discussion and conclusion accordingly. Additional comments: Results: You seemed to lump all providers together thereby failing to acknowledge the varying levels of training received. Community health workers (CHWs) receive far less training than clinicians. Moreover, given the level of training received by CHWs, should they be expected to be familiar with the administration and/or use of NGs for rehydration or even the related WHO guidelines? There are arguably 3 or 4 levels of providers with regard to training. However, the results section, as currently written, does not seem to consider the extent to which provider training may influence NG use. You essentially treat all providers as equal leaving me with several answered questions: Were specific provider types more likely to describe NGs as less effective? Were specific provider types more likely to be familiar with the WHO guidelines? Did this change according to practice type (i.e. providers at a referral hospital are more likely to use the procedure)? Other similar considerations are offered below. Line 266: How many providers out of 44 were unfamiliar with the guidelines? Again, did you look at this according to provider type? Or practice type? Further, is it possible that the results were impacted by other factors? How long did each provider practice? How certain are you that these approaches/techniques were taught? At some point prior to the study, did all providers receiving training on the administration of NGs? Was this influenced by provider type? Additionally, nurses represented almost half of the participants included. How might this impact the study results? Additional general comments: Title: Rehydration can occur both within and outside of healthcare settings. Your title should reflect your focus on healthcare providers/healthcare settings. Line 104: You suggest that NGs can be applied before arrival to the hospital. Given the settings and the more typical modes of transportation used when trying to access healthcare, who would be applying the NGs before arrival. As written, it implies that non-healthcare providers, those involved in transport, could perform the procedure. Line 164: Consider writing out emergency department rather than using the acronym “ED”. Line 175: Every interview is not likely to be a “key-informant” interview in the strictest interpretation of the term. I would encourage you to remove that language as it does not add anything to the sentence and may distract the reader from the main point you are trying to make. Line 183: See comment regarding line 126. Line 204: Be specific about the type of triangulation referenced (investigator triangulation). Line 209: In general, you should avoid beginning a sentence with a number. Spell out the number 44. Line 313: Consider this statement in concert with the statement regarding line 110. None the studies you allude to are clearly references above.

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.

Title: QUALITATIVE STUDY OF HEALTHCARE PROVIDERS’ CURRENT PRACTICE PATTERNS AND BARRIERS TO SUCCESSFUL REHYDRATION FOR PEDIATRIC DIARRHEAL ILLNESSES IN KENYA Review round: 1 Reviewer: 2

Basic reporting: The English language is good, fluent, correct and clearly understood. Experimental design: There are 3 main problems with the study: 1. According to Good Clinical Practice (GCP) one needs an approval for a study, and informed consents, for studies dealing with patients. This study does not deal with patients: it deals with caregivers. This study is a questionnaire for caregivers. Well, it is the same as examination, so it needs neither approval, nor informed consents. 2. The sample size is 44 caregivers. The reader has no idea about the population size of caregivers out of which the sample was taken, and its distribution. It could be that the sample was too small, and hence the study may not make any conclusion regarding the population of caregivers in Kenia. Moreover, the authors did not explain how they chose the participants in the sample, so even the authors have no idea if the sample that they chose is representative. 3. According to WHO guidelines, one does NOT use NG tube after failing an attempt placing IV line; rather, one uses NG tube BEFORE even trying the first attempt placing IV line! So, the questionnare should be changed, all the study should be redone. Validity of the findings: The findings can not be discussed, as there are the study is mal-designed. Additional comments: I am sorry, but the manuscript is not acceptable for publishing anywhere, due to serious design mistakes.

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.

Title: MECHANISMS OF ACTION AND IN VIVO ANTIBACTERIAL EFFICACY ASSESSMENT OF FIVE NOVEL HYBRID PEPTIDES DERIVED FROM INDOLICIDIN AND RANALEXIN AGAINST STREPTOCOCCUS PNEUMONIAE Review round: 1 Reviewer: 1

Basic reporting: ... stated in 'General comments for the author' Experimental design: ... stated in 'General comments for the author' Validity of the findings: ... stated in 'General comments for the author' Additional comments: In the manuscript #18224v1 by Jindal and colleagues “Mechanisms of action and in vivo antibacterial efficacy assessment of five novel hybrid peptides derived from Indolicidin and Ranalexin against Streptococcus pneumoniae”, authors aim to investigate the mechanisms of action and the therapeutic efficacy of the hybrid peptides. Among other things authors assessed the effects of those peptides on the integrity of the pneumococcal cell wall/membrane. They also measured by DNA retardation assay, the impact of the peptides on the integrity of, and binding to genomic DNA of those microbes. The in vivo-toxicity and antibacterial activity was evaluated on ICR male mice. Authors have found that the hybrid peptides caused significant morphological alterations in Streptococcus pneumoniae and disrupted the integrity of the cell wall/membrane. The rapid release of ATP from pneumococcal cells after one hour of incubation suggests that the antibacterial action for the hybrid peptides is based on membrane permeabilization and damage. The DNA retardation assay revealed that at 62.5μg/ml all the hybrid peptides were capable of binding and preventing the pneumococcal genomic DNA from migrating through the agarose gel. In vitro synergy was observed when pneumococcal cells were treated with combinations of hybrid peptides with each other and with conventional drugs erythromycin and ceftriaxone. The in vivo therapeutic efficacy tests revealed that the hybrid peptide RN7-IN8 could improve the survival of pneumococcal-infected mice, as 50% of the infected mice survived up to 7 days post-infection. In vivo antibacterial efficacy of the hybrid peptide RN7-IN8 was significantly improved when combined with the standard antibiotic ceftriaxone. The presented results suggest that breaching the cell wall/membrane is likely the principal mechanism of action of the hybrid peptides. Furthermore, the hybrid peptides may affect DNA synthesis and gene expression. This is potentially-interesting manuscript, however it requires a lot of improvements. Improvements suggestions: 1.) Manuscript’s English language is so bad that often it is difficult to understand the meaning of the sentences (even in the summary). The manuscript MUST BE edited by a native English scientist prior to resubmission. 2.) Please state in the figure legends 3 and 4, from how many experiment-repetitions the data has been obtained. 3.) The data displayed in figure 3 is poorly presented. It should be rearranged and preferably split into 2 subfigures (with controls included in both), so that the ATP-release is seen as time kinetics. 4.) The discussion needs to be expanded, especially regarding possible molecular mechanism(s) of the interactions of the dipeptides with bacterial membranes. Authors may get inspired by recent papers by Ghavami et al (doi: 10.1111/j.1582-4934.2008.00129.x), and Savelyeva et al., (doi: 10.1007/978-1-4471-6458-6_10).

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.

Title: MECHANISMS OF ACTION AND IN VIVO ANTIBACTERIAL EFFICACY ASSESSMENT OF FIVE NOVEL HYBRID PEPTIDES DERIVED FROM INDOLICIDIN AND RANALEXIN AGAINST STREPTOCOCCUS PNEUMONIAE Review round: 1 Reviewer: 2

Basic reporting: Overall, the English is acceptable. However, the first sentence shows an unsual structure. Reference style needs to be optimized (capitals in journal titles) The authors use hybrid AMPs but do not explain what sort of hybrids they used; a reference to a previous paper is not enough information for a reader. Introduction and Discussion need to be optimized; do not repeat information in both parts. Experimental design: Quality of EM pictures is low and not very informative. The effect of AMPs is time and dose dependent; provide such data. ATP release: provide time and dose dependent data AMPs can work within minutes; proide data for 10, 20, 40 min DNA binding: why do you assume that AMPs bind to DNA; explain your rationale; Fig 4 is not convincing; do you have a positive control for a DNA binding agent? Kaplan Meyer curves: Better than control would be: healthy animals without infection Not untreated: better infected animals without antimicorobial treatment Validity of the findings: Without dose and time dependent data, this paper is rather premature. It needs more laboratory work to be conclusive. Additional comments: Without dose and time dependent data, this paper is rather premature. It needs more laboratory work to be conclusive.

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.

Title: MECHANISMS OF ACTION AND IN VIVO ANTIBACTERIAL EFFICACY ASSESSMENT OF FIVE NOVEL HYBRID PEPTIDES DERIVED FROM INDOLICIDIN AND RANALEXIN AGAINST STREPTOCOCCUS PNEUMONIAE Review round: 1 Reviewer: 3

Basic reporting: The authors of this report present us with an interesting study. The contribution of this paper proves significant in determining the action mechanism of new antimicrobial peptides. The investigation about new molecules with antimicrobial activities is currently under study. Introduction to this paper is comprehensive, and it provides a clear support for both the theoretical aspects and the research objectives. Literature reference is sufficient to support the study. The article structure is clear and figures and tables are appropriately used. Some suggestions and points that need clarify are in the revised version. Experimental design: The article is an original study, the experiments assays are appropriately designed, however in some cases the materials and methods section requires more detail and more clarity in order to be useful to future readers. The authors should be providing information about the reproducibility of findings. How many experiments were carry out? Validity of the findings: No comment Additional comments: The article is original and was appropriately discussed. The comments and suggestions to make it better are in revised version (pdf).

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.

Title: MECHANISMS OF ACTION AND IN VIVO ANTIBACTERIAL EFFICACY ASSESSMENT OF FIVE NOVEL HYBRID PEPTIDES DERIVED FROM INDOLICIDIN AND RANALEXIN AGAINST STREPTOCOCCUS PNEUMONIAE Review round: 2 Reviewer: 1

Basic reporting: . Experimental design: . Validity of the findings: . Additional comments: I am satisfied with the revision.

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.

Title: MECHANISMS OF ACTION AND IN VIVO ANTIBACTERIAL EFFICACY ASSESSMENT OF FIVE NOVEL HYBRID PEPTIDES DERIVED FROM INDOLICIDIN AND RANALEXIN AGAINST STREPTOCOCCUS PNEUMONIAE Review round: 2 Reviewer: 2

Basic reporting: My main concern and criticism has not been adressed. Introduction and discussion are not consice enough; omit information which is not necessary to understand this paper Experimental design: My concern addressed the quality of the experiments. It demands that some experiments need to be repeated and proper controls need to be used. Validity of the findings: without controls the results remain difficult to interpret Additional comments: the quality of the figures is still very bad; colours cannot be seen. Figures are very crowded and not well designed

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.

Title: MECHANISMS OF ACTION AND IN VIVO ANTIBACTERIAL EFFICACY ASSESSMENT OF FIVE NOVEL HYBRID PEPTIDES DERIVED FROM INDOLICIDIN AND RANALEXIN AGAINST STREPTOCOCCUS PNEUMONIAE Review round: 2 Reviewer: 3

Basic reporting: The basic report is include in general comments for the author Experimental design: No comments Validity of the findings: No comments Additional comments: The second version of the paper “Mechanisms of action and in vivo antibacterial efficacy assessment of five novel hybrid peptides derived from Indolicidin and Ranalexin against Streptococcus pneumoniae” was appropriately revised. However, some improvements are required. 1. Figures: The figure 2 should be described appropriately on the figure legends. In the actual figure 3 the authors should be include standard deviation and explain carefully the data of significant differences. 2. I suggest that the authors review the use of abreviations of Units following the international system of Units. Por example: abbreviation for “hora” should be “h”. Use the space between number and unit.

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.

Title: MECHANISMS OF ACTION AND IN VIVO ANTIBACTERIAL EFFICACY ASSESSMENT OF FIVE NOVEL HYBRID PEPTIDES DERIVED FROM INDOLICIDIN AND RANALEXIN AGAINST STREPTOCOCCUS PNEUMONIAE Review round: 3 Reviewer: 1

Basic reporting: I am satisfied with the revision Experimental design: I am satisfied with the revision Validity of the findings: I am satisfied with the revision Additional comments: I am satisfied with the revision

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.

Title: INTEGRATION OF LNCRNA–MIRNA–MRNA REVEALS NOVEL INSIGHTS INTO OVIPOSITION REGULATION IN HONEY BEES Review round: 1 Reviewer: 1

Basic reporting: The manuscript is flawed because it did not make clear the major question and the hypothesis of the study, neither in the abstract nor in the introduction and/or discussion sections. The authors did not cite literature already published in Apis mellifera, which make important relationships for the robustness and support of this manuscript: (1) ovarian activity and microRNAs (Macedo et al., 2016 - Insect Mol Biol. 25(3):216-26.); (2) vitellogenin and microRNAs (Nunes et al., 2013 - J Exp Biol. 216(Pt 19):3724-32.); (3) EcR-mRNA-microRNAs (Mello et al., 2014 - Front Genet. 5:445.); (4) caste and microRNAs (Guo et al., 2013 - PLoS One. 8(12):e81661.; Shi et al., 2014 - Apidologie. 46:35); (5) ovary, caste, mRNA, long noncoding RNAs (Humann and Hartfelder, 2011, Insect Biochem Mol Biol. 41(8):602-12.; Humann et al., 2013. PLoS One. 8(10):e78915.; Jayakodi et al., 2015 - BMC Genomics. 16:680.; Lago et al., 2016 - Insect Biochem Mol Biol. 79:1-12.). Figures, tables and legends are not clear and should be improved. For example: What does A, B and C mean in Figure 1? Experimental design: In my opinion, the research would fill an identified knowledge gap if question was well defined (but it is not well defined) and if data were appropriately analyzed. Methods are not described with sufficient information to be reproducible by another investigator. For example: (1) how lncRNA's target genes were predicted? (2) how many genes were validated by qPCR (5 lncRNA as in line 165 and 298? or 9 as in "result sheet" of Table S1? or 10 (used actin as reference gene?) as in "primer sheet" of Table S1? (3) What does microarray mean in line 170? (4) Line 152: What does "trans role is to identify each other by the expression level" mean? Validity of the findings: Sequencing data appear to be of sufficient quality and preliminary analyzes were well performed. However, the experimental design of comparisons to know whether a gene is differentially expressed is unclear. What experimental design was used: reference design? loop design? saturated design? others? They used DESeq to compare any two groups (lines 132-135) / discriminate between two comparison groups (lines 179-180), however I'm not convinced that this is the best form of analysis. I would have agreed with authors if they had been performed all possible pairwise comparisons. If they have done all them, this is not clear from the text of the manuscript. Finally, the authors performed an unsupervised hierarchical clustering analysis, which is fine (great job!). But they need to clarify in more detail about the use of each approach and the parameters used in each of them. How lncRNA's targets were predicted? About the construction of networks, the authors assumed two expression patterns: (1) up-regulated lncRNA / mRNA versus down-regulated miRNA or (2) down-regulated lncRNA / mRNA versus up-regulated miRNA. In what literature have the authors based themselves on assuming such premises or supporting such expression patterns? In my view, conclusion are not well stated and not limited to supporting results. The queen's reproduction regulation was not elucidated (lines 375-378). The conclusion does not present the answer of a scientific question (knowledge gap). There are many speculations, but they have not been identified as such. Additional comments: Dr. Xiao Chen and colleagues investigated the expression of genes (mRNA, miRNA and lncRNA) in queen ovaries undergoing four different contexts () in order to understand the molecular events associated with prolificacy. Despite the quality data, the written information in the manuscript is vague, generic, confusing or inconclusive. In general, it seems more a making-list + long descriptions than the expected interpretive deepening of the data. Below, some points and suggestions: 1 - Prolificacy is an argument used in the abstract, in the beginning of the introduction and in line 340 of the discussion. I suggest that the authors use the data generated to explain this term "molecularly". Throughout the text, it is very confusing to understand in which situation a gene is up- or down-regulated. The authors should make clear which is the reference sample, and always declare something like that: "up-regulated in situation A compared to situation B (or situations B, C and D)", for example. It is not clear from the text what the new insights are. The found GO terms are too vague and / or generic (for example, system and organism development / metabolic process / etc.) so that the knowledge can not be advanced/deepen. Line 129: Did miRNA target prediction use only 3'UTRs of target genes? Please, clarify it. Lines 160-170: About Quantitative qPCR, how many genes were really validated? I observed primers designed for Gene ID 726542 (histone H3) but no results. Also, if authors used oligodT primer, it means that all validated genes are polyadenylated, are not they? Line 290: network of ovaries activation or network of oviposition recovery? Please, clarify it. Lines 331-352: microRNAs such as let-7, miR-316, miR-252, miR-1, miR-133, miR-375, miR-31a, miR-92a, miR-3049, miR-3718 should be better (Nielsen et al., 1994), and the results obtained in the present study were similar to those reported in the present study (Macedo, et al., 1991, Insec Mol Mol Biol 25 (3): 216-26). Exp Biol. 216 (Pt. 19): 3724-32.). In addition, please also observe all the criticisms / comments / suggestions presented in the sections above: 1. Basic reporting, 2. Experimental design, 3. Validity of the findings.

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.

