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

Input:
Title: FORELIMB JOINTS CONTRIBUTE TO LOCOMOTOR PERFORMANCE IN REINDEER (RANGIFER TARANDUS) BY MAINTAINING STABILITY AND STORING ENERGY
Review round: 2
Reviewer: 2

Output:
Basic reporting: As before, in general, the grammar and writing are acceptable. There are a few instances where corrections are advisable, primarily in sentences added to this revision. The authors have met criteria for references, background, professional article structure, figures and tables. They have also provided the raw data. The results are self-contained and relevant to the introduction in the article. In response to reviewer comments, the authors have added questions to frame what they did in their study. I would have preferred specific hypotheses, but these are acceptable.
Experimental design: This is original primary research that is within the aims and scope of the journal. The research questions are relevant and meaningful. The authors state how this study fills a gap in the literature. The authors provided a rigorous investigation of reindeer forelimb mechanics using inverse dynamics and appear to have met institutional ethical requirements. The methods have been described sufficiently to allow for replication and improvements were made in their description in response to reviewer comments.
Validity of the findings: The findings in this study are valid, robust, and are sound. The authors have indicated what statistical methods they used (F-test), but did not offer a lot of detail. The authors conclusions are now connected to specific research questions posed in the introduction.
Additional comments: Overall, I feel that the authors have improved their manuscript with the suggested changes from the reviewers. However, there are a number of grammatical issues, primarily with the sentences that were added in this revision which will require some proofreading. I have made a few comments by referencing line numbers in the manuscript that I added for easier reference. I will upload this file.

Abstract

[Line 22] Unnecessary ‘the’. Should read ‘increased with rising locomotor speed’

[Line 24] Missing ‘the’. Should read ‘…net joint power related to the vertical GRF’

Introduction

[Line 46] Trunk shouldn’t be plural. Should read ‘…in their elastic feet to drive the trunk forward…’

There are still some grammatical and proofreading issues with this manuscript. I have pointed out the first few but many are in sentences that have been added to this revision.

[Line 167] This is likely a translation issue, but I think it would be more appropriate to say that the females were ‘excluded’ rather than ‘abandoned’.

[Line 447] The ‘Wrist joints as a stabilizer and a pusher’ section didn’t need to be completely removed, I just suggested removing references to human locomotion since I didn’t feel it was relevant.


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

Input:
Title: GENE SIGNATURE FOR PROGNOSIS IN COMPARISON OF PANCREATIC CANCER PATIENTS WITH DIABETES AND NON-DIABETES
Review round: 1
Reviewer: 1

Output:
Basic reporting: I would recommend that the authors seek professional language services to improve their writing.

The terms ‘protective’ and ‘risky’ genes may be replaced with more suitable terms. Please refer to other articles describing prognostic biomarkers.

The use of ‘generally speaking’ should be avoided.
Experimental design: Experimental design appears to be appropriate.
Validity of the findings: The authors identified a set of prognostic biomarkers for pancreatic cancer patients from TCGA. They have validated these markers using a dataset from GEO. It would be useful to validate the markers in another dataset (with more patient numbers) from ICGC.
Additional comments: No additional comments


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

Input:
Title: GENE SIGNATURE FOR PROGNOSIS IN COMPARISON OF PANCREATIC CANCER PATIENTS WITH DIABETES AND NON-DIABETES
Review round: 1
Reviewer: 2

Output:
Basic reporting: The English language should be improved to ensure that an
International audience can clearly understand your text.

The authors do not discuss the clinical significance of their findings and the utility and nature of markers they found. The markers are correlative and not necessarily causative.

The conclusion section needs to be improved and the authors need to elaborate what are the main findings of their study.

Specific comments are added in the annotated PDF file.
Experimental design: no comment
Validity of the findings: Impact and novelty not discussed
Conclusions are not well stated and need to be rewritten
Additional comments: No additional comments


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

Input:
Title: GENE SIGNATURE FOR PROGNOSIS IN COMPARISON OF PANCREATIC CANCER PATIENTS WITH DIABETES AND NON-DIABETES
Review round: 1
Reviewer: 3

Output:
Basic reporting: The manuscript is unfortunately poorly written. The use of Scientific English and the grammar is poor. Throughout the manuscript the given information, findings and discussion of the data is not intelligible.

Writers do not link their hypothesis and their findings in the text and often make confusing jumps to other findings. The coherence is severely lacking.
Experimental design: Clinical findings on tumor state and histological grading is not novel, what authors here try to show is the difference in diabetes groups however there is no highlight to diabetes and in the manuscript it feels like they are repetitively mentioning well established findings.

The results and their discussion should be focused on the novel findings.
Validity of the findings: There are mislabeled figures and table numbers in the main text body, which is misleading and confusing (e.g in lines 203 and in 204 the label says Table 2 however it must be Table 1)

Some findings that are essential to the hypothesis are completely untouched, not mentioned.

Authors should give more conscience explanation of each result.
Additional comments: I advise for thorough editing of the use of English. From the introduction to the discussion it is very hard to understand and follow the logic of the findings.

also more detailed explanation of the rationale of the study and the findings must be given.


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

Input:
Title: GENE SIGNATURE FOR PROGNOSIS IN COMPARISON OF PANCREATIC CANCER PATIENTS WITH DIABETES AND NON-DIABETES
Review round: 1
Reviewer: 4

Output:
Basic reporting: pass
Experimental design: pass
Validity of the findings: pass
Additional comments: The authors reported that the integrated gene prognostic biomarker systems are identified in PC with non-diabetes or diabetes

1.	Are there relation or differences between two prognostic biomarker systems？
2.	The diabetes were newly discovered in PC patients？was there any difference between the newly and old diabetes？
3.	TNM staging is most important for prognosis, why the author did not include TNM staging?


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

Input:
Title: GENE SIGNATURE FOR PROGNOSIS IN COMPARISON OF PANCREATIC CANCER PATIENTS WITH DIABETES AND NON-DIABETES
Review round: 2
Reviewer: 1

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


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

Input:
Title: GENE SIGNATURE FOR PROGNOSIS IN COMPARISON OF PANCREATIC CANCER PATIENTS WITH DIABETES AND NON-DIABETES
Review round: 2
Reviewer: 2

Output:
Basic reporting: This manuscript presents an interesting analysis of publicly available PDAC datasets and presents a gene signature that can be utilized for a prognostic assessment. However, the use of academic English is