Title: INTEGRATION OF LNCRNA–MIRNA–MRNA REVEALS NOVEL INSIGHTS INTO OVIPOSITION REGULATION IN HONEY BEES Review round: 1 Reviewer: 2

Basic reporting: The English in the manuscript needs to be improved. In many instances the language does not make the meaning of the sentences unclear, but it would still be improved considerably by having a native English speaker review the manuscript before resubmission. An example is lines 346-347: “Ame-miR-315 down regulated in ovaries activation and target 253 mRNAs in the network.” This should say: “Ame-miR-315 is down regulated in the ovary activation samples and targets 253 mRNAs in the network” It could also say “… regulated in activated ovaries and targets…” Along these lines, while “ovaries” is the plural of ovary, when referring to a sample type (e.g. ovary activation) it should be singular because it is a category. This is done in the introduction and the figure legends, but not in the rest of the manuscript. There are some cases in which it is unclear what the meaning of a sentence is. Line 152: “Trans role is lncRNA to identify each other by the expression level.” This sentence doesn’t make sense. A google search reveals that this is used multiple times, virtually verbatim in several publications over the last 3 years. These other papers seem to be making the distinction between cis and trans effects that lncRNAs can have (acting on neighboring genes, cis, or acting on distant genes, trans). However, “identify each other by the expression level” is completely unclear. How does a lncRNA “identify” something based on expression level? The remainder of the paragraph says that the authors looked for correlations between the expression levels of lncRNAs and coding genes. If the first sentence is meant to state this, it would be better stated as something like: “The potential trans role of lncRNAs (acting on non-neighboring genes) can be assessed by correlating expression levels between lncRNAs and mRNAs.” Importantly, this sentence and the sentence following it are nearly identical to those used by other authors, particularly Lu et al., (2016). I understand that some technical phrases are difficult to rewrite and I am not accusing the authors or plagiarism, but it is important to rework these ideas into your own words. This case is particularly poignant because the sentence is completely ambiguous and has been passed down over several papers. I have also found the interpretation to be difficult due to potentially ambiguous use of the term “target.” Targeting has a directionality to it and finding that X targets Y doesn’t mean that Y targets X. Lines 74-75: “…lncRNAs can also be directly targeted by miRNAs for cleavage.” This line is clear and is directly supported by the citations, they show that miRNAs target the lncRNAs (miRNA acts upon the lncRNA), not the other way around. Lines 155-156: “…lncRNAs and mRNAs which linked by the same target miRNAs, with…” In this case, the sentence states that the lncRNAs and mRNAs both target (act upon) one particular mRNA. As far as I am aware this is not the direction in which this works, mRNAs and lncRNAs are targeted by miRNAs (miRNAs act upon the others) and not the other way around. The Fan et al., (2015) paper supports this, the lncRNAs can influence mRNA expression level by interfering with the miRNAs that normally act upon that mRNA, but they interfere by being targeted by the miRNAs, not by targeting the miRNAs. I think that the Fan et al., 2015 reference does a good job in making this clear in their language (“lncRNAs acting as miRNA targets”). The article contains many other grammatical, spelling, and formatting problems. Some of these should be caught by spell-checking software (e.g. line 156 “up-regualted”). There are a number of cases in which spaces are omitted (lines 126, 128, Figure 1 and Table 2 legends). These may be due to pdf formatting, but should be checked. Similarly, in the note of the Table 2 legend, the “Q” group seems to have a copy/paste error “Q, group of virgin queens normal oviposition queens;”. Results section, Lines 234-296. These 6 paragraphs are tedious to read, if only because they are largely the same format and just report the two main patterns (up/down/up or down/up/down) in the three ovary states (activated, inhibited, recovery). Could these be incorporated into a table rather than being written out? If this can be done, it would make the results, and therefore the paper, much more concise and would make it easier to focus. Line 259-260: What is meant by “lncRNAs target miRNAs from 2 to 3” or “miRNAs target mRNAs from 9 to 18”? Does it mean that the lncRNAs have from 2 to 3 target miRNAs? (however, see note above about use of “target”) If this is the case, then it should be spelled out. E.g. lncRNAs act as targets for 2 to 3 miRNAs. Figure 1: Why does the mRNA heat map show all 3 samples for each treatment, but the others only show a single column for each treatment? Supplemental files: I appreciate the supplemental files as it makes it much easier to assess the results. However, it would be helpful to have a single file that includes all the details about the supplemental tables, including explanations of headers in those files. The header explanations could be included as a separate sheet in the table workbooks. I didn’t look at every entry in every supplemental file, but I found two errors. The first sheets in Supplemental Tables 3 and 5 have rows that only contain FBgn names and they look like a pasting error (S3 row 2687, S5 rows 1644 and 1646). The rest of the files should be checked for these types of errors. Experimental design: This work is original primary research that fits within the aims and scope of PeerJ. Overall, the experimental design and data collection appear rigorous. However, the methods need to be more clearly spelled out. I lay out some questions that should be addressed in a new version. Lines 151-159. What are some of the specifics of constructing a network? 1) Line 155-156: This states that lncRNAs and mRNAs were linked by the same target miRNA (presumably they are targeted by the same miRNA). However, lines 224-229 states that the network was constructed based on the correlation analysis, which looked just at expression levels. Which is the case? 2) What does it mean to have correlated expression? Do they have to have the same pattern (e.g. both upregulated), or do they have to have the same magnitude within a pattern (e.g. each shows a 3.5 fold upregulation)? 3) How were targets determined? Line 154 says that miRNA targets were determined using miRanda, but what parameters were used? 4) Why wasn’t GSTAr.pl run like Fan et al., 2015? That seems like an important analysis to determine whether a lncRNA is a decoy vs a target of a miRNA. While the manuscript isn’t explicitly about decoy vs target, the authors ultimately want to conclude that lncRNAs are acting to regulate mRNAs through this network, and they seem to rely on the lncRNAs acting as decoys. 5) Lines 93-96: Were these ovaries from a single queen? It seems like this is the case, but explicitly stating it would be helpful. 6) Lines 131-138: It is never explicitly stated what comparison is used for the DE analysis. Based on the categories of DE used in Figs 2-4, it seems that over- and under-expression are based on comparisons with the virgin queens (i.e. overexpression means: Q>V, C>V, R>V and underexpression means: Q<V, C<V, R<V). This should be made clear in the methods. Validity of the findings: Apart from the language issues and making the results more concise, my chief concern is the network analysis interpretation. Here is my understanding based on the manuscript: RNAs were considered to be in the same network if they showed one of the two patterns lncRNA-miRNA-mRNA (up-down-up or down-up-down). lncRNA and mRNA seem to also be linked by being targeted by the same miRNA (line155-156, see comment in experimental design section). I think that this network analysis makes sense when considering the expression levels, but I think it goes awry when trying to look at functional classifications because it begs the question (i.e. it is circular). GO terms and KEGG pathways were determined for lncRNAs and miRNAs by looking at the functions of their targets, which are mRNAs. These mRNAs should be already in the network, since the network is defined in part by the shared targets (one miRNA targeting a lncRNA and an mRNA). If this is correct, then that means that any functional analysis of lncRNAs or miRNAs should find considerable overlap between these RNAs and the mRNAs. This leads to spurious conclusions, like lines 204-205: “The result revealed that the lncRNAs and mRNAs interacted with each other and promoted the ovaries activation and the subsequent oviposition process.” This is exactly what you would expect based on the analysis regardless of any actual interaction. I’m not saying they didn’t interact, but the only evidence of that is the shared miRNAs and the expression pattern, the functional analysis adds no information. I think that the functional analysis can stay, if done solely at the mRNA target level and not attempting to compare it with the lncRNA or miRNA. This will still be helpful for understanding what the genes in these networks do, but it can’t be used to support the existence of the network itself. More broadly speaking, I think the manuscript can be improved with a bit more discussion of what these processes mean for ovary activation and development. There are a lot of GO terms and KEGG pathways stated in the results and it would be helpful to have a more in depth biological discussion. The current manuscript relies heavily on the network analysis, but I think this should play a smaller supporting role. It seems that the main story should be about the biological processes involved in ovary regulation and the network analysis is a method of finding what functions these ncRNAs might have. Additional comments: In the manuscript, “Integration of lncRNA–miRNA–mRNA reveals novel insights into reproductive regulation in honey bees,” Chen et al. report on expression levels of long-noncoding RNAs, miRNAs, and mRNAs in the ovaries of queen honey bees in four conditions of egg laying. These are: virgin queens not laying eggs, normal queens laying eggs, caged queens that are restricted from laying eggs, and recovering queens that were restricted but have been allowed to begin laying eggs again. They analyzed expression regulatory networks by comparing expression levels and potential targets between these categories of RNAs and by comparing the predicted functions of these genes (using GO and KEGG analyses). I think that this project is very exciting, that the experiments and data are solid, and that the results will be very useful for our understanding of honey bee reproductive biology and how it is regulated. However, I have concerns about the analyses and presentation of the work. I recommend that the manuscript undergo major revisions to address these concerns and to make the manuscript more concise.

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.

Title: INTEGRATION OF LNCRNA–MIRNA–MRNA REVEALS NOVEL INSIGHTS INTO OVIPOSITION REGULATION IN HONEY BEES Review round: 2 Reviewer: 1

Basic reporting: The manuscript was substantially improved. However, language still needs many tweaks and improvements. Several sentences remain vague, confusing or meaningless. This makes it difficult to read and consequently to understand the manuscript. Experimental design: Language needs to be improved to allow full understanding of the text. Validity of the findings: Language needs to be improved to allow full understanding of the text. Additional comments: Language needs to be improved to allow full understanding of the text. Also, please provide nucleotide sequences (or GenBank accession number) of all data named as TCONS_? How can authors know that many TCONS_ sequences are genuine lncRNA? To my knowledge, 20E is a symbol used for 20-hydroxyecdysone.

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.

Title: INTEGRATION OF LNCRNA–MIRNA–MRNA REVEALS NOVEL INSIGHTS INTO OVIPOSITION REGULATION IN HONEY BEES Review round: 2 Reviewer: 2

Basic reporting: 1 The English has been improved in the manuscript, removing many ambiguous sentences. However, there are still many grammatical errors throughout. 2 Lines 42-44: The first sentence makes a very broad statement with no citation included. What are the biological constrain(t)s that are going to be ameliorated? If it’s the complex genetic architecture, then this is circular reasoning. These sentences together state that the way to ameliorate the problem of complex genetic architecture of reproduction is to better understand the genetics of reproduction. 3 Lines 65-66: This is another very broad statement with no citation. 4 Figures and tables: The network figures (Figures 2 and 3) are large, crowded and not readable. If these kinds of network figures are included, they should be scaled down or altered to guide the reader through the network. As it stands, they are simply too complex to serve as a main figure. If the full network needs to be included, then it should go in the supplement. Figure 4 is still very large, but this one at least shows that many of the genes in the pathways are DE in this study. Table 3 isn’t a table, it’s a list. Again, if the authors feel this is necessary then it should be in the supplement. In that case, it can be converted into a useful format (spreadsheet) so that each gene has it’s own row, so that readers can easily sort or copy IDs. 5 In my previous review, I brought up concerns about performing functional analyses (GO and KEGG) on the lncRNA and miRNA, because those analyses rely on using networked partners of those RNAs (they use the mRNAs). This was point #19 in the previous review. While the authors did clarify their methods for making the network and don’t use the functional analyses to support the existence of a network, they still performed the functional analyses on both lncRNAs and miRNAs and proceed to discuss how similar the results of those analyses are to the results of the mRNA analysis. I don’t see any way that the results could not overlap because the three RNA types have already been shown to be in a network (based on expression correlation). The function of the lncRNAs and miRNAs are defined by the mRNAs with which they interact, so of course they overlap. If there is something I’m missing on why this analysis was performed and why it’s discussed, then that needs to be made very clear in the manuscript. It currently is not. 6 Lines 329, 365, and 431: None of these sentences should use the term “proved”, these are scientific hypotheses that have been supported. Experimental design: The authors have improved the description of the methods significantly. Knowledge Gap. I think that the work attempts to fill a gap in our knowledge of the genetic regulation of oviposition in honey bees. I’m not so sure that they spell out that knowledge gap very well (see comments above regarding lines 42-44). The manuscript fits within the aims and scope of the journal. Validity of the findings: I appreciate the addition of the QTL for ovariole number, I think it improves the strength of the current conclusions. However, the language makes it hard to determine what are new conclusions and what are conclusions from previous work. Lines 179-181 make it sound like this was a QTL analysis performed for this manuscript, but it was the work of Linksvayer and Graham. I don’t think this is intentional, but simply stating that it was “previously localized to…” would clarify. This same issue comes up a lot in the discussion. I appreciate that the authors are discussing their findings in relation to what is known, but it is not always clear when new conclusions are being made and when they are simply stating the previous conclusions. The use of the past tense also makes this more difficult (e.g. lines 587-588, “Nej was considered…”) Additional comments: I appreciate the fuller discussion of the the genes in relation to previous work, however I still feel that the manuscript could be improved by having a common thread for the reader to focus on throughout the discussion. In some ways the discussion feels like a list of facts about these pathways without being tied back together into a proper conclusion. I understand that a lot of this work results in new candidate genes to be further investigated, but more comprehensive conclusions would help to guide the reader.

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.

Title: INTEGRATION OF LNCRNA–MIRNA–MRNA REVEALS NOVEL INSIGHTS INTO OVIPOSITION REGULATION IN HONEY BEES Review round: 3 Reviewer: 1

Basic reporting: no comment Experimental design: no comment Validity of the findings: no comment Additional comments: The authors adequately addressed all of my questions/suggestions (as well as those of the other reviewer), clarifying them satisfactorily and significantly improving the quality (and also the language) of the manuscript. The study increases our understanding of the molecular relationships between the non-protein-coding layers of bee genome and the reproductive biology of Hymenoptera. I recommend that the revised version of the manuscript be accepted for publication at PeerJ.

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.

Title: A RANDOMIZED CONTROL TRIAL FEASIBILITY EVALUATION OF AN MHEALTH INTERVENTION FOR WHEELCHAIR SKILL TRAINING AMONG MIDDLE-AGED AND OLDER ADULTS Review round: 1 Reviewer: 1

Basic reporting: no comment Experimental design: no comment Validity of the findings: no comment Additional comments: Authors have done a wonderful work describing their feasibility study which would help them carry out a larger randomized controlled trial in future. The manuscript is very well written in professional English. I have few minor specific comments which are detailed below: L40: Please mention if the values in L41 are Mean+/-SD or Mean+/-SEM or something else? L47: "questionnaire." L148: Consider replacing "reticence" with something simple. L219: "was randomly assigned" L224: "the group" Figure1: Consider reducing the size of Figure 1 L258: "were 44" L306: "wheelchair usage might" Table 2: What is DC1, DC2, HUI. Consider describing them in a footnote of this Table. L366: "training) were tabulated" L371: Use the Greek symbol alpha L390: Why there is n=7 in Table 3's title and not n=9? Also in Table 3, to be consistent consider using 8.0 and 26.0 for the range. L455: "expositions" L464: "identifies that" L482: "Moving forward," L516: "frustration with his/her" L517: "attend the post-treatment"

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.

Title: A RANDOMIZED CONTROL TRIAL FEASIBILITY EVALUATION OF AN MHEALTH INTERVENTION FOR WHEELCHAIR SKILL TRAINING AMONG MIDDLE-AGED AND OLDER ADULTS Review round: 1 Reviewer: 2

Basic reporting: 1. The introduction of this paper reviewed sufficient past related research and have a clear field background for this study. 2. Article structure, figures, tables, and data are provided appropriately. Experimental design: 1. This study which used 2 x 2 factorial design RCT, recruited 18 participants with a retention rate of 94% and practiced their mHelath MWC skills training program for older adults met the good technical standard in experimental design. 2. The method of this study was also described clearly with detail information to replicate. Validity of the findings: 1. The results of EPIC Wheels group post-treatment questionnaire showed that participants had high satisfaction of the program, however, there is a need to state how those questionnaires were distributed and administrated to clarify if participants felt free and comfortable to give their feedback without being afraid of ruining their patient-trainer relationship. Additional comments: 1. According to the definition of WHO and ageing and aged research, most developed world countries have accepted the chronological age of 65 years as a definition of 'elderly' or older person. However, in this study, participants were aged 55 and older. Please reconsider if the title still remind the use of "older adults" or only use the participants who are aged 65 and older. 2. Gender always plays an important role on the level of involving interventions or programs in ageing and aged related studies. Please add gender information and possible demographic background (age distribution, race,...etc.) to understand the overall look of the participants.

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.

Title: RELATIONSHIP BETWEEN HEMOGLOBIN GLYCATION INDEX AND EXTENT OF CORONARY HEART DISEASE IN INDIVIDUALS WITH TYPE 2 DIABETES MELLITUS: A CROSS-SECTIONAL STUDY Review round: 1 Reviewer: 1

Basic reporting: Results: - The authors might consider to create a flowchart to visualize the process of the number of patients screened, number of patients excluded, excluded - Can the authors show which antidiabetic drug were the patients on? - The last sentence (line 173) should be deleted from the result section and inserted in the discussion. - The authors presented a univariate regression analysis for the association between MVD and LAD disease with HGI. The authors need to perform a multivariate regression analysis including well known confounders for CAD severity (e.g. hypertension, hyperlipidemia etc.). Otherwise this is a strong limitation for the study. - The authors might consider to OR and 95% CI for MVD and LAD disease using fasting glucose or HbA1c alone in comparison to HGI. Experimental design: Methods: - Patients had measurement of HGI 3 months before coronary angiography. What were the qualifying reasons for enrollment? How many patients had angina, angina equivalent, SOB (eg. According to CCS and NYHA classification)? What measures were used to schedule an angiography (e.g. positive stress test, high likelihood of CAD estimated by pre-test probability). - Please clarify, whether patients with existing CAD were excluded or not. If not excluded, add a raw in table 1 showing the percentage of these patients in each HGI category. - Describe in detail who you calculated the predicted HbA1c. - Describe the statistical methodology in more detail. Specifically, the regression analysis Validity of the findings: The findings described by the authors are of particular finding given the fact that HGI easy to calculate and widely available. However, the authors have to address some issues raised by this reviewer (see below). Additional comments: Drs. Cheng et al. performed a nice cross sectional study of patients with DM scheduled for angiography. The authors calculated the HGI before angiography and found an association to CAD severity (MVD and LAQD disease). The paper is well written and carefully performed. Nevertheless this reviewer has the following comments: Abstract - Describe the method for calculation of HGI in the abstract. Methods: - Patients had measurement of HGI 3 months before coronary angiography. What were the qualifying reasons for enrollment? How many patients had angina, angina equivalent, SOB (eg. According to CCS and NYHA classification)? What measures were used to schedule an angiography (e.g. positive stress test, high likelihood of CAD estimated by pre-test probability). - Please clarify, whether patients with existing CAD were excluded or not. If not excluded, add a raw in table 1 showing the percentage of these patients in each HGI category. - Describe in detail who you calculated the predicted HbA1c. - Describe the statistical methodology in more detail. Specifically, the regression analysis Results: - The authors might consider to create a flowchart to visualize the process of the number of patients screened, number of patients excluded, excluded - Can the authors show which antidiabetic drug were the patients on? - The last sentence (line 173) should be deleted from the result section and inserted in the discussion. - The authors presented a univariate regression analysis for the association between MVD and LAD disease with HGI. The authors need to perform a multivariate regression analysis including well known confounders for CAD severity (e.g. hypertension, hyperlipidemia etc.). Otherwise this is a strong limitation for the study. - The authors might consider to OR and 95% CI for MVD and LAD disease using fasting glucose or HbA1c alone in comparison to HGI. Discussion: - The authors mentioned that HGI reflects an excess of advanced glycosylation end products. Since there is a difference to HbA1c levels, the authors should devote some lines to address these differences. Essentially he relation between HbA1c on one side and advanced glycosylation end products on the other side with CVD severity. - Since the marker easy to calculate, the authors should describe clinical implications of the study findings.

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.

Title: RELATIONSHIP BETWEEN HEMOGLOBIN GLYCATION INDEX AND EXTENT OF CORONARY HEART DISEASE IN INDIVIDUALS WITH TYPE 2 DIABETES MELLITUS: A CROSS-SECTIONAL STUDY Review round: 1 Reviewer: 2

Basic reporting: Clear and unambiguous English is used throughout the text. References are relevant, but most are older than 10 years. More recent references should be used, if available in the literature. Overall, the paper is clear and make simple and significant correlations. Other commentaries may be found in the .pdf document provided. Experimental design: Experimental design is overall clear and reproducible. Other commentaries may be found in the .pdf document provided. Validity of the findings: Other commentaries may be found in the .pdf document provided. Additional comments: The work is simple and very clear. If reproduced broadly, this correlation between HGI and CHD in T2D patients may provide an efficient tool for diagnosis purposes worldwide. Please refeer to my other commentaries in the .pdf document for further suggestions.

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.

Title: RELATIONSHIP BETWEEN HEMOGLOBIN GLYCATION INDEX AND EXTENT OF CORONARY HEART DISEASE IN INDIVIDUALS WITH TYPE 2 DIABETES MELLITUS: A CROSS-SECTIONAL STUDY Review round: 2 Reviewer: 1

Basic reporting: No comments. Experimental design: The study design is accurate. However the description of the estimation of predicted HbA1c is still not clear. The authors need to provide regression coefficients etc. in the graph (figure 1) and explain the where d the numbers in the equation (predicted HbA1c = 0.008 x FPG + 6.28) come from. Furthermore the reviewers recommendation of insertion of a multivariate OR 95%CI was only commented by the authors and not inserted in the revised version of the paper. Validity of the findings: The study addresses an important patient population. In such patients developing tools for better patient care are crucial. Additional comments: The reviewers comments were only partly addressed in the revised version of the manuscript. Essentially the authors should avoid discussing their finding in the result section (165-171 of the PDF version of the revised manuscript). Despite attempts to address the reviewers comment regarding the predicted HbA1c equation, the authors did not provide an understandable explanation in the manuscript. Please provide information where the numbers in the equation come from!

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.

Title: RELATIONSHIP BETWEEN HEMOGLOBIN GLYCATION INDEX AND EXTENT OF CORONARY HEART DISEASE IN INDIVIDUALS WITH TYPE 2 DIABETES MELLITUS: A CROSS-SECTIONAL STUDY Review round: 2 Reviewer: 2

Basic reporting: Clear and unambiguous English is used throughout the text. References are relevant, authors removed older references and added more recent ones. Corrections suggested directly into the submission .pdf file were sufficiently fullfiled. All tables are structured accordingly. Supplementary (raw) data is provided separatedly. Despite being clearly presented in Table 2, I believe that there's no need for presenting "yes" or "no" numbers for "LAD disease". By simply indicating "LAD disease" on the left colum and data related to "yes" group (presence of LAD disease) would be enough to imply that the remaining individuals from the groups did not present with the disease. It, however, is not an essential modification, and authors may opt not to make further changes in the refered table. Figures are good and improve undertanding of the data, but I'm not certain about resolution; authors should make sure to provide files with at least 600dpi resolution. If such files were already provided separately, please disregard this recommendation. Experimental design: Experimental design is overall clear and reproducible. I have no further comments on this section. Validity of the findings: "no comment" Additional comments: Please verify my comments on the "Basic reporting" section. Other minor sugestions are presented at the .pdf file. New recommendations are of minor relevance and authors may or may not accept such changes, considering that they refeer only to the manuscrit, not the study itself. After that, I acept the paper with no further changes.

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.

Title: RELATIONSHIP BETWEEN HEMOGLOBIN GLYCATION INDEX AND EXTENT OF CORONARY HEART DISEASE IN INDIVIDUALS WITH TYPE 2 DIABETES MELLITUS: A CROSS-SECTIONAL STUDY Review round: 3 Reviewer: 1

Basic reporting: The article is presented in high quality. This reviewer has no further comments Experimental design: Design is adequate. This reviewer has no further comments Validity of the findings: The authors responded to all comments and suggestions made by the reviewers. This reviewer has no further comments Additional comments: Thank you for addressing all comments and suggestion made by the reviewers. This reviewer has no further comments

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.

Title: RELATIONSHIP BETWEEN HEMOGLOBIN GLYCATION INDEX AND EXTENT OF CORONARY HEART DISEASE IN INDIVIDUALS WITH TYPE 2 DIABETES MELLITUS: A CROSS-SECTIONAL STUDY Review round: 3 Reviewer: 2

Basic reporting: There is a highlight mark in the word "glycation" (left and right) from figure 2 - bottom boxes, I believe it's not supposed to be there. Other than that, no comment. Experimental design: no comment Validity of the findings: no comment Additional comments: no comment

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.

Title: CLONE WARS: ASEXUAL REPRODUCTION DOMINATES IN THE INVASIVE RANGE OF TUBASTRAEA SPP. (ANTHOZOA: SCLERACTINIA) IN THE SOUTH-ATLANTIC OCEAN Review round: 1 Reviewer: 1

Basic reporting: Please see General comments below. Experimental design: Please see General comments below. Validity of the findings: Please see General comments below. Additional comments: In this paper, the authors examined the genetic population structure of invasive Tubastraea species in the South-Atlantic Ocean by using microsatellite markers which have been newly developed by their study. Their analyses seem good and the interpretation of the results is also valid. Thus, I recommend the publication in PeerJ. I show some comments below. When reading the manuscript first, it seemed difficult to understand the present distribution patterns of Tubastraea species used in their study. I recommend that the authors add the information in the map (Figure 1). In the result, the lower genetic diversity around IGB for both species seems intriguing. Is IGB the marginal area for their distributions? If some environmental data such as temperatures are available, it would be informative to interpret the results (for example, please see the paper below). Reference: Dimond, J. L., Kerwin, A. H., Rotjan, R., Sharp, K., Stewart, F. J., & Thornhill, D. J. (2013). A simple temperature-based model predicts the upper latitudinal limit of the temperate coral Astrangia poculata. Coral Reefs, 32(2), 401-409. L323: About the possibility of cryptic species, more descriptions may be helpful for the discussion. Please refer to the paper below too. This paper also discusses clonal diversity and the existence of cryptic species in coral shown by microsatellite analysis. Reference: Nakajima, Y., Nishikawa, A., Iguchi, A., Nagata, T., Uyeno, D., Sakai, K., & Mitarai, S. (2017). Elucidating the multiple genetic lineages and population genetic structure of the brooding coral Seriatopora (Scleractinia: Pocilloporidae) in the Ryukyu Archipelago. Coral Reefs, 36(2), 415-426. Other comments: L130: the authors should add the citation for FASTX-Toolkit. L139 and other lines: "uL" -> "micro-L" L139: "10ul" -> add space L142: "MgCl2" -> "2" should be subscript. L144 and other lines: "52°C–62°C" -> 52–62°C L199: "hypothesis" -> "hypotheses" L256: "were similar" -> "were higher"? L305: "to a thoroughly examine" -> "to thoroughly examine" Table 2: "," -> "."

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.

Title: CLONE WARS: ASEXUAL REPRODUCTION DOMINATES IN THE INVASIVE RANGE OF TUBASTRAEA SPP. (ANTHOZOA: SCLERACTINIA) IN THE SOUTH-ATLANTIC OCEAN Review round: 1 Reviewer: 2

Basic reporting: This manuscript is well written. However, there are a few things that should be revised or clarified before this manuscript is published. Line 26: azooxanthellate Line 87: monobuoys Line 88: citation please Lin 165: MLL, term needs to be specific and identical in whole manuscript Line 298-299: this sentence is not appropriate here. It is better to cite cases from animals instead of plants. There are some other examples from animals such as cnidarians that have been reported. Table 2. He values of T. tagusensis should be with dot. Experimental design: There are 4 collection sites (Buzios Island, monobuoy, Todos-os-Santos Bay (northeastern Brazil), and Ilha Grade Bay (southeastern Brazil)) as described in the Materials and Methods. Why only the result of the genetic connectivity between two localities have been shown in the study? What are the genetic structures of samples from Buzios Island and monobuoy at Sao Sebastiao channel? Line 193: The initial burn-in is 500,000 cycles followed by 500,000 additional cycles. How the number of burn-in cycles and run length have been determined? Line 194: why the test of K varied from 1 to 3 only? Validity of the findings: Since the asexual reproduction is the nature of these species which could cause the low genetic diversity overall. I would suggest the authors to increase the sample size to get more understanding regarding the origin of the invasive Tubastraea which is more relevant to the goal here. For example, if these species were transferred by the vessels, is it possible to collect some samples from the harbors and compare the data with those from the other areas, so we may see a picture of how these species migrate or spread? Since the Tubastraea was first reported in the Caribbean and is now in Gulf of Maxico and Brazilian coast. It would be great if the authors perform the phylogeographic analyses to show the relationship of these species in the Brazilan coast and other places. By doing this, this will be clear to see how the invasion affect the local population. The Tubastraea coccinea has been shown high dispersal potential (at least on the scale of tens of kilometers) (Mizrahi et al. 2014). Although this impact might be ruled out from the sites between TSB and IGB, can it affect the populations from the locations nearby, such as the localities in the south? Also, I would suggest the authors to add an inset in Figure 1 to illustrate the collection sites in the south, so it could be helpful to understand the geographic distance among these sites. The results also indicated at least two different populations of T. coccinea have been introduced to the southwestern Atlantic. Is it possible to detect the gene flow? By doing this, it will be helpful to understand where is this species from. I also suggest authors to describe the asexual reproduction mode (s) of Tubastraea in the Introduction. Do T. coccinea and T. tagusensis have the same asexual reproduction strategies? Are these modes important for the expansion of these species? Additional comments: The study aimed to identify the origin of the invasive Tubastraea spp. along the Brasilian coast by using microsatellite approach. The results showed the high proportions of clones at two analysed localities on Brazilian coast, indicating the dominate asexual reproduction which leads the conclusion that no significant population structures have been observed. The authors also concluded that the vector transport is the cause for spreading the species along the coast. I appreciate that the authors share these dataset and analyses which will be important for the marine conservation management in the future.

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.

Title: CLONE WARS: ASEXUAL REPRODUCTION DOMINATES IN THE INVASIVE RANGE OF TUBASTRAEA SPP. (ANTHOZOA: SCLERACTINIA) IN THE SOUTH-ATLANTIC OCEAN Review round: 2 Reviewer: 1

Basic reporting: 1. The authors should check the English before the publication. Line 26: azooxanthellate 2. From the response, it seems the goal of this study is not to identify the origin of the invasive population as what is said in the manuscript. To make less confusing, I suggest the authors should remove this sentence in the Introduction "and the origins of the invasive populations remains a mystery", or modify it to something like "dispersal of the invasive population remains unclear". Experimental design: no comment Validity of the findings: no comment Additional comments: no comment

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.

Title: NEW WHAITSIOIDS (THERAPSIDA: THEROCEPHALIA) FROM THE TEEKLOOF FORMATION OF SOUTH AFRICA AND THEROCEPHALIAN DIVERSITY DURING THE END-GUADALUPIAN EXTINCTION Review round: 1 Reviewer: 1

Basic reporting: This paper describes two new whaitsioids, and the description is informative. I agree on the identification of new taxa, but I think it is better to compare them with relative taxon to show their autopomorphies and other diagnostic characters. This part can add after the phylogentic analysis. Figure 5 A the light is too bright B The position of this one seems not match the direction of light ONLINE SUPPLEMENTARY INFORMATION is nearly identical to 2015 JVP paper, you need adjust it for this paper. Really, only the codings of new taxa are needed here. Experimental design: For the diversity study, my opinion is similar to that of Frobisch and Irmis. You can do it and you need think about the number of specimens as well. Your main point is the earlier appearance of Hofmeyria and other species. It is better you provide a photo of SAM-PK-K10525 and other specimens (L635-L637), because they are your key evidence. Validity of the findings: no comment Additional comments: You said the age of Pristerognathus Assemblage Zone is (late Capitanian–Wuchiapingian) (Table 2, L917), but your figure 1 and figure 11 it is only in Capitanian. Also, you state Hofmeyria atavus in Pristerognathus Assemblage Zone, why do you not show it on Fig 11? Table 3 Dalongkou fuae We showed the age is latest Permian in Fig. 1 L130 miss a ‘.’ L172, 173 References: Broom 1905, 1903 L266, L 274 repeat ‘bars the lacrimal from contacting the nasal’ L278 Fig. 3C? L289 bar is more so than in other? L301 dorsalmost L432 anterior face that (should) overlap the It is disarticulated! L437 enclosure forming the pineal duct?? L582 Ancestral state reconstructions of early eutherocephalian dentition The title is too big for the following content, you only state morphology and texture, change it L588 precanine maxillary teeth? Figure captions: remember change the taxonomical names in italic L603 For this kind of study, my opinion is similar to Irmis. You can do it and you need think about the number of specimens as well. L692 Micro

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.

Title: NEW WHAITSIOIDS (THERAPSIDA: THEROCEPHALIA) FROM THE TEEKLOOF FORMATION OF SOUTH AFRICA AND THEROCEPHALIAN DIVERSITY DURING THE END-GUADALUPIAN EXTINCTION Review round: 1 Reviewer: 2

Basic reporting: No comment Experimental design: No comment Validity of the findings: No comment Additional comments: This manuscript names and describes for the first time two previously mentioned but unnamed whaitsioid therocephalian taxa, which were in the past years collected from the upper Permian Teekloof Formation of the South African Karoo Basin. The manuscript is well written and requires only very minor editorial corrects (see track changes in doc file). The content is clearly articulated and well structured. The illustrations are sufficient and properly referenced throughout the manuscript. Beyond the description, the authors discuss the new specimens' relationships within Therocephalia and their impact on the general phylogeny of the clade. Moreover, the Discussion includes a brief treatment of ancestral therocephalian dentition (as new anatomical information from the new material provides new insights) as well as a short review of therapsid and specifically therocephalian diversification across the end-Guadalupian Extinction. All of these aspects are well integrated and provide new insights into therocephalian evolution. The only concern that I have with regards to this contribution concerns the fact that the authors assume a mostly undistorted nature of the holotype and only specimen of the second new genus, Ophidostoma. The authors mention that the type skull is slightly crushed but on the other hand disregard the possibility that the overall unique skull proportions of being as wide as long is a result of distortion rather than genuine. I don't generally doubt the validity of this taxon or the overall anatomy as described, but the images and drawings clearly indicate that the skull is massively distorted. This should be made more clear in the text and overall proportions should be taken with caution. Other than that and the few suggestions for changes (see doc file), I strongly recommend publication of this manuscript in PeerJ.

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.

Title: THE PLASMODIUM BERGHEI RC STRAIN IS HIGHLY DIVERGED AND HARBORS PUTATIVELY NOVEL DRUG RESISTANCE VARIANTS Review round: 1 Reviewer: 1

Basic reporting: No additional comment; the paper satisfies all the criteria. Experimental design: No comment. Validity of the findings: No comment. Additional comments: In this study, Songsungthong et al use whole genome sequencing to examine the P. berghei RC (PbRC) strain that in earlier work by Peters was selected to be chloroquine resistant. The RC strain is deficient in haemozoin formation, has increased levels of glutathione, and was more recently identified as resistant to artemisinin. Here, the authors start by validating the reduced susceptibility to artesunate, with the RC strain approximately 8-fold less sensitive than PbANKA. Whole genome sequencing of PbRC revealed a high level of divergence from the reference PbANKA, as well as several other sequenced P. berghei genomes. The high number of variants that are unique to PbRC, almost 10-fold higher than any other strain with unique or private variants, confounds the identification of possible resistance loci that could contribute to the observed phenotypes. The authors survey possible candidates, and report SNVs in several, including PbCRT, PbMDR1, the deubiquitinating enzyme PbUBP1, and PbPI3K, but not the P. berghei ortholog of Kelch13. None of these SNVs is an obvious smoking gun, and the inability of the RC strain to form gametocytes precludes the use of genetic crosses to further dissect the resistance phenotypes. Overall the study by Songsungthong et al demonstrate the high level of divergence of the RC strain from other sequenced strains of P. berghei, and provide a foundation from which further genetic validation of drug resistance candidates could be based. There are a few additional points that the authors should consider. i) For the analysis of “private” variants in PbRC, it would be informative if the authors described in more detail the proportion of SNVs that are located in coding regions, are non-synonymous, and are in the core genome. ii) Gene amplification is a common mechanism of drug resistance in Plasmodium sp. Could the authors comment on whether gene amplifications could be detected, and might be a mechanism of resistance in this strain? iii) Given the inability of the RC strain to form gametocytes, were there mutations detected in AP2-G and other candidate loci involved in gametocytogenesis?

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.

Title: THE PLASMODIUM BERGHEI RC STRAIN IS HIGHLY DIVERGED AND HARBORS PUTATIVELY NOVEL DRUG RESISTANCE VARIANTS Review round: 1 Reviewer: 2

Basic reporting: Well written paper that is clear and easy to understand. All the data has been shared. Experimental design: Antimalarial drug resistance is an emerging global health issue as malaria has become untreatable in some parts of the world due to failure of drug treatment, including the current frontline therapy, artemisinin. Recent evidence show that artemisinin resistance is multi-factorial though mutations in one gene, kelch 13, show a clear association with resistance. The authors of this study attempt to identify the genetic basis of antimalarial resistance using the murine malaria parasite, Plasmodium berghei. A specific strain of P. berghei, PbRC was known to be resistant to several antimalarial drugs, including artemisinin (ART) and chloroquine (CQ). The authors hypothesize that PbRC may share some of the resistance mechanisms with drug resistant human malaria parasites. The authors use whole genome sequencing to answer this question and compare the genome of PbRC to published genomes of several P. berghei strains to identify SNPs unique to PbRC. They use Tajima's D score to identify genes undergoing selection. The methods are described in detail and the work is well done. The story of artemisinin resistant malaria is still evolving and we don't have the complete picture. The authors identify numerous genes that may play a role in drug resistance. Validity of the findings: The authors show that PbRC is resistant to artesunate, a water-soluble derivative of ART, with an increased ED50. Using whole genome sequencing, the authors identify several unique SNPs in the PbRC strain and convincingly demonstrate that it is quite divergent from other sequenced P. berghei strains. Unfortunately, the SNPs identified in PbRC do not match with SNPs found in artemisinin resistant P. falciparum. This does not take away from the study though as artemisinin resistance is known to be multi-factorial and our understanding of it is incomplete. The authors are careful about using the literature to make an educated guess as to which sets of genes are likely responsible for drug resistance. However, one issue is that the PbRC strain does not form gametocytes and strains that do not form gametocytes often have associated mutations with them. The authors do not compare PbRC genome with the genome of the ANKA 2.33 strain (gametocyte non-producer) to narrow down their targets. Another potentially useful method to narrow down the list of candidate genes responsible for drug resistance would be to assess essentiality. The authors do that for one candidate (Pbdpap3) but not for others. As the authors may be aware, this information is available for nearly 3000 genes in the P. berghei genome (Plasmogem phenotype database: http://plasmogem.sanger.ac.uk/phenotypes). These comparisons may help the discussion to focus on drug resistance and curate the list of candidates further. Additional comments: Please discuss why you think that host cell invasion genes are changing due to balancing selection. Its unclear how they would help/hurt drug resistance. Why would PbRC need to evade the immune system more than other strains? Maybe it has more to do with being a gametocyte non-producer than drug resistance? It may also be useful to compare the unique PbRC SNPs with the transcriptional dataset for ART-resistant P. falciparum (Mok et al Science 2015, 347:431-35).

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.

Title: ASSESSING THE LIVING AND DEAD PROPORTIONS OF COLD-WATER CORAL COLONIES: IMPLICATIONS FOR DEEP-WATER MARINE PROTECTED AREA MONITORING IN A CHANGING OCEAN Review round: 1 Reviewer: 1

Basic reporting: English is not clear in some cases, I have annotated the pdf to highlight ambiguous statements and in some cases I commented or provided suggestions of how I think the English can be improved. Yet I did not revise the entire manuscript thoroughly as I believe the authors should do this before submitting a manuscript for revision. Also the manuscript uses many acronyms and it is difficult to keep track of all of them – I suggest authors should consider spelling out as many terms as possible rather than using acronyms.Other minor suggestions are noted on the pdf. My expertise lies with tropical corals, remote sensing and climate change, not cold water corals. I am not aware of any important references that were missed, but the manuscript seems to have many self-citations. I advise another reviewer with cold water coral expertise comments on this point. The structure of the manuscript is good, However, several figures are sub standard they need significant improvement – please see specific comments on pdf This is a self-contained manuscript – but hypothesis are not explicitly explained. Experimental design: Original primary research within Aims and Scope of the journal. Yes Research question well defined, relevant & meaningful. It is stated how research fills an identified knowledge gap. The submission should clearly define the research question, which must be relevant and meaningful. The knowledge gap being investigated should be identified, and statements should be made as to how the study contributes to filling that gap. Yes Rigorous investigation performed to a high technical & ethical standard. The investigation must have been conducted rigorously and to a high technical standard. The research must have been conducted in conformity with the prevailing ethical standards in the field. No – my biggest concern about this manuscript is that it is not possible to determine the precision of the measurements made. Based on both the text and the figures provided it seems to me that the lines used in Image J to measure coral size could have stopped anywhere, they do not seem to stop at the top of the coral and from previous experience with similar measurements I know it can be very tricky to do this in a standardized fashion. Unless more detail and exact method of standardization of these measurements is provided I cannot recommend publication. Methods described with sufficient detail & information to replicate. Methods should be described with sufficient information to be reproducible by another investigator. See comment above Validity of the findings: Impact and novelty not assessed. Negative/inconclusive results accepted. Meaningful replication encouraged where rationale & benefit to literature is clearly stated. Decisions are not made based on any subjective determination of impact, degree of advance, novelty, being of interest to only a niche audience, etc. Replication experiments are encouraged (provided the rationale for the replication, and how it adds value to the literature, is clearly described); however, we do not allow the ‘pointless’ repetition of well known, widely accepted results. No, see comment under experimental design and validity of measurements. Data is robust, statistically sound, & controlled. The data on which the conclusions are based must be provided or made available in an acceptable discipline-specific repository. The data should be robust, statistically sound, and controlled. No, see comment under experimental design and validity of measurements. Also, images for all 18 colonies and measurements should be provided in supplementary material – not just the table of derived values. Given the rarity of these ecosystems and data, it is essential to provide the imagery. Conclusion are well stated, linked to original research question & limited to supporting results. The conclusions should be appropriately stated, should be connected to the original question investigated, and should be limited to those supported by the results. NA – I did not finish reading the manuscript as I don’t think it has merit unless details are provided regarding the methodology and measurements. Speculation is welcome, but should be identified as such. NA – I did not finish reading the manuscript as I don’t think it has merit unless details are provided regarding the methodology and measurements. Additional comments: This manuscript investigates the live vs dead coral tissue of 18 coral colonies in deep cold water reefs. Cold water corals are not studied enough and it is important to improve our understanding of these systems and their related organisms. My biggest concern about this manuscript is that it is not possible to determine the precision of the measurements made. Based on both the text and the figures provided it seems to me that the lines used in Image J to measure coral size could have stopped anywhere, they do not seem to stop at the top of the coral and from previous experience with similar measurements I know it can be very tricky to do this in a standardized fashion. Unless more detail and exact method of standardization of these measurements is provided I cannot recommend publication. The manuscript also needs significant improvement on its English grammar, at present it is not very clear and in some instances it is difficult to understand what the authors mean. I recommend authors ask a good scientific writer (preferably whose first language is English) to revise their manuscript prior to resubmission. I have made detailed comments on the pdf. The manuscript figures are of low quality and need to be significantly improved as well. I have made detailed comments on the pdf.

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.

Title: ASSESSING THE LIVING AND DEAD PROPORTIONS OF COLD-WATER CORAL COLONIES: IMPLICATIONS FOR DEEP-WATER MARINE PROTECTED AREA MONITORING IN A CHANGING OCEAN Review round: 1 Reviewer: 2

Basic reporting: The structure of the paper meets required standards. The language is clear and the literature cited was fairly extensive given the relatively short paper. Figures etc were clear and of good quality. The discussion is rather disjointed in places with statements that do not seem to have a context. A little more work is needed on the discussion The addition of a hypothesis would improve the paper. Since the authors are comparing two different sites, it would be relatively easy to formulate a hypothesis about expected differences between the sites. Experimental design: The survey design was unbalanced, which reduced the comparative value of the study but did not invalidate it. The study is focused on the application of a new method for assessing coral growth, and as such is useful. I think there needs to be more detail on the abiotic factors: what the values were and how they were derived from the model. They are superficially described but the reader has no information with which to assess the colony measurements or the discussion. The absence of temperature as an abiotic correlate is surprising as it is one of the more important factors that influence growth. Temperature should be included in the analysis, or if the data is not available, it should be considered in the discussion. There is no abiotic data for the Pisces sites, which is a flaw as it does not allow site comparisons. If this data is not available, the effects on the analysis should be acknowldged in the discussion Validity of the findings: Additional information the aboitic factors is needed to determine whether the data used is robust and statistically sound. The statistical results for these analyses should be presented fully - a table would suffice. Additional comments: A valuable paper but needs some work on the discussion and data analysis. I have made some additional comments and suggested edits in the attached PDF file.

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.

Title: ASSESSING THE LIVING AND DEAD PROPORTIONS OF COLD-WATER CORAL COLONIES: IMPLICATIONS FOR DEEP-WATER MARINE PROTECTED AREA MONITORING IN A CHANGING OCEAN Review round: 2 Reviewer: 1

Basic reporting: This is a second review and the authors have addressed all my initial concerns appropriately. I recommend accepting the re-submitted version of this manuscript. Experimental design: No comment Validity of the findings: No comment Additional comments: No comment

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.

Title: IMPACT OF LAND RECLAMATION AND AGRICULTURAL WATER REGIME ON THE DISTRIBUTION AND CONSERVATION STATUS OF THE ENDANGERED DRYOPHYTES SUWEONENSIS Review round: 1 Reviewer: 1

Basic reporting: Seems very good. Relatively good English with a few small errors or stylistic problems as noted in the General Comments section. Well structured in standard format. Experimental design: Seems fine to address most of the questions asked. Probably not adequate for some, though--extreme spatial autocorrelation of sources of water means that many other grographical variables may be confounded with water source; see comments on Figure 2 in general comments section and comment on lines 229-235 in "Validity of the Findings" Validity of the findings: 229-235 Not sure I see the significance of this? I assume that the percentages of sources of water just relate to the species’ geographic distribution. Sources of floodwaters are highly spatially autocorrelated and cannot be regarded as independent for the pruposes of things like chi-squared tests. See also Figure 2 comment in general comments section and comment on 287-297 Additional comments: Most of these are simple English comments, however a few are more substantive. 54 should either be “a species’ range and habitat preferences” or “species’ ranges and habitat preferences” 60 “The amphibia class -> “The Class Amphibia” 64 “Besides, those farmlads” -> “Farmlands” 76 “lead to expect” -> “led us to expect” lead is a metal, or something one does to guide someone to a place (pronounced "leed"), led is the past tense of the guiding meaning. Good old English... 79 “karyotype warranting” -> “karyotype, warranting” 88 “negative” -> “negatives” 100 “absence for” -> “absence of” 110 “study for” -> “study of” 139 “presence of the” -> “presence of” 174 needs a rewrite. Do you mean you hypothesized that it was important that water be linked to the Gxxx river? I say Gxxx because here you refer to the Geum river and above (156) you referred to the Gum river, and I am on an aeroplane with no access to a map to check which is correct. 176-177 “We subsequently assessed the random distribution of D. suweonensis in relation of the agricultural flood water.” -> “We subsequently assessed whether distribution of D. suweonensis was random in relation to that of agricultural flood water.” 184 “and variables such as water quality are important the” -> and because variables such as water quality are important in the” 195 “were” -> “where” 202-203 “surveyed three” -> “surveyed over three” 218 This is important—did the range decrease by 729 km2 or from 729 km2 to some lesser area? Just a typo I’m sure 241 “temporal” -> “temporary” if that is what you mean; temporal is definitely wrong, anyway. Dr. Who’s TARDIS is a temporal structure; I don’t know of any others. 254-255 “The known range of Dryophytes suweonensis has doubled over the three years of this study” -> “The known range of Dryophytes suweonensis was doubled by the data collected over the three years of this study” 258-259 “This new knowledge is, however, sobered down by several potential local extirpations” -> “The apparent expansion of the species’ known range is, however, countered by several potential local extirpations” 260 “”Besides, the” -> “The” 262 “Namely, because of an extent” -> “It has an extent” 265 “Besides, the” -> “At present, the” 267 “It is however” -> “This is” 272 “Accordingly” -> “Additionally” 276 “Encroachment had been” -> “Encroachment on the species’ range by development has been” 277 “carried at” -> “carried out at” 283 if there are numerous major landscape barriers within potential metapopulations, they probably are not single metapopulations, but either groupings of isolated populations or groupings of isolated metapopulations. This needs to be revised. Just adding the word “potential” preceding “metapopulations” would do the trick. 287-297 See my comment in the “Validity of the Findings” section about this. If this is a real thing it needs to be explained more thoroughly and justified better. Otherwise it may just be the result of any of the many factors that might be restricting geographic distribution of the species. 306-307 “neither than introduction at new sites” is completely wrong wording; I really am not sure what you mean; do you mean you do not recommend ex-situ conservation other than introduction at new sites outside the present range (commonly called translocation), or do you mean that you recommend neither ex-situ conservation nor translocation? Figure 1 legend I would suggest saying (blue dot) Dryophytes suweonensis present (red dot) Dryophytes suweonensis absent i.e. reverse the wording for the present and absent categories. Not really necessary but clearer and better English 449 “was” -> “were” Figure 2—note the extreme degree of spatial autocorrelation, i.e. each point sampled is very non-ndeendent from many others in terms of the source of water, so that regarding them as independent and conducting a chi-squared test, which assumes that, is incorrect. 452 “Bird view” -> “Bird’s-eye view” or “Overhead view” 457 “Lateral view the” -> “Lateral view of the”

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.

Title: IMPACT OF LAND RECLAMATION AND AGRICULTURAL WATER REGIME ON THE DISTRIBUTION AND CONSERVATION STATUS OF THE ENDANGERED DRYOPHYTES SUWEONENSIS Review round: 1 Reviewer: 2

Basic reporting: I found this to be a very well written paper, based on several years of survey data. Clearly a lot of work was done to gather the data. I make some minor suggestions below to improve the grammar. Line 28: replace “evidences” with “evidence” Line 30: replace “Besides,” with “In addition,” Line 33: delete “ultimately” as this word is not needed in the sentence Line 41: I don’t know if you mean “extinction risks” (plural) or “extinction risk”. You should say either “extinction risks depend on” or “extinction risk depends on”” Line 44-45: Change so it reads “The lack of knowledge of the distribution of a species has already resulted in extinction that could have been easily avoided”. Line 45: Change to “For example, the Tecopa pupfish….”. Line 48: Change “Knowledge on” to “Knowledge of” Line 50: Say “subspecies of Ursini’s viper”, not “subspecies of the Ursini’s viper” Line 51: Change to “was known to occur only in Greece and at a single locality in Albania.” Line 57: Change to “The occurrence of a species ….” Line 58-59: you finish the sentence with “and despite the low occurrence of endangered species within protected areas (Brooks et al. 2004).” I don’t understand what you mean by this, in relation to the rest of the sentence. Can you clarify? Line 60: Change “amphibian class” to “Class Amphibia” Line 64: Change “Besides” to “Furthermore” Line 66: Say “where rice production has decreased …” Line 72: Change “occur on” to “occur in” Line 76-77: Change to “lead to the expectation of a broader distribution for the species” Line 79: Change “enterprised” to “aimed” Line 99: this is the first time you mention D. japonicus. You should say something about why this species is important to your study. Line 110: Change to “occurrence of this species” Line 116: Change “before” to “adjacent to” Line 125-126: Do you mean you had previously measured the detection range of frog calls, or you did this at the end of each site survey. I suspect the former, but please clarify. Line 160: Change “traceability in the origin” to “traceability of the origin” Line 171: Insert a comma after “species” Line 177: Change “in relation of” to “in relation to” Line 179: Change “From presence data from the surveys” to “From survey presence data” Line 182: Say either “the species’ preferences” or delete “the” and just say “species preferences” Line 184: Change “the ecological preferences” to “ecological preferences” Line 191 to 193: I would make this sentence more concise by saying something like “That is, the sites were beyond the ecological requirements of the species as there was no standing water; instead the sites were mostly greenhouses, apartment complexes or dry crops.” If that is what you mean. Line 195-196: Change “from 94 of the 100 sites were the species was detected” to “from 94 of the 100 sites where the species was detected” Line 198: delete the comma in the sentence as it is not necessary. Line 204” I’m not sure “aggravated threats” is the best phrase. Perhaps change to “significant threats” or similar. Line 215: Change “Besides” to “In addition” Line 218: you say “the species decreased by from 729 km”. It should either be “by 729 km” if that is what you mean, or “from 729 km to XXXX” if that is what you mean. But don’t say “by from” in the same sentence. Line 247: I would change “bird-eye view” to “aerial view” Line 258: Change “sobered down” to “tempered by” or similar Line 260: Replace “Besides” with “Furthermore” Line 262-265: The sentence “Namely, because of an extent of occurrence < 5000 km2, a severely fragmented population, with a continuing decline observed, estimated, inferred or projected for extent of occurrence; area of occupancy; area, extent and/or quality of habitat; and the number of locations or subpopulations” is a bit confusing. I am not sure exactly what you mean, but this should be clarified. For example, what does “and the number of locations or subpopulations” refer to? Is that referring to a previous part of the sentence regarding continuing declines (observed, estimated, inferred, projected), or are you just talking about the actual number of locations and subpopulations being the issue? Line 270: Change “strive” to “exist” Line 271: Change “knowledge on” to “knowledge of” Line 277: Change “carried at” to “implemented at” Line 302: Change “southern part on” to “southern part of” Line 306: Change “neither than” to “nor” Line 447: You need a better Figure heading. Keep what you have, but start with something like “Relationship between water type and species presence”. Experimental design: My main question relates to the use of a single 5 minute survey each year to assess presence or absence of the frog species at a site. This may be justifiable - I just don't know. See my comments below in the Validity of the Findings Section. Validity of the findings: I have some questions about the data analysis. The authors detected frogs at 114 sites, and never found frogs at something like another 250 sites. Each site was surveyed during a single 5 minute survey (listening for frog calls) during the breeding period. My first question relates to detection probability. How confident are the authors that a single 5 minute survey is sufficient to detect the presence of the species at a site? There is a large literature on this for amphibians in general. For some species, detection probability is quite high (close to 1; thus a single survey is an adequate sampling period), but for other species it is low and so multiple surveys in a short time frame are required to be confident of species presence or absence. This obviously can have significant implications for identifying the distribution range of the species, as well as identifying habitat correlates. Do the authors have any data on the detection probability of their target frog species? If so, and it is high, that would help justify the use of a single 5 minute survey at a site once a year. I agree that the authors have expanded the range of the species, I’m just not sure if the actual range is even larger than they suspect – if the species has low detection probability. As I understand it, to identify important habitat variables for species presence, the authors focused on the 114 sites where the species was present, and did not include data from the other 250 sites (approximately) where the species was never detected. Is that correct? If yes, I don’t understand why the authors didn’t use the data for all 350+ sites (species detected or not detected) in an analysis like logistic regression to see which of the habitat variables was significant in explaining the detection or non-detection of the species at sites? Unless you think detection probability is low, and therefore sites where the species was not detected are too questionable to use to identify habitat correlates. Or there were other reasons to not expect the presence of frogs at some sites independent of local habitat conditions. There may be a reason for their approach to analysis, but the authors should clarify their decision. Or if I have misunderstood the analysis, the authors should be more explicit in describing the analysis they used. Additional comments: No additional comments.

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.

Title: IMPACT OF LAND RECLAMATION AND AGRICULTURAL WATER REGIME ON THE DISTRIBUTION AND CONSERVATION STATUS OF THE ENDANGERED DRYOPHYTES SUWEONENSIS Review round: 1 Reviewer: 3

Basic reporting: Nicely conceived maps and figure. I would like to see a few clarifications of them as specified in the manuscript enclosed. I was confused by your use of ancestral and current distribution and your suggestion that rice seedlings are necessary for the species. If this is true ancestral has a very restricted meaning. Experimental design: I think your a priori selection of field sites to survey is excellent, but you need to highlight how excellent it is by telling us more about how you went about drawing the boundaries on the original area that you are focused on and how you the selected the individual sites within this larger area. Validity of the findings: I suggest you develop your figure 3 more clearly. It is based on other work in large part (e.g. vegetation characteristics) so it kind of drops out of the blue. But, it is a beautiful figure. Additional comments: This is an important study that has many good features associated with it. Primarily the extensive field survey work which documents the occurrence of this endangered species and data associated with positive impacts of both rice paddy development and negative impacts of urban encroachment. A particularly strong component of the study is the large effort that went into gathering presence absence data and correlating this with human land use modifications. The paper is difficult to read and I have made many suggestions, corrections, and comments on the enclosed manuscript. I edited in MSword so the track change comments came across with MOU and REV associated with them. They have not meaning. They are all my comments.

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.

Title: EXCEPTIONAL SOFT TISSUES PRESERVATION IN A MUMMIFIED FROG-EATING EOCENE SALAMANDER Review round: 1 Reviewer: 1

Basic reporting: The report is written mainly clear English, but see below. Literature is appropriate. Figs are excellent. No clear hypotheses but not necessary in this report. Considerable speculation but generally ok with me. Experimental design: Not relevant Validity of the findings: I think the descriptive information and especially the visuals are of high quality. Additional comments: I would tuck the information gathered from this manuscript in that part of my brain labeled “Cabinet of Curiosities”. The phosphorized fossil is remarkably well preserved and the authors have derived a lot of information from the scans. I am interested in salamander anatomy, and accordingly enjoyed reading about there methods and interpretations. What I take home from this manuscript is that under certain circumstances, not well understood, amazing preservation of anatomical detail is possible in fossils. That is it. It is not known exactly where the fossil was found and so taphonomic details are lacking and speculation results. Line 69 is an incomplete sentence and the entire complex sentence would be better as several sentences. Technical English is best in short declarative sentences. I think the anatomical inferences are generally good and justified. I think the authors are correct regarding muscles, lung, cloacal glands and even the partial frog in the gut. The frog in the gut of a relatively small salamander is a surprise. Salamanders generally do not eat frogs or even tadpoles (I can add Ambystoma gracile to the short list of salamanders known to eat metamorphosing frogs). In this instance the frog seems to be partially dismembered, or has been digested in a peculiarly selective manner). Even without the lung evidence, this is not a plethodontid on the basis of vertebral anatomy and especially the very long and well-developed ribs.

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.

Title: EXCEPTIONAL SOFT TISSUES PRESERVATION IN A MUMMIFIED FROG-EATING EOCENE SALAMANDER Review round: 1 Reviewer: 2

Basic reporting: No comments Experimental design: No comments Validity of the findings: The paper is a valuable contribution and I strongly support its publication in the PeerJ (after very minor revision). Additional comments: I have no major suggestions on the paper. I enjoyed the opportunity to read this MS and I look forward to see it published. My minor suggestions for the improvement of the MS are listed below: “Abstract”, line 26, I recommend to add “juvenile” before “frog”. It is important to provide a characteristic of a prey (= the swallowed frog was juvenile). “Introduction”, line 47. Please, clarify why “external features” are “notable” (e.g. notable for taxonomic identification?) “Results”, “Lung”, line 156. I recommend to remove the phrase “(i.e. within the rib cage)” because salamanders do not have a rib cage (this structure is typical for amniotes) “Discussion”, “Exceptional preservation”, line 193, I recommend to use the name Vertebrata (Latin) or vertebrates (English), but not Vertebrates. I waive all anonymity for my review. The authors are welcome to contact me directly should they have questions or anything they wish to discuss. Pavel Skutschas

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.

Title: PERCH, PERCA FLUVIATILIS SHOW A DIRECTIONAL PREFERENCE FOR, BUT DO NOT INCREASE ATTACKS TOWARD, PREY IN RESPONSE TO WATER-BORNE CORTISOL Review round: 1 Reviewer: 1

Basic reporting: The article is largely well-written, with sufficient references and background in most places. The raw data are available. I do not think that, given the controls in the experiment, that the hypotheses are truly being addressed as they are currently worded. Experimental design: The research aims are within the scope of the journal. The research question is well defined, although I think more motivation is needed. I think that additional controls, such as behavior of a non-predatory fish, would have been useful. The ethics and methodological details are sufficient, though there are some things that should be clarified with the statistics. Validity of the findings: There are some major statistical concerns (see general comments) that should be addressed. I think that, given that more or different controls would be useful for identifying if the change in behavior is indeed predatory, the discussion should be more speculative. Additional comments: General comments The manuscript is generally well written and clear. I think that it presents an interesting and relevant topic, as the idea that reduced stress responses may have evolved as a mechanism to avoid predators is an intriguing idea and worth exploring. However, I have some major concerns about the manuscript in its current state: 1. I have some major statistical concerns that I think need to be addressed to make the results more convincing. a. You found that perch tended to show increased lunges toward stickleback during the cortisol treatment, but the majority of trials that were removed because the perch did not move or lunge at all were cortisol trials. It seems likely to me that removal of those cortisol trials (with zero lunges) may have biased the dataset, and it would be useful to run the stats with and without those trials. I understand that using those trials for proportion of time oriented or spent in a zone is biased by including them, but I don’t think that the same is true for the lunges. b. I am somewhat confused by your statistical methods. I assume you ran 4 separate models, each with one fixed effect (treatment) and one random effect (perch ID)? If so, you should specify that in the table legend and replace “fixed effects” in the top row to “treatment”. Additionally, because the proportion of time spent in the chemical stimulus zone, proportion of time oriented in the chemical stimulus zone, and proportion of lunges are likely not independent, it would be more statistically rigorous to control for multiple tests. c. I don’t understand why you used the proportion of lunges and not the total number of lunges. It seems to me that the total number of lunges would be a more accurate reflection of predatory behavior, and it would be useful to justify why proportions were used in this case. Further, how often did the perch lunge in the opposite stimulus zone and do you know what was the purpose of that behavior is? 2. Since you used stressed levels of cortisol (0.20), you need to justify why you used acclimated sticklebacks in your experiment or reframe your question. It seems to me that, to understand if cortisol itself is responsible for giving cues of prey vulnerability, a better control would be to compare cortisol alone (at stress induced levels) to visual cues of stressed prey and/or natural chemical cues of prey that are stressed. 3. Given that, as you state in the introduction, prey can respond to the cortisol emitted by conspecifics, how do you know that predators interpreted the cortisol as cues of prey and not as a signal of danger to themselves? Given that some predators froze, which is an antipredator behavior in many species, is it possible that this is the case? On the other hand, predators might be less willing to stay in the area with the cortisol if it was a cue for danger (the lunging results would also provide further proof of prey cues if the results still hold after redoing the statistics). I think it would have been extremely useful to have additional controls- to understand if a non-predatory fish reacts to the cortisol release in a different way or is this behavior specific to predators. I think that further discussion of this is necessary (and additional tests with this control, if possible, would definitely improve the manuscript). Suggestions for other possible controls are given below. 4. Clarification on what type of cortisol (free or conjugated) is dropped into the water would be useful, as receptors for conjugated steroids tend to be much more prevalent than receptors for free steroids and therefore, perch would have different abilities to detect free versus conjugated steroids. Specific comments Lines 30-32: Here and elsewhere, it feels like there are more commas than are needed in the text. Reducing the number of commas would improve the readability in sections. Line 32: relative to presentations of natural chemical cues… Line 33: In addition, perch tended to show increased… Line 52: If you bring up the life and dinner principle, it would be useful to readers to define it here. Line 74: Citation? Lines 70-76: Here and elsewhere (e.g. lines 82-84), you repeatedly motivate the study by stating that we do not know that much about responses of predators to prey. There should be more motivation to do the study than the fact that it merely has not been done before, and indeed, I do think that it is an important topic of interest. I would remove these statements and instead be more specific in how “predators respond to the chemical cues released by prey would provide insight into the selective forces that shape the chemical ecology of predator-prey interactions”. For example, you allude generally to something in lines 371-372 of the discussion that is worth expanding on in the introduction. Line 79: Such as? And given that there are lots of potential predatory cues, is it possible that putting any of them into the water at high concentrations would produce the same results? As in, I think it is worth discussing (and further investigating for someone) whether or not these results are associated with cortisol alone or if these results can be replicated by having high concentrations of any alarm pheromone in the water. Line 98: influence Line 112-113: I think part of this sentence is missing? Line 113: Or cortisol might just provide information about location of prey, not necessarily vulnerability. I think a control where you run cortisol plus natural cues on one side, with just natural cues on the other, would differentiate between these. Line 143: Did you reuse sticklebacks? If so, were there any notable changes in stickleback behavior over time? As in, sticklebacks could be responding behaviorally different to perch the first versus third time they see the predator. Line 158: Again, what type of cortisol is important information. Lines 160: Since that is not natural levels, but stressed levels of cortisol, I don’t understand why you used acclimated sticklebacks in your experiment, rather than stressed sticklebacks who would give visual cues of vulnerability and natural chemical cues of vulnerability. It seems to me that, to understand if cortisol itself is responsible for giving cues of prey vulnerability, that a better control would be to compare cortisol alone (at stress induced levels) to natural cues of prey that are stressed. Lines 216-221: Again, why proportions? How often did they lunge not at stickleback? Lines 224: Again, I assume that these are not independent. If they are not independent, controlling for multiple tests would be beneficial. Line 228: Degrees of freedom are needed here and throughout Line 234: Package? Lines 251: Final sample sizes for each treatment after removals would be useful. Also, I think removing 12 tests from models looking at lunging behavior likely biases the results. Lines 260: Including the statistics here and in the table is redundant- please choose one. Lines 264-265: I’m not quite sure what you mean by “the chemical stimulus was dripped in the cortisol test relative to the water control”. Lines 265-267: I may have missed this, but how did you measure movement? Just movement between zones? Line 270: Range or standard error? Lines 270-271: Again, this is a large difference, which makes me question why comparing unstressed visual and chemical cues to stressed cortisol levels helps you test “the hypothesis that the disturbance cue cortisol informs predatory behaviour when presented with prey.” I think you need to reframe and/or clarify your question, making it clear that you aren’t testing visual versus chemical cues of stress. Lines 276-285: See my general comments- I think that alternative explanations for altered behavior should be considered here. Lines 298-305: It is a little unclear how these sentences connect to the rest of the discussion- transitions would be useful Line 307-308: But prey who have been repeatedly exposed to predators may have dampened cortisol responses Line 316-317: I think that you can really only say this if you look at whether or not non-predatory fish behave in the same way. Line 333-334: Affect behavior in what way? Also, “but” at the beginning of the next sentence might be replaced by “however”. Line 335: that were elevated Lines 337: What do you mean by undirected changes? References: Make sure you italicize scientific names Figure 2: I don’t understand what the numbers in the bars are- please include that in the figure legend. Also, it would be useful to see both sides of the standard error bars, especially since the data are not normally distributed and therefore, the error bars should not be symmetrical. 2C should have letter b instead of c over the cortisol treatment.

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.

Title: PERCH, PERCA FLUVIATILIS SHOW A DIRECTIONAL PREFERENCE FOR, BUT DO NOT INCREASE ATTACKS TOWARD, PREY IN RESPONSE TO WATER-BORNE CORTISOL Review round: 1 Reviewer: 2

Basic reporting: This is, in its majority, a very well written manuscript. It flows well and everything is backed up with what seems to be a very comprehensive literature review. The results obtained are relevant to the hypothesis and interpreted in a rigorous and informed way. In L335: change 'we elevated' to 'were elevated'. I would encourage the authors to avoid using bar graphs and replace them with boxplots. This is because boxplots show the distribution of the raw data and, thus, are more informative. Experimental design: The design is simple and well thought through. It is clear that the research question(s) fills a knowledge gap. Methods are described in detail. The passage were the hypotheses are stated, however, needs to re-worded. Paradoxically, this is the least clear paragraph in the whole manuscript. Validity of the findings: Data analysis is mostly appropriate. Taking into account the 'repeated measures' nature of the experiment, the authors rightly included predator id as a random factor in their statistical models. However, I'd suggest they reanalyse their data regarding number of movements and number of attacks with a GLMM with a Poisson distribution, which tends to be the distribution that fits counts best. The authors acknowledge the limitations of their study, and state the conclusions that derive strictly from the data presented. Additional comments: I enjoyed reading the manuscript!

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.

Title: PERCH, PERCA FLUVIATILIS SHOW A DIRECTIONAL PREFERENCE FOR, BUT DO NOT INCREASE ATTACKS TOWARD, PREY IN RESPONSE TO WATER-BORNE CORTISOL Review round: 2 Reviewer: 1

Basic reporting: It is well-written and clear, sufficient background and literature review. Experimental design: Well-designed, research question clear and relevant, methods are sufficiently detailed. Validity of the findings: Some minor statistical changes would be useful, but generally, the data are sound. Additional comments: This version of the manuscript is greatly improved over the last version, both in terms of readability and in terms of robustness of the data. The abstract, introduction, and discussion all read very well, although some minor changes to the introduction and discussion (see below) would clear up a few remaining ambiguities. The additions to the methods were useful, although I still have some remaining statistical concerns. Specific comments Lines 60-62: It would be useful to give an example here of how this is the case. Lines 67: information regarding the presence of predators? Lines 81-82: Additional fitness benefits of releasing alarm cues? This paragraph is a bit unclear to me, as it starts with saying the predators use alarm cues to locate prey (which seems bad for the prey), but you then say that it is beneficial because it attracts more predators. Expanding and clarifying this paragraph would improve the readability. Lines 83: Remove the comma between injury and attract Lines 89-90: It would be useful to define disturbance cues here. Lines 135-141: You make predictions here with respect to visual cues versus cortisol, but it would be useful to understand what differences you expect to see (if any) in natural cues versus cortisol. Lines 263-264: I think it would be useful to clarify that these are (as I interpret it) lunges toward the stickleback on the side of the chemical stimulus versus lunges to sticklebacks on the visual cue only side. Lines 276: Did you check to see if your data were overdispersed (i.e. if you should use Poisson or negative binomial distribution)? Lines 272-275, 328-332: Does the total number of lunges and movements include the trials in which perch did not lunge? I agree that, in some cases, excluding individuals who do not react to the stimulus presented by the researchers is appropriate. For example, removing the samples in which the perch were immobile when trying to evaluate proportion of time spent in the chemical stimulus zone seems appropriate because, as you say, it is possible that they spent 100% in the chemical stimulus zone merely because they never moved. With the proportion of lunges, it is also appropriate. However, I do think that it is important to include those zeros in the trials examining total numbers of lunges/movements- if you remove them, you effectively remove many more zeros from the cortisol treatment group than you did from the other two treatment groups and this almost certainly biases the data. As it is written, it is unclear if you did or did not include the trials with immobile perch in your current analysis, and you should show your data with and without those trials removed. In the minimum, you should show the additional analyses that you put in your letter “we do not think this is the case for our data because when we re-analyse the perch behaviour including only those perch who are active in all three conditions (9 perch, and 27 trials) our result is the same: Proportions: Duration, GLMM t = 3.62, P = 0.002; Orienting, GLMM t = 3.91, P < 0.001, Lunges, GLMM t = 4.34, P = 0.08. Total lunges: GLMM z = 4.50, P < 0.001.” I think this does provide support for the robustness of your results. Lines 354-357- I would expand this a bit, or at least cite studies showing that immobility is a deterrent behavior. Lines 360: Instead of “reduced predator avoidance”, I would say “a reduced ability to avoid predators”. Line 366: I think a more specific transition here would make this a bit more readable. Instead of “Context can influence the behavioral response of fish to chemical cues”, it would be useful to say something along the lines of “Predators may respond differently to chemical cues of prey depending on the social or ecological context or depending on their own attributes” as the example you use primarily refers to differences in size/age in how perch respond to cues. Lines 433-434: It would be useful to explain this. E.g. further tests with a non-predatory fish would be needed to confirm that changes in behavior are specific to predatory fish and not a general response to cortisol. I think that this would be interesting because it would likely confirm that this is indeed something specific to predator-prey interactions.

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.

Title: PERCH, PERCA FLUVIATILIS SHOW A DIRECTIONAL PREFERENCE FOR, BUT DO NOT INCREASE ATTACKS TOWARD, PREY IN RESPONSE TO WATER-BORNE CORTISOL Review round: 2 Reviewer: 2

Basic reporting: I have had a look at the response letter and the amended manuscript and I am happy to say that the authors did a great job with their revisions in agreement with my, and the other reviewer's, suggestions. Thus, I am happy for this MS to be accepted. Experimental design: As I said in the previous review, I think the experimental design is well thought through. Validity of the findings: Conclusions are backed up by the results of the experiment Additional comments: The authors did a serious and thorough job with the revisions!

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.

Title: A PRELIMINARY SURVEY OF ZOANTHARIAN ENDOSYMBIONTS SHOWS HIGH GENETIC VARIATION OVER SMALL GEOGRAPHIC SCALES ON OKINAWA-JIMA ISLAND, JAPAN Review round: 1 Reviewer: 1

Basic reporting: 1- Line 28: The verb “prefer” is not only too anthropomorphic, but also implies experimental manipulations were performed to investigate shifts in Symbiodinium communities. I strongly recommend that it be revised to “be associated with” as their study is purely one of associations between diversity and the environment. 2- Line 29: “slight” is awkward. Suggest “less than those usually investigated….” 3- Line 32: Suggest “accurately determine species diversity and their distributions.” 4- Intro, paragraph 2: Microsatellite markers have also been develop to investigate fine-scale diversity in Symbiodinium (see, Pettay & LaJeunesse 2007 [Molecular Ecology Notes], Grupstra et al. 2017 [Coral Reefs]). 5- Line 58: Suggest specifying “this work,” perhaps as “As a result of the development and implementation of finer resolution molecular markers…” 6- Line 65, for consistency, revise “reexamine” to “re-examine.” 7- Line 92: Another crucial aim was to explore potential associations between diversity and the environment, and is equally important to include at the end of the Introduction. 8- Line 122: Revise “Nucleic acids were extracted” to “DNA was extracted.” 9- Line 139: The psbA *gene* was not sequenced; the non-coding region was. 10- Line 142: Which previously reported sequences? Provide accession # for these here (as well as later in the Results as has been done). 11- Line 197: First sentence is awkward. Suggest, “We next examined the distribution of Symbiodinium lineages across locations.” Experimental design: 1- Line 86-87: Clarification is needed here (as well as later in the Methods for PCR and sequencing ITS2). If ITS2 sequences were already obtained for Symbiodinium within P. tuberculosa in southern Japan (*including Okinawa*), what was acquired in this study? New samples? 2- Line 124: According to TableS1, not all samples have a pending accession # for ITS2, implying that only a subset of samples were PCRed and sequenced for ITS2? Please clarify. 3- Line 131: Primer concentrations are needed. 4- Environmental data results: Were there any significant differences in temperature and chl-a between sites, for example, were southern sites *significantly* warmer than northern sites? A simple statistical test to test for significance between sites could add a great deal to explaining how/why the environment could shape the diversity observed. In contrast, if the sites are not significantly different, more caution should be made in declaring that the environment shapes diversity. Alternatively, address whether these environmental differences (whether significant or not) are biologically meaningful to the coral/symbiont relationship? Does a 0.95°C difference in temperature make a big difference in the coral/symbiont world? Furthermore, “Yearly average chl-a concentration values were generally low” (Line 172 and later in Discussion), but no mention of whether there are apparent differences between sites to consider. Finally, are there available software that could be used in this study to better test for diversity/environmental data associations? The program Geste and R ‘vegan’ package come to mind in using generalized linear modeling to investigate whether genetic structure relates to environmental factors. However, I am unsure whether the sample sizes in this study are sufficient or whether sequence data are able to be analyzed by these programs. Worth looking into these and perhaps other options. 5- Why the ITS2 marker was used at all needs additional justification (in addition to Line 277- the end of the paper). Its results are hardly ever mentioned/discussed in the discussion and understandably so; however, it needs to be made more clear why this marker was used in the first place (in Methods), and why the results from psbA noncoding region were the focus of the conclusions and discussion. Validity of the findings: Overall, the findings and conclusions drawn are sound. I appreciate their recognition of the limitations of their study Lines 252-260. As pointed out in this paragraph, host variation could explain Symbiodinium diversity. While they cite cases of mismatch between host and symbiont, they should also reference studies that have found a match suggesting Symbiodinium diversity/distribution is simply due to host-specificity (examples include Bongaerts et al. 2010 [PLoS One], Prada et al. 2014 [Mol Eco]). Additional comments: Noda et al. use a faster evolving gene to elucidate the diversity of Symbiodinium within Palythoa tuberculosa in Japan, and combine it with fine-scale environmental data to explore any associations between this diversity and the environment. This work is crucial to the field in joining a few other recent papers that advocate for such a finer-scale approach to better understand the biogeography of Symbiodinium. Overall, the study is sound and robust. The paper is overall well written and this review presents minor comments and revision suggestions.

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.

Title: A PRELIMINARY SURVEY OF ZOANTHARIAN ENDOSYMBIONTS SHOWS HIGH GENETIC VARIATION OVER SMALL GEOGRAPHIC SCALES ON OKINAWA-JIMA ISLAND, JAPAN Review round: 1 Reviewer: 2

Basic reporting: The authors present a clear and unambiguous analysis of Symbiodinium diversity in Palythoa tuberculosis using a highly resolving genetic marker. Using satellite derived environmental data, the authors discuss the fine-scale zonation of different zoox haplotypes and discuss this in the context of the importance of fine-scale surveys to accurately capture Symbiodinium diversity in corals. I am glad to see more manuscripts moving away from the classical single-marker ITS2 approach and towards a multiple-marker approach. Based on the improvements made to this version of the manuscript (I reviewed a previous version), I am happy to recommend this manuscript for publication in PeerJ. The next obvious question that arrises from this work is what are the functional differences between psba haplotypes? Experimental design: no comment Validity of the findings: no comment Additional comments: No additional comments

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.

Title: ELEVATED SERUM OSTEOPROTEGERIN MAY PREDICT PERIPHERAL ARTERIAL DISEASE AFTER KIDNEY TRANSPLANTATION: A SINGLE-CENTER PROSPECTIVE CROSS-SECTIONAL STUDY IN TAIWAN Review round: 1 Reviewer: 1

Basic reporting: the article is written in a clear way references are sufficient (but could be improved) the structure is acceptable the results are clearly expressed, though they do not fully support the hypothesis Experimental design: the research is within the aims of the Journal as reported in the comments to the AA, the methods are not fully adeguate to add knowledge to the topic No problem by the ethical point of view, but there are some methodological inconsistencies the description of the methods should be improved Validity of the findings: no particular novelty The Data and their statistical handling are far from being satisfying the conclusions should be resized on the basis of the limited information given by the presented data Additional comments: This is a prospective observational study intended to evaluate the relationship of osteoprotegerin (OPG) serum levels and peripheral arterial disease (PAD) in a cohort of kidney transplanted (KT) patients (n. 74). The main conclusion was that higher OPG levels are associated with the presence of PAD in KT patients. There are some critical points which should be better addressed: - The first point to be faced is to better explain which could be the possible pathogenic link between high OPG levels and vascular calcifications, given that all the experimental data indicate that OPG mainly acts as an inhibitor of the calcification process. Is OPG simply a marker of VC or a counteracting compensatory response? - A second point which deserves a more in-depth discussion is the assumption that an elevated ankle-brachial index (ABI) is sufficient to make a diagnosis of PAD. In fact, though I can easily agree that ABI is highly related to the vascular calcification process, it cannot be assumed that all patients with higher ABI have for sure a PAD, given that no other clinical and instrumental tool have been used for a specific diagnosis of PAD. It could be more appropriate to talk of a relationship between OPG and arterial stiffness and not PAD. - It is not clear whether the KT patients included in the study were all the patients followed in the medical center in Hualien or only part of the whole cohort. If the latter was the case, it could be worth describing the criteria for choosing the studied cohort. - The authors should describe more in detail the methods used for the measurement of OPG and PTH, giving the normal values for their laboratory, the coefficient of variation, the intra- and interassay CV.

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.

Title: ELEVATED SERUM OSTEOPROTEGERIN MAY PREDICT PERIPHERAL ARTERIAL DISEASE AFTER KIDNEY TRANSPLANTATION: A SINGLE-CENTER PROSPECTIVE CROSS-SECTIONAL STUDY IN TAIWAN Review round: 1 Reviewer: 2

Basic reporting: The manuscript would benefit from professional editing. In particular, a few sentences are really ambiguous and rather difficult to read. Examples: line 66 "the arterial-venous shunt preservation on the limb for prior renal replacement therapy", lines 82-84 "the ABI is the ratio of the systolic blood pressure of the ankle divided by the systolic blood pressure of the arm with lower one of ankle systolic blood pressure value to calculate ABI. As previous our study we set...", lines 170-171 "OPG plays a protective role in term of vascular calcification of animal studies but also being detrimental effect on the progression of atherosclerosis in clinical consideration". Additionally, there are several typos and syntax and grammar mistakes (e.g. line 72, "The informed consent" rather than "Informed consent"; line 86, "part of sample" rather than "part of the samples" etc.) Table 3 does not show (or mention) the different variables included in the model, apart from the variables which reached statistical significance. Experimental design: The research question is well defined and potentially meaningful. Methods are reproducible with any statistical software. Ethical standards were respected. Validity of the findings: The Authors should have compared the sensitivity and specifity of osteoprotegerin with the sensitivity and specificity of ankle-brachial index, in order to emphasize the potential relevance of the new essay. For example, a recent review from the Cochrane collaboration (Crawford F et al. Cochrane Database Syst Rev. 2016. 9:CD010680 showed that only one study out of 49 met the eligibility criteria, thus underscoring that evidence about the accuracy of ankle brachial index is sparse, as also highlighted by other Authors (sensitivity 15-79%; Xu D et al. Vasc Med. 2010. 15: 361-369. Additional comments: At the moment, the discussion is excessively focused on evidence from animal models. The Authors should make an effort to try to reconcile evidence from experimental studies (protective role of OPG) and evidence from human studies (detrimental role of OPG). Although they acknowledge that the observational nature of the study does not allow to establish a causal relationship, they should try to discuss possible explanations of this discrepancy. Is OPG an "innocent bystander", rather than the cause of peripheral arterial disease? Is it possible that the increased level of OPG triggered by inflammation is actually a compensatory mechanisms that tries to limit the harmful effects of endothelial dysfunction? This is a crucial point, as inflammation markers have not been tested in the study. Finally, are the different actions of OPG potentially mediated by different receptors, like in the case of AT1 and AT2 receptors in the renin-angiotensin system? Also, there is an apparent contradiction between the statement (lines 144-145) that "the renal function of patients who undergo KT unfortunately remains within the range of chronic kidney disease (which is often true, but not always) and the subsequent statement (line 197) that "the KT patients in the present study had relatively good renal function". Also, how would an average eGFR of 42 (stabe 3b CKD, moderate CKD) represent a "relatively good renal function"? Line 143: please substitute "multivariable" with "multivariate" (Am J Public Health. 2013 January; 103(1): 39–40) Line 92: please provide a reference for the MDRD formula; did you use the 4-variable MDRD or the 6-variable MDRD? Line 53: please substitute "PAD is not an uncommon manifestation" with "PAD is relatively common" Line 54. please substitute "was shown to significantly associate" with "has been associated with" Conclusions (line 208). The statement "Management of biological markers of vascular risk" should be substituted with "Timely detection of biological markers of vascular risk"

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.

Title: ELEVATED SERUM OSTEOPROTEGERIN MAY PREDICT PERIPHERAL ARTERIAL DISEASE AFTER KIDNEY TRANSPLANTATION: A SINGLE-CENTER PROSPECTIVE CROSS-SECTIONAL STUDY IN TAIWAN Review round: 2 Reviewer: 1

Basic reporting: all the four issues are satisfied Experimental design: all the four issues are satisfied Validity of the findings: all the four issues are satisfied Additional comments: the authors fully answered the raised questions

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.

Title: ELEVATED SERUM OSTEOPROTEGERIN MAY PREDICT PERIPHERAL ARTERIAL DISEASE AFTER KIDNEY TRANSPLANTATION: A SINGLE-CENTER PROSPECTIVE CROSS-SECTIONAL STUDY IN TAIWAN Review round: 2 Reviewer: 2

Basic reporting: No concerns Experimental design: No concerns Validity of the findings: No concerns Additional comments: Please modify the sentence : “Fourth, lack of study concerned of sensitivity and specificity of ABI for diagnosis PAD in KT patients and no angiographic image was also applied in this study” with "Fourth, the lack of data on the sensitivity and specificity of ABI for the diagnosis of PAD in KT patients and the absence of any angiographic images may pose additional limitations to our study".

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.

Title: DIVERSE RESPONSES OF SYMBIODINIUM TYPES TO MENTHOL AND DCMU TREATMENT Review round: 1 Reviewer: 1

Basic reporting: The introduction is too sparse. For example at L60 the authors state the mechanisms of menthol bleaching are “not clear,” but the cited references discuss the potential for menthol to interfere with the action of membrane receptor TRPM8 and lead to calcium-stimulated expulsion. These ideas are included in the discussion but they should be addressed in the introduction as well. The authors state in L64 of the introduction: “Whether the PSII breakdown in endosymbiotic Symbiodinium in corals is directly or indirectly caused by menthol irritation of cnidarian hosts was also evaluated.” However, these results aren’t reported or discussed. To fit the data presented, this line needs to be removed, but if the authors did investigate it, I would rather they include the data. The introduction could do a better job of establishing that no other studies have compared the responses of different Symbiodinium to both menthol and DCMU, and that this work investigates the important but underexplored topic of functional diversity among Symbiodinium species. Across all figures and tables, please use the same order for the Symbiodinium types. Right now each figure has a different order. For Figure 2, I recommend including a label of the Symbiodinium type and menthol concentration in each panel. Experimental design: The replication scheme should be reported in the materials and methods, not in the results. Three of the Symbiodinium types are taxonomically described, and these names should at least be included at the first mention of each type: B1 = S. minutum, D1a = S. trenchii, C1 = S. goreaui Validity of the findings: It appears that the ROS assay fluorescence data (presented in Table 3) was not analyzed statistically, even though replicates were measured. Such an analysis should be performed (or the reason it was not performed should be stated explicitly). Also, Table 3 should include the background control fluorescence values (reported in L167). In the results (L155), the authors note that DCMU-treated Symbiodinium showed no ROS generation. I feel like this point should be addressed in the discussion, specifically tying back to the Jones 2004 study where the working model was that DCMU drove ROS production and subsequent bleaching. The idea presented in L192 that clades B and D are more closely related to each other than either are to clade C is incorrect. B and C share a more recent common ancestor (take a look again at Pochon et al. 2014). In L194-200, the authors discuss the mechanisms of menthol bleaching. As I mention earlier, this should be addressed a bit more in the introduction. In the discussion, I would like to see more explanation of how the results of this study inform our understanding of the mechanism of menthol bleaching, especially with respect to ROS production. The results run counter to previous ideas (e.g. Jones 2004) and this should be highlighted. In L204, the authors suggest Symbiodinium minutum (B1) may lack a TRPM8 homolog. This would be easy to check, as the S. minutum genome is published and easily BLAST-able at reefgenomics.org, as are the genomes of S. microadriaticum (A1) and S. kawagutii (F1). Unfortunately, those other species were not tested here, but I think it would be valuabe to check all three anyway. The discussion ends abruptly and does not adequately address the implications of the results. What does this mean for researchers considering using menthol or DCMU for future studies? Additional comments: Wang et al. exposed several types of Symbiodinium to two common chemicals used in studies of coral bleaching (DCMU and menthol) to investigate their effects on photochemical efficiency and reactive oxygen species production. The authors establish functional variation among species with respect to the point at which 50% photosystem II inhibition occurs and the level of ROS that is produced. This is useful data, but the manuscript is incomplete in several ways. The introduction is brief and presents an idea that is not tested, a key result lacks statistical analysis, and the discussion does not do a good job of exploring the implications of the findings. A major revision would be required to make the manuscript acceptable. John Parkinson Oregon State University Minor Comments Abstract L23: change to “have been studied extensively” L24: “cell depletion” is ambiguous. Later on it becomes clear you mean coral ‘bleaching’ (in the broadest sense, any loss of symbiont cells or reduction in photosynthetic pigmentation). I would use this term instead of “cell depletion” throughout the manuscript. L31: as is, the sentence implies that B1 is most sensitive, while C3 and C1 are the least sensitive, when in fact the result is the opposite. Just change “PSII sensitivity” to “PSII tolerance” to fix this. L35: could use a summary/implication statement here Introduction L41: change to “nine Symbiodinium clades (A-I) and numerous subcladal types” L48: include additional examples and references for other types of functional variation (as you do in L183-187). L48-49: rephrase or delete the clause that starts with “which provides a chance…” As written, the statement is host-centric and an oversimplification. L54: change to “evidence has been given” L57: change to “cells have been studied extensively” and provide references Materials and Methods L93: start at new paragraph at “When determining…” L95: need to indicate that Fv/Fm values >0.6 are generally considered healthy/normal L98: spelling. “photosynthetically” L110: delete “Practically,” L111: explain why values of 1.73 mM menthol and 0.13 mM DCMU were used (also, everywhere else DCMU concentration is reported in pM—why not do the same here?) Results L126: replication should be described in the methods. Unless it’s really important, I don’t think indicating triplicate sampling should be done so often in the results. L160: change “significant” to “measurable” or some other word; I prefer to use ‘significant’ only in the context of statistical results. L173: move the clause that begins with “indicating a typical ROS burst” to the discussion, talk about it more, and include references. Discussion L180: again, I’d avoid “algal-depletion” and use coral bleaching L183: change to “Diverse physiological performances among different Symbiodinium types have been observed with respect to thermal tolerance…” L206-209: Rephrase. This is a bit misleading, because D1a did produce ROS, while B1 did not (but B1 isn’t considered to be as stress-tolerant as D1a). Moreover, one of the most heat resistant Symbiodinium is S. thermophilum, a C species. This is why broad generalizations about clades should be avoided.

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.

Title: DIVERSE RESPONSES OF SYMBIODINIUM TYPES TO MENTHOL AND DCMU TREATMENT Review round: 1 Reviewer: 2

Basic reporting: I commend the authors on a clearly written and professionally composed manuscript. If there were any areas where the text could be improved, lines 54-55 “…indicate coral bleaching caused by ROS generated by DCMU-treated Symbiodinium” reads a little unclear, and perhaps the term coral bleaching needs to be defined for the wider reader. Also Line 50 “…Symbiodinium depletion” may need to indicated that this is depletion from cnidarian host cells. The references are generally sufficient, although I would like to suggest some more recent references in some areas: Line 41 – there are many more detailed and recent references on the translocation of photosynthetic products and nitrogen cycling, e.g. Hillyer et al 2017, and Pernice et al 2015. A more relevant review of compound exchange than Douglas 2010 is Davy et al 2012. Line 49 – needs a reference e.g. the review by Blackall et al 2015. Lines 50-53 – there are many more recent papers investigating the mechanism of Symbiodinium depletion from host cells, e.g. the review by Weis 2008. Line 80 – “Exaiptasia” needs the reference Grajales and Rodrigues, 2014. Line 173-174 – need a reference for this statement. The raw data are complete and the tables and figures are all necessary to better understand the results. My only comments are that sample numbers would be helpful in the figure and table legends for readers to better interpret the results. Experimental design: The methods are well explained and utilise well-established methods of investigating Symbiodinium functionality. However, I think some justification as to why all Symbiodinium types were tested under the same 25˚C temperature is required in the text. While I appreciate this is a traditional reductionist approach (to target PSII differences in response to the chemical treatments), this decision needs to be alluded to in the text. As shown recently by Silverstein et al (2017), there are variable effects of temperature on photodamage between Symbiodinium types. The 25˚C may have been suitable to the B1 symbionts (as this is presumably, the temperature the anemone hosts were maintained under?), but this may not have been the case for the other types e.g. we know D1a photosynthetic performance is hindered under low temperatures (e.g. Silverstein et al 2017). For example, line 148-149 – are B1 the most tolerant because this is their optimal temperature? When considering Symbiodinium type responses in terms of photosystem function and ROS production, some consideration of the experimental temperature conditions will make these methods and interpretation more robust. Other areas where the clarity of the methods could be improved are: Line 79 – “corals were acclimated to lab conditions…” could be improved if there was a reference to another section of this manuscript or previous publication where these are detailed. The same point can be made for line 97. Line 86 – DGGE is not currently thought to be the most effective way of identifying Symbiodinium type composition (e.g. Wham and LaJeunesse 2016), particularly if there may have been some background types, as suggested in line 86 with the description that only the “dominant” type was identified. This is particularly important in regards to the overall functionality of the Symbiodinium population, as background populations are functionally active (e.g. Cunning and Baker 2014, Cunning et al 2015). Line 110 – A reference for how the Symbiodinium cells were counted would support these methods. Validity of the findings: This study provides impactful information regarding the functional variability between Symbiodinium types. Specifically, this manuscript provides intriguing findings regarding the variability in sensitivity and ROS production between types. The statistics are well conducted and valid. Additional comments: This manuscript by Wang et al provides a very important and interesting first step to understanding the mechanisms of menthol-induced Symbiodinium depletion in host cells, with vital comparisons to the traditional DCMU treatment. Furthermore, this paper is of great value to our understanding of the diverse responses between Symbiodinium types, particularly in ROS response. Additional areas that may improve the flow of the text they are: Lines 131-125 – The equations were very useful, although the authors may wish to consider whether they would be better placed in the methods section. Line 194 -200 – This paragraph provides a useful background understanding of the cellular responses to menthol, and provides context for the following paragraph. For this reason, the authors may wish to consider combining the two paragraphs into one.

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.

Title: DIVERSE RESPONSES OF SYMBIODINIUM TYPES TO MENTHOL AND DCMU TREATMENT Review round: 2 Reviewer: 1

Basic reporting: no comment Experimental design: no comment Validity of the findings: no comment Additional comments: This revision is much improved—thank you for addressing most of my concerns. While I would still like to see a statistical analysis for the data presented in Table 3 to formally test for differences among species (as it is one of the key findings in of the experiment), I realize you have a small sample size and the effect is fairly obvious. I’d personally run a few more trials to boost the numbers for an ANOVA, but I leave that choice to you. Regarding the TRMP8 blast, I think you used a human nucleotide sequence to query the Symbiodinium nucleotide databases using a nucleotide blast (blastn). Instead, you’d want to use a protein blast (blastx). There’s likely far too much divergence between humans and Symbiodinium to get any decent nucleotide hits. Instead, blastx will translate the human nucleotide sequence to an amino acid sequence, which is more likely to be conserved across distant taxa. When I tried it there were no good hits to S. minutum, which supports the idea that TRMP8 is absent. But there weren’t any good hits to S. microadriaticum or S. kawagutii either, so this may not explain the differences in ROS production among species. It was worth checking but the results are inconclusive because you don’t have ROS data for the other two species so I’d probably leave the blast analysis out. The rest of my comments are all minor text suggestions. John Parkinson Oregon State University L38: Awkward grammar. Do you mean “This study further confirmed that Symbiodinium have diverse photochemical responses to stress even within the same clade.” L57: “coral host” to “coral hosts” L58: “(reviewed in Weis 2008, from cnidarian host cells, is generally attributed” to “(reviewed by Weis 2008) from cnidarian host cells is generally attributed” L81-84: It’s a bit strange to reference a result of this study in the introduction, but ok. L98: “clades” to “types” L104: “Symbiodinium was isolated” to “Symbiodinium were isolated” L118: “were identified as Symbiodinium C1, C3, C15, D1a, and B1” to “were identified as Symbiodinium ITS2 types C1 (S. goreaui), C3, C15, D1a (S. trenchii), and B1 (S. minutum)” Sorry to nitpick on how you include species names, but I really think you want to make sure to link ITS2 type to the binomial when they are first introduced in the manuscript (excluding the abstract). L127-128: reference Fitt et al. 2001 Coral Reefs for the normal/healthy Fv/Fm value L145: “S. minimum” to “S. minutum” L168: delete “data indicated that” L176: “S. minimum” to “S. minutum” L180: delete “Table 2 indicates that” L182: delete “A statistical analysis suggested that” L195: “S. minimum” to “S. minutum” L198: delete “Table 3 indicates that” L201: consider replacing “B1” with “S. minutum” just to be consistent. L220: “observed to thermal tolerance” to “observed with respect to thermal tolerance” L222: “expressions” to “expression” L240: “Symbiodinium C clade” to “clade C Symbiodinium” L244: “huge” to “large” L244: “defense strategy to stress” to “defensive response to stress” L245: “no ROS generation” to “the lack of ROS generation” L249: “the most “stress-tolerant” nature of the D clade among Symbiodinium types” to “the stress-tolerant nature of members of Symbiodinium clade D” L255: “symbiophagy (Dani et al. 2016) in certain Symbiodinium types” to “symbiophagy (Dani et al. 2016) in the hosts of certain Symbiodinium types”

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.

Title: DIVERSE RESPONSES OF SYMBIODINIUM TYPES TO MENTHOL AND DCMU TREATMENT Review round: 2 Reviewer: 2

Basic reporting: Generally, I am satisfied with the authors rebuttal to the concerns raised in the previous reviews. There are some spacing issues that need to be rectified (e.g. different hanging indent space, line 137), and font issues in the legend of table 2. Lines 81-84, I am confused by this sentence – are these results from this current study? If so, it does not belong in the introduction, and if not, which study is it from? Line 58 – missing parenthesis Line 126 – delete one “the” References – missing Davy et al 2012 reference Experimental design: I am satisfied with the changes made following the previous review. Validity of the findings: The new manuscript is improved in a number of sections here, especially if the TRPM8 homolog comparison can be included Additional comments: This is much improved on the previous submission.

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.

Title: HARNESSING MTDNA VARIATION TO RESOLVE AMBIGUITY IN ‘REDFISH’ SOLD IN EUROPE Review round: 1 Reviewer: 1

Basic reporting: The scope is interesting sine the adequacy or not of COI universal code must be taken into account. A suitable genetic marker selection is a big concern in traceability issues, to assure the transparency across the complete food chain, and sustainibility. Table 1 & 2 are very confused In general the presenting of results is confused, including Fig 1 &2 Experimental design: Materials and Methods Sequence analysis: -line 135; subset of previously samples...which included (Shum et al, 2015): how many? -line 143; see results?? in Mat&Meth section?? what do you mean? In the analysis a number of previously published sequences have been included in the analysis. Include them in a table includeingthe reference etc... Results: line 160; Clarify and specify the meaning of "h" and "π" data line 168-174; Percentages are confused: 30% I guess 12/41 70% neither 27/41 nor 27/48 how do you calculate? 92% I guess 44/48 8% I guess 4/48 Please make easier the presentation of results. Improve Table 1 & 2 defining "ambigous results" in order to make understandable. Is very diffiult to identify the mislabeled samples line 170; Define what is for authors one "ambiguous results" In the species id you conclude that an unknow sample is shallow or deep (S. mentella). However, you conclude this with only 6 reference samples (Table 2) In my iponion you can not resolve this aspects with this reduced number of samples Sequence analysis (Fig 1&2): You can not included in the building of a NJ phylogenetic tree all samples including, in the same analysis, "market" and "blind/references" sequences. This fact produces a very important and significant bias. First, you must build a tree only with the reference samples and after doing this analysis you can include the unknown samples but "one by one" to resolve with accuracy the species i.d. In Fig 2, you say that red group is S. mentella (shallow). Attending to Table 2 classification; B6, B19 and B23 are S. mentella (shallow) but none of these reference samples are aligned in the red branches, can you explain it?, I'm confused Discussion: line 216; (see below) where? be more specific line 285; "visual eye", do you mean "attending to taxonomic keys"?? Please modify it line 298; "alarming discovery"" This statement is to strong. You have analysed only 5 samples from UK!!!! You demonstrate nothing with this small sampling size Validity of the findings: Some statements are very ambitious and are not demonstrated with the findings Additional comments: References can be improved. I miss a number of important references concerning to authentication of seafood products and the use of genetic markers.

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.

Title: HARNESSING MTDNA VARIATION TO RESOLVE AMBIGUITY IN ‘REDFISH’ SOLD IN EUROPE Review round: 1 Reviewer: 2

Basic reporting: This paper takes focused look at the identification and labeling of commercially exploited North Atlantic Sebastes species. It addresses the difficulties of genetic and morphological identification in a recently diverged group of fishery species, proposes an alternative marker (d-loop) to the COI standard in BOLD, and examines labeling accuracy in northern Europe. Overall, the paper is well-written and well-organized, and the figures and tables are good. I have a few minor grammatical changes (suggested below), and a few larger suggestions to expand the methods and discussion (suggested in appropriate subheadings). However, all of these are fairly minor, and with some revision I believe this manuscript meets PeerJ’s standards and would be a solid addition to the literature. Specific comments: I found it surprising that the authors made little reference to the issues inherent in Pacific Sebastes identification. There are very similar complications in this group, particularly with regard to vague commercial labeling of multiple species. A brief comparison between the Atlantic and Pacific groups would be a nice addition to the discussion, and help to broaden its context. The authors already reference a paper on this (Logan et al. 2008); it would be nice to see it discussed in a little more detail in the discussion. Experimental design: The experimental design was generally fine. The authors did a nice job of laying out the difficulties and importance of identifying north Atlantic Sebastes species, and their sampling was good. Their inclusion of blind genotyping of 29 samples morphologically identified by outside experts was a particularly strong element of the study. Validity of the findings: The conclusions were generally well-supported and the authors addressed the study’s limitations fairly. In particular, they clearly acknowledged the potential for hybridization to confound any approach based on mitochondrial markers. Specific comments: How many sequences listed as your four target species were present in GenBank and BOLD for d-loop / COI at the time of your search? I would like a bit more reassurance that the difference in identification between the databases wasn’t confounded by very different reference availability. Likewise, I would like to see a GenBank search for COI; this would allow more direct comparison between the databases. I do agree that the phylogenetics presented later suggest that d-loop is just a better marker for this group. However, since the paper directly compares the two databases, it would be helpful to have a more direct comparison and a brief discussion of the impact of sequence availability on database-specific results. Deep-vs-shallow S. mentella: Is there really such a bright line between these groups? Your d-loop tree suggests a lot of diversity. Would like to see a little more discussion of what might be causing the ambiguity – eg, since it is classified as an “incipient” divide, is there potential ongoing introgression between “deep” and “shallow” S. mentella beyond occasional hybridization? Additional comments: Figures 1 & 2: This is kind of a chronic issue with large trees, but it is pretty difficult to make out/interpret the sample names, even printed full-page. Would really like to see something done to improve the size and clarity of the names. More context on the sample names would also help – for example, including the blind sample morphological ID or given market name in the sample name. Suggested grammatical changes: L20: Change “comprises of” to “is comprised of”. L38-39: Change “as well as monitoring” to “and monitoring”. L41: Change “and the complex supply networks” to “and complex supply networks”. L66: Change “is deposited” to “are deposited”. L89: Change “respectively, 2)” to “respectively, and 2)”. L91: Change “diversified” to “diverged”. L136-137: Change “as well as shallow” to “and shallow”. L157: Change “among four European” to “in four northern European”. L182: Change “Both mitochondrial” to “The two mitochondrial”. L226: Change “affected by” to “compromised by”. L250: Change “and/or lacks the diagnostic polymorphisms to accurately identify them.” to “and/or a lack of diagnostic polymorphisms for identification.” L265: Change “a good source of reference” to “a reliable reference”. L270: Change “, in those groups known to hybridize” to “in hybridizing groups”. L285: Change “by visual eye” to “visually”. L341: Change “amounts” to “numbers”.

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.

Title: COMPARATIVE STUDY OF CD UPTAKE AND TOLERANCE OF TWO ITALIAN RYEGRASS (LOLIUM MULTIFLORUM) CULTIVARS Review round: 1 Reviewer: 1

Basic reporting: 1. Basic Reporting • The submission must adhere to all PeerJ policies (see: 'Journal Policies'). • The article must be written in English using clear and unambiguous text and must conform to professional standards of courtesy and expression. The level of English used in this article is not good enough to be published. It should definitely be reviewed and rewritten by a native speaker with a scientific background. It cannot be published in this condition. • The article should include sufficient introduction and background to demonstrate how the work fits into the broader field of knowledge. Relevant prior literature should be appropriately referenced. Short introduction: the introduction could be improved by providing more relevant background. Also, specify the physiological stress markers you are going to address in your article. • The structure of the submitted article should conform to an acceptable format of ‘standard sections’ (see our Instructions for Authors for our suggested format). Significant departures in structure should be made only if they significantly improve clarity or conform to a discipline-specific custom. Standard sections: OK The results of the effect of Cd on plant mineral concentrations and TFs are badly structured and it is hard for the reader to comprehend. My advice is to split up the results for root, shoot and TF and discuss the cultivars separately for each of the minerals. It is not needed to introduce new titles, but you can work with paragraphs here. • Figures should be relevant to the content of the article, of sufficient resolution, and appropriately described and labeled. The labels of the figures are incomplete. A part of the text is missing at the end. Figure 3 also does not mention anything about the p-values or levels of significance, nor does it mention the nutrients (metals) shown in the graphs. There is also no information about the error bars in each of the figures. Are they STDEV or SE or … ? In some figures, there is only “g” as unit (for example in figure 3), where it should be “mg / g FW”). The image quality of the graphs is OK. • The submission should be ‘self-contained,’ should represent an appropriate ‘unit of publication’, and should include all results relevant to the hypothesis. Coherent bodies of work should not be inappropriately subdivided merely to increase publication count. In my opinion, the submission is self-contained. The paper informs me on the tolerance of two Italian ryegrass cultivars. It also addresses one mechanism to cope with the Cd stress. The article has no revolutionary conclusions (in my opinion). Experimental design: 2. Experimental Design • The submission must describe original primary research within the Aims & Scope of the Journal. To my knowledge, it is original primary research. • The submission should clearly define the research question, which must be relevant and meaningful. The knowledge gap being investigated should be identified, and statements should be made as to how the study contributes to filling that gap. No stated knowledge about the effect of Cd on Italian ryegrass in other literature. This article gives information about the effect of Cd on Italian ryegrass. • The investigation must have been conducted rigorously and to a high technical standard. The number of replicates for each treatment is quite small. Still, the error bars are small. BUT: The authors do not indicate which calculation they have used for the error bars. • Methods should be described with sufficient information to be reproducible by another investigator. Information about the calculation of distribution proportion is not given. Information about the correlation test is not given in M&M. The formulas of the TFs and BCFs are incomplete. It is not stated whether Cd concentration, Cd accumulation, … is used for these calculations. • The research must have been conducted in conformity with the prevailing ethical standards in the field. OK. Validity of the findings: 3. Validity of the Findings • The data should be robust, statistically sound, and controlled. Only 3 biological replicates for each treatment. Yet, they have achieved to obtain small error bars. BUT: The others do not indicate which calculation they have used for the error bars. • The data on which the conclusions are based must be provided or made available in an acceptable discipline-specific repository. Data are supplied. I did have insufficient time to check whether these data and calculations are correct. • The conclusions should be appropriately stated, should be connected to the original question investigated, and should be limited to those supported by the results. Conclusion is in line with the research question. • Speculation is welcomed, but should be identified as such. / • Decisions are not made based on any subjective determination of impact, degree of advance, novelty, being of interest to only a niche audience, etc. Reconsider the use of Italian ryegrass for cultivation and phytoremediation. Looking at fundamental knowledge about the Cd tolerance mechanisms is interesting. If you want to look at these mechanisms for cultivation of Italian ryegrass for fodder, fuel, ... you should first consider the fact that it is grown on Cd contaminated soil. The use of Italian ryegrass for phytoremediation should be reconsidered since accumulation levels in root and shoot are about the same. For example, in contrast to maize, see: Wang, Aiyun, Minyan Wang, Qi Liao, and Xiquan He. 2016. “Characterization of Cd Translocation and Accumulation in 19 Maize Cultivars Grown on Cd-Contaminated Soil: Implication of Maize Cultivar Selection for Minimal Risk to Human Health and for Phytoremediation.” Environmental Science and Pollution Research 23 (6): 5410–19. doi:10.1007/s11356-015-5781-z. In this article, they have shown that maize accumulates about 80% in the straw and grain. • Replication experiments are encouraged (provided the rationale for the replication, and how it adds value to the literature, is clearly described); however, we do not allow the ‘pointless’ repetition of well known, widely accepted results. No replication, since the authors state that it is not yet tested for their cultivars / Italian ryegrass. • Negative / inconclusive results are acceptable. I would reconsider being positive about the cultivation purposes for Italian ryegrass (phytoremediation, fodder, …). You might want to add a negative advice for that. You could consider fundamental research concerning the mechanisms in tolerance, damage, uptake and transportation. Additional comments: I have added the PDF with markings and annotations. The red markings concern syntax errors. I have not marked all the syntax errors. The orange markings are questions or remarks. The questions/remarks are added to these markings. The large blue marking (of Effects of Cd on plant mineral concentrations and TFs) concerns a block of text which is hard to comprehend. I advise a new structure for that text in order to make it easier to comprehend. If you cannot read these remarks with your pdf-viewer, please open the pdf with "Adobe Acrobat Reader" and open the remark tab. Please do read the remarks. Also: In my opinion, Italian ryegrass is not suitable for phytoremediation or cultivation for fodder or fuel (on Cd contaminated soil) (based on your results about accumulation). Since I'm quite new in this field of research, I do advise you to seek out more peers and discuss this matter with them. I do think your research is worth being published, since you compare a tolerant and a sensitive cultivar and their response to Cd. Maybe address their variation in response in the conclusion and be specific about it (mention the parameters you've measured). I hope you find my review valuable and I also hope you are able to use this feedback in improving your manuscript.

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.

Title: COMPARATIVE STUDY OF CD UPTAKE AND TOLERANCE OF TWO ITALIAN RYEGRASS (LOLIUM MULTIFLORUM) CULTIVARS Review round: 1 Reviewer: 2

Basic reporting: The manuscript describes the effect of Cd exposure in 2 Italian ryegrass cultivars with regard to Cd uptake and translocation as well as the sensitivity/tolerance characteristics of both cultivars. The authors performed an extensive study with plenty of data but at present there are some major remarks that should be addressed by the authors. Concerning the English language, the manuscript should be carefully checked for grammatical errors, such as use of tenses, plural/singular... It is recommended to have the manuscript checked by a native English speaking person to improve the readability of the manuscript. Experimental design: Whereas the authors want to investigate the underlying mechanisms of Cd responses in 2 different ryegrass cultivars in relation to biomass production and Cd uptake for phytoremediation purposes, a hydroponic setup might be questioned. The authors should be careful with stating that high Cd accumulation occurs in hydroponics, this might definitely not be the case when plants are grown on Cd-contaminated soils. Extrapolation of lab results (controlled environmental setup) to field experiments is not straightforward. It is highly recommended to add an experiment on these cultivars grown on Cd-contaminated soils to confirm or not the possible extrapolation of the current data. In the majority of the M&M section, the authors describe very well the methodology, only for Cd distribution proportion, a description is lacking. This should be added by the authors. Validity of the findings: Although the authors performed a lot of work, the novelty of the manuscript is rather restricted as the analyses performed under different Cd exposures have been explored in multiple studies. Nevertheless, the work published on Lolium multiflorum is limited. Although the figures are clear, the authors should add the statistical analysis in the figure legends (as was done for figure 1). With respect to phytoremediation purposes, the current manuscript should be considered with care because hydroponics and metal uptake are completely different from field conditions on Cd-contaminated soils. The authors should revise this way of description of the context for there research questions. Additional comments: No additional comments

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.

Title: COMPARATIVE STUDY OF CD UPTAKE AND TOLERANCE OF TWO ITALIAN RYEGRASS (LOLIUM MULTIFLORUM) CULTIVARS Review round: 3 Reviewer: 1

Basic reporting: • English language: OK, but need minor changes here and there (be sure to look at my specific remarks for the individual lines). The editorial editing from AJE greatly improved the readability. • Literature references: OK, sufficient. • Raw data: o raw_data1: translate foreign language + Re in first column is unclear + file is coupled to other file, please make file work on its own o raw_data2: OK o raw_data3: translate foreign language o raw_data4: name sheet 1 o raw_data5: name sheet 1 o raw_data6: translate foreign language o I was unable to find absorbance levels for NPT at 412 nm. I might have overlooked it. • Figures and tables: o Figure 1: Isn’t it: “Duncan’s multiple range test”? “(n = 3) of three replicates”  don’t they mean the same? o Figure 2: Isn’t it: “Duncan’s multiple range test”? “(n = 3) of three replicates”  don’t they mean the same? o Figure 3: Isn’t it: “Duncan’s multiple range test”? “(n = 3) of three replicates”  don’t they mean the same? It would might help to add Zn, Fe, Mn and Mg above the graphs and Root concentration, Shoot concentration and TFs vertically to the left of the graphs. Just so the reader gets a quick overview of the contents of all the graphs. o Figure 4: Isn’t it: “Duncan’s multiple range test”? “(n = 3) of three replicates”  don’t they mean the same? o Figure 5: Isn’t it: “Duncan’s multiple range test”? “(n = 3) of three replicates”  don’t they mean the same? o Table 1: DW, dry weight Declare what the values mean (i.e. 153.73, 11.04, 38.32, …) Isn’t it: “Duncan’s multiple range test”? o Table 2: inhabitation? o Table 3: Duncan… o Table 4: OK Experimental design: • Identified knowledge gap: OK • Research question is relevant and meaningful. • High technical and ethical standard: OK • Methods: be sure to look at my specific remarks for the individual lines. Validity of the findings: OK + be sure to look at my specific remarks for the individual lines. Additional comments: Dear authors, Your current article has improved a lot compared to the previous version. The text was clear and the level of English was very good (except for a few sentences). Please consider the changes and adaptations which I suggested, but also feel free to argument why something should not be changed or adapted, since it is just my opinion. The approach to a more fundamental report of the response of the two cultivars to Cd was an improvement and most of the discussion is well written. I have chosen for a major revision, even though the revision needed is of a much smaller scale compared to the previous version. Since the article has changed a lot compared to the previous version, I would like to go through it once more after you have dealt with the remarks of the reviewers. By opting for a major revision, I get a chance to see how you interpreted my comments for improvement and where you do not agree (which of course is possible as well ! ). Good luck and thank you for your effort. Kind regards. IMPORTANT: I have specific remarks for individual lines. These remarks concern basic reporting, experimental design & methods and validity of the findings & discussion. These remarks are: Line 65: It is not just because a substance is toxic to plants, that plants have evolved mechanisms to cope with it. Line 73: Cd-PC, this abbreviation is stated or explained elsewhere. Line 121: “to the constant weight” --> the? Line 134: “the middle part of 100 mg of fresh leaves” --> what do you mean? Line 135: Link wave lengths to what you measure at these wave lengths Line 139: What is extracted? Line 142: What do you measure at these wave lengths? Line 142: Don’t you just measure MDA as a parameter for lipid peroxidation. Here it seems like you have measured both. Line 158: “some physiology parameters” --> to vague Line 165 – 166: “biomass reductions with increasing Cd dose from 5 μM to 100 μM were more distinct in Harukaze (P<0.01). --> ANOVA Cdxcultivar is not significant, meaning that both cultivars do not react differently to the cadmium treatment. Cultivars are significantly different, but they already differ in starting weight, which might be the most important factor why the weight of the cultivars differs. Line 173: Is it EC or IC50? I’m not sure. Please check. Line 178-179: Please nuance! Idyll is not tolerant, but MORE tolerant to Cd compared to Harukaze. Line 183: Why is it obvious? Sounds like you were expecting this or sounds like there is a specific reason for this. Line 185: Correlation is significant? Table 4 shows correlation both cultivars taken together. Could it be interesting to check correlations for both cultivars separately? Line 187: TFs --> TF Line 211: Reduction is not significant Line 217 – 219: More importantly: The 2 cultivars only respond significantly different for Fe and Mn in the root. There, the interaction of Cd and cultivar is significant. For the other parameters, there is no significant interaction, meaning there is no significantly different response. Line 227: Inhibited or reduced? / replace: that --> the Chl a/b ratio Line 234 – 240: The two-way ANOVA also showed a significant interaction, meaning that both cultivars responded significantly different to Cd treatments for NTP production in root and shoot. Line 241: Is it interesting to add a correlation analysis for both cultivars separately? Or is this a bad idea? Line 248 – 253: Please inform the reader of Cd concentrations in the crops mentioned. Line 259 – 260: Biomass reduction and TI give the same information, yet they are handled as different parameters. Line 262: Bad syntax. Line 267 – 268: Can you add shortly how Cd diminishes the capacity for ROS removal? Line 273: You report oxidative damage. Please specify --> oxidative damage: oxidation of lipids Line 273 – 274: How can a reduction in biomass be linked to oxidative stress? Line 294: Specify which cultivar has constant or increased Cd TF. Line 302 – 305: For me, the second part of the sentence is a harder to comprehend (…, whose tendency was the opposite of NPT content.). It might be just me, but maybe you want to have another look at it. Line 306 – 308: Just thinking out loud: If high NPT in Idyll promotes Cd sequestration and cause it stay in the root, then you would expect Idyll to have higher Cd concentrations in the root compared to Harukaze. This is not the case! The higher NPT content might protect, but maybe Idyll also is an excluder which does not take up and transport Cd easily. This is just a suggestion and I am not stating this is correct, but you might want to look into some more literature. Also, could it be that a lower translocation of Cd and higher amounts of NPT in the shoot are beneficial for the photosynthetic pigments? This might lead to a better photosynthesis, more energy and maybe better stress response? By making this reasoning, I am assuming a lot of stuff and maybe the connections are not that straight or even true... It just thinking out loud. Maybe you want to dig into some more literature and also think about it. Please don't feel obligated. Line 310 – 311: Unclear sentence.

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.

Title: COMPARATIVE STUDY OF CD UPTAKE AND TOLERANCE OF TWO ITALIAN RYEGRASS (LOLIUM MULTIFLORUM) CULTIVARS Review round: 3 Reviewer: 2

Basic reporting: The resubmitted manuscript is of excellent English quality, which is a major improvement in relation to the initial submission. In addition, the authors addressed the research question in a different way, not from a phytoremediation perspective but rather from a Cd tolerance/sensitive perspective and the mechanism behind this observation, which is much better when using a hydroponical setup for fundamental research. Experimental design: OK Validity of the findings: OK Additional comments: The manuscript improved a lot with regard to the initial submission.

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.

Title: COMPARATIVE STUDY OF CD UPTAKE AND TOLERANCE OF TWO ITALIAN RYEGRASS (LOLIUM MULTIFLORUM) CULTIVARS Review round: 4 Reviewer: 1

Basic reporting: Line 136-137: Are the wavelengths of 665, 649 and 470 respectively for chlorophyll a, b and carotenoids? Would be nice if this is clear for the reader by using "respectively" if it is. Line 143: To what substances do these wavelengths correspond? Line 141: "extraction" sounds like you are extracting MDA, where actually MDA is reacting with TBA (if I'm not mistaken...). Line 143-145: "Lipid peroxidation was estimated with the content of MDA that is thiobarbituric acid-reacting substances (TBARS) as described by Ali et al. (2014)." --> I know what you mean, but the sentence is unclear. A suggestion: "MDA content was estimated through a TBARS assay, as described by Ali et al. (2014)." + I would consider making this sentence the first sentence of the paragraph. Line 175: IC50 is underlined. Line 280-281: "The oxidative lipids" sounds strange. Line 309-311: "In our study, Cd concentration ..., the tendency of which was the opposite of NTP content...". This sentence is unclear to me. You might want to consider rephrasing. Experimental design: No comment. Validity of the findings: No comment. Additional comments: In my opinion, using Cd contaminated biomass for biofuel might only be possible if filtering of the combustion gases takes place (or if Cd is not there in the biofuel after processing of the biomass). If not, isn't there a chance for rereleasing the Cd back into the environment during combustion of the Cd contaminated biofuel? I enjoyed reviewing your article. Thank you for your effort in rewriting and considering my remarks.

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.

Title: GROWTH OF HYBRID OPEN ACCESS, 2009–2016 Review round: 1 Reviewer: 1

Basic reporting: This is a short report of a study that has attempted to estimate the increase in the numbers of hybrid OA journals and articles in the period 2009-2016. It is a fairly straight-foward account but in a number of places (highlighted with comments in the annotated PDF), the mode of expression could be clearer. In its present form, the manuscript is not an easy read. Greater clarity and attention to sentence structure will make it more accessible for an international audience. Experimental design: The methods used to estimate numbers of hybrid OA journals and articles include a mix of requests to publishers, estimates derived from examination of publisher web-sites and information gathered from other similar studies. In some places the description of the methods could be clarified for the benefit of the reader (see annotated PDF for details). The study aggregates estimates from 20 leading publishers (including the big five). It would be useful to know what is their total market share (in publishing and in OA).This would provide a useful sense of scale that cannot be derived from the numbers reported in Figures 1 and 2. Validity of the findings: The estimates produce appear reasonable to me. However, what is lacking – and this is highlighted by the mention of OA mega-journals in the final paragraph of the discussion – is a fuller sense of context. What fraction of all OA articles (in repositories, in hybrid OA journals and in OA journals) are hybrid? This seems an important and relevant point, especially if one wants to understand the importance and the potential future of hybrid options (and given the concerns expressed by some national funders). Overall also, I would like to see more references cited in support of some of the claims made (e.g. that authors perceive an APC of $3000 as 'high'; that starting hybrid options involves 'very low cost'; that hybrid APCs prices are 'rather uniform' - see annotated PDF). Otherwise, the tone risks becoming overly rhetorical. Additional comments: No further comments.

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.

Title: GROWTH OF HYBRID OPEN ACCESS, 2009–2016 Review round: 1 Reviewer: 2

Basic reporting: (1) There is a problem with table two. The column headings do not match the data in the table for the the first two columns. This must be fixed. (2) The author spends a lot of time in the methods discussing the challenges to collecting the full cadre of data that would be ideal for a study like this, and does an appropriate job conveying that because of the limitations the results are a lower bound. However, it is somewhat confusing to make sense of this in the method and I think that to address this the authors should include a THIRD table that lays out the methods used for each year of the study (and of course the citations where appropriate). This information is present in the text but I think it would be more clearly conveyed as a table that is organized by year. Grammar / Clarity - - In the abstract the abbreviation OA is used but introduced. This can be done in line 10. - In line 14 of the abstract what exactly “scattered” is referring to is unclear. Is it that the small numbers are scattered across many journals? This should be stated explicitly. - Line 28 the “one” should be removed - The sentence that starts on line 31 is awkward. The point of laying out the different types of OA is important and should be stated clearly. The sentence could be split into two sentences, the first that addresses the “timing” and the second that address the “read vs. re-use rights”. - Comma after “but in addition” in line 45 - The sentence that starts on line 49 should be rewritten to “ Due to the price level of typically around $3,000 USD, which many authors and their institutions perceive as high, the uptake of the option is low, on average only a couple of percentages of eligible articles” - Should Entemological Society of America on line 53 have its first letters uppercased? - Line 59, “starting” should be removed - Line 62, “Since” instead of “Starting from” - Line 64, replace “very low cost” with inexpensive - line 65, consider changing systems to platform - line 67/68 - end sentence with “since the subscription revenue remains” - line 90 - almost always or usually instead of “tend in particular” - line 92 - Secondly, - line 143 - it is unclear what is being refered to with “That share” on line 143. Is it what fraction of the number of hybrid-OA articles come from the major 5 publishers? - line 189 - English should be adjusted to “from either personal funds, departmental funds, or external research grants” - Sentence starting on line 194 - not clear whether the 3.8% average share refers to the percentage of total articles that are OA in a journal over a year or the fraction of journals that have atleast 1 OA article per year - Sentence starting on line 203 — Not entirely sure if this meaning is correct but if not the sentences can be adjusted. “First, several leading research funders set up centralized funds to pay for APCs. Second, a new type of consortial electronic subscription license has been introduced in which APC costs are included in the price.” Experimental design: (1) It is necessary to report the total number of article published by the journals along with the number of hybrid-OA articles. Without the total number of articles published we cannot distinguish between whether the rate of uptake has increased or whether the rate of uptake has remained the same but the total number of articles published has increased. (2) The author mentions that PNAS is a unique publication in that close to 1/3 of its articles are hybrid open-access. This raises the concern that perhaps it is such an outlier compared to the other publishers/journal groups that is skewing the overall statistics. Validity of the findings: The paper “Growth of hybrid open access, 2009-2016” provides an overview of the development of the hybrid open access sector of publishing and presents new data about both the number of journals that offer hybrid open access and the number of hybrid open access articles. The author does a good job conveying the challenges of acquiring the necessary data about the hybrid-OA sector. And given the clear challenges to acquiring this data, it is important that this work be published so that these numbers are available to the public. My concerns related to this section covered in the sections above. Additional comments: No comment

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.

Title: GROWTH OF HYBRID OPEN ACCESS, 2009–2016 Review round: 1 Reviewer: 3

Basic reporting: No comment, in general, this paper presents solid information regarding the Hybrid Open Access model that might be relevant to many scholars focusing on academic publishing. Experimental design: This is a descriptive manuscript with interesting statistics which were obtained using data that is not public which makes it hard to replicate. I would negotiate with the publishers to also allow access to the underlying data. Validity of the findings: Besides the fact the there is a growing number of publishers using the Hybrid Model which, in itself, is relevant, it lacks substance as to what the impact this phenomenon has in the publishing world as a whole. Additional comments: It is a nice manuscript but a little more argumentation would make it even better.

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.

Title: GROWTH OF HYBRID OPEN ACCESS, 2009–2016 Review round: 2 Reviewer: 1

Basic reporting: Since this is a review of a revised manuscript I will confine my comments to a single section. Overall, I am satisfied that the author has addressed all the comments made on the first version of this article and I think it is now suitable for publication. There are one or two residual points – all minor – which I leave to the author's discretion. (For detailed see the comments in the attached manuscript file). Experimental design: Please see above. Validity of the findings: Please see above. Additional comments: No additional comments

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.

Title: DOES SILVOAGROPECUARY LANDSCAPE FRAGMENTATION AFFECT THE GENETIC DIVERSITY OF THE SIGMODONTINE RODENT OLIGORYZOMYS LONGICAUDATUS? Review round: 1 Reviewer: 1

Basic reporting: No particular comments. There are minor grammatical problems throughout the text that will require minimal corrections. One issue is a need for additional punctuation (commas) to divide sentences. Experimental design: The study focuses on the effect of landscape fragmentation on the genetic structure of Oligoryzomys longicaudatus that acts as a main reservoir of Hantavirus in South America. They realized their sampling at a small scale (3.5km2) in a region constituted of different landscapes. One important concern of this study is the type of molecular marker that authors used (mtDNA). Even if they argue that, this marker can be used for population genetics studies, at this geographical scale, I am not confident about the obtained results. This represent a problem since other confounding factors can also have a major impact (such as demography, differences between male and female). Microsatellites markers should have been used to answer to this question. The authors also chose to sample Oligoryzomys populations during one season (during the period of low mobility). It would have been interesting to compare their results with the period of high mobility to see how landscape influences the genetic structure of this species. Validity of the findings: The results of landscape analysis could be given in more details (explaining clearly figure 4). As isolation by resistance analysis is not significant why comparing it to least-cost path results. Authors should discuss about the factors that cannot be identified using mitochondrial markers as it is more adapted to large scale studies and long evolutionary process. Additional comments: In their manuscript, the authors highlight the importance of landscape genetic studies of reservoir species in order to better understand the viral circulation and risk of emergence. The purpose of this study is really interesting and could correspond to the scope of the "PeerJ". A more detailed justification for the results and addition of those obtained by Ortiz (2016) would improve the manuscript. The authors could add reference: Ortiz N, Polop FJ, Andreo VC, Provensal MC, Polop JJ, Gardenal CN, Gonzalez-Ittig RE. 2016. Genetic population structure of the long-tailed pygmy rice rat (Rodentia, Cricetidae) at different geographic scales in the Argentinean Patagonia. Journal of Zoology. In the abstract : Line 47: replace “hanta” by “hantavirus” and “contagion” line 49 by “emergence”.

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.

Title: DOES SILVOAGROPECUARY LANDSCAPE FRAGMENTATION AFFECT THE GENETIC DIVERSITY OF THE SIGMODONTINE RODENT OLIGORYZOMYS LONGICAUDATUS? Review round: 1 Reviewer: 2

Basic reporting: The objective of this paper is to evaluate the effects of landscape fragmentation and matrix features on the genetic diversity and structure of Oligoryzomys longicaudatus, a sigmodontine rodent species, natural reservoir of Hantavirus in southern South America. The writing is simple and easy to understand. The theoretical framework is properly formulated and framed on the centrality of the issue. The references are appropriate and updated. More than 45% of the references correspond to the last 10 years. The article is correctly structured in its organization. The tables are self-explanatory and provide appropriate information. The figures are pertinent but I suggest that the metric scale be incorporated in Figure 1. This will allow a better interpretation of the distance relationship between the sampling sites (patches) of O. longicaudatus. The results obtained in the study, although they contribute to describe the use of the environment by O. longicaudatus through the use of corridors that communicate the patches with the native forest, these have already been described in the bibliography. The most interesting contribution of the article is its methodology focused on the use of 3 distances (IBD, LCP and IBR) to describe the effect of the landscape matrix on the genetic structure of O. longicaudatus. Experimental design: The experimental design is appropriate and the questions are addressed with original methodologies. The identification of the effect of the landscape matrix on the genetic structure of O. longicaudatus allows to evaluate the effect of landscape changes on the migration and connectivity of these populations of high epidemiological importance. Although the methodology in general is well explained I suggest that it is necessary to go into more detail in the description of the structure of the landscape. It is understood from the map legend that land use is classified into 6 different types of coverage but missing elements to interpret the layout of distances depending on each model. The methodology describes in detail the statistical package used and, from the technical point of view, the solution to certain difficulties in determining the distances. However, it is not clear how the environmental variables of each capture location are weighted in the sampling grid and how the data matrix was constructed to determine the different types of distances. Therefore, I suggest that the explanation be extended at this point in order to achieve a better interpretation of the results. Validity of the findings: The results presented indicate that O. longicaudatus shows high haplotype diversity, but a general low nucleotide diversity, suggesting recent differentiation and potential changes in population size. Although these findings are robust, it is not clear the interpretation of the differentiation in the genetic structure of the 2 clusters as show the significant LCP analysis. It is not interpreted from Figure 4A that the shortest paths traced for LCP are through grassland /shrubland to connect patches of native forest. The same difficulty arises from FIG. 4B in which it is not interpreted that the shortest paths by means of the IBR are that individuals are moving through native forest only, but through longest path. I suggest improving Figure 4 so that the differences between the traces obtained by the 2 approaches (LCP and IBR) can be more clearly observed. I would also recommend that data of average distances (or other variables) be included to justify in each case the alternative use that the species makes of the landscape elements, either from the matrix or from the corridors. On the other hand, the authors interpret that the use that would make the species of grassland/shrubland as corridors could be explained from their diet. However, the captures were made in winter and in this time of the year the availability of seeds is limited by the plant's own phenology. The authors conclude that, taking into account the effects of the surrounding matrix, O. longicaudatus individuals would preferably use grassland / shrubland to connect native forest patches. Although the conclusion is well established and relates to the original question I find certain limitations since it is not clear the interpretation of the results obtained from the analysis of the distance of least cost, on which it is based. Additional comments: The subject matter is of interest in the area of ecoepidemiology since it contributes to understand the spatial context of silvoagropecuary risk of Hantavirus contagion. Although it presents clarity in the objective of its investigation and good articulation between the conceptual framework and the development of its different sections, I consider that the results are partially supported by the data. I suggest that the article needs to specify, in more detail, the criteria used to characterize the landscape structure, to reformulate Figures 4 (A and B) in order to show differences in LCP and IBR tracings and to provide average distance data in order to interpret the differential use of the environment according to both analyzes. I believe that these modifications could contribute by giving greater reliability and validity of the results and to a greater support of the discussion.

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.

Title: DOES SILVOAGROPECUARY LANDSCAPE FRAGMENTATION AFFECT THE GENETIC DIVERSITY OF THE SIGMODONTINE RODENT OLIGORYZOMYS LONGICAUDATUS? Review round: 1 Reviewer: 3

Basic reporting: The manuscript is well written, and structured, but, the English language should be improved.. Literature references are well selected. Enough background is given in relation to the problem being addressed. All appropriate raw data have been made available. Experimental design: This paper reports a landscape genetic study of the sigmodontine Oligoryzomis longicaudatus (“Colilargo”) based on the analysis of 5 populations sampled in 5 patches located in a fragmented landscape with different types of soil use. The aim of this study is to evaluate the effects of landscape fragmentation on genetic diversity, and to investigate the most probable landscape characteristics of commuting “least-cost corridors” among patches The research question is well defined. The fact that the studied species is a reservoir of the Hantavirus emphasizes the importance of the results for regional sanitary controls, in order to reduce the risk of humans to contract HPS. However, data are neither robust enough nor statistically sound. The main conclusion drawn from the least-cost corridors used by this species is confusing because the results are not clearly presented. Besides, the statistical analysis should be improved. Methods have been described with sufficient information to be reproducible by other investigators. However, the methodology used to describe geographic distribution patterns of genetic variation (i.e. GENELAND) was not appropriately chosen. Geneland is a statistical computer program for population genetics data analysis. Its main goal is to detect population structure in the form of systematic variation in allele frequency by departure from Hardy-Weinberg and linkage equilibrium. Geneland requires individual multilocus genetic data that are optionally geo-referenced. It implements several models that use both geographic and genetic information to estimate the number of populations in a dataset and delineate their spatial organization (Guillot et al., 2005). In the present study, the authors use a single molecular marker, instead of multilocus data, thus preventing the assessment of allelic frequencies, which are necessary to detect departures from HW equilibrium. Furthermore, if each nucleotide position were considered as a different locus, then all positions would be linked and not in linkage equilibrium. This analysis should not have been performed with only one sequence marker. Validity of the findings: With respect to the validity of the conclusions I have some doubts about the interpretation of the data. They authors inferred correlations between geographic distances (calculated as IBD, LCP and IBR distances) with genetic distances through Mantel tests. They found that the only significant correlation was “genetic distance vs. least-coast distance”, and then, they calculated the shortest paths based on LCP and IBR. Based on these analyses, they concluded that the shortest path traced for LCP and IBR indicate that individuals of Oligorizomis are moving through “grassland/shrubland” and “native forests”, respectively, to connect patches of native forests. However, Figures 4 A and B do not show this difference. Instead, both figures show very similar results, with quicker paths occurring either across plantation forestries or native forests. This conclusion should be supported by providing better explanations and figures. The authors apply the raster of land use only to show its distribution on a map with the least cost path superimposed. They do not use this variable to analyze the landscape resistance as a function of dispersal probabilities. Perhaps it would be appropriate to perform some kind of simulation analysis (i.e. CDPOP, Landguth and Cushman, 2010) that takes into account landscape heterogeneity, to see if the pattern of distribution of genetic variability obtained with empirical studies, is similar to that recovered from simulated data. Additional comments: No comments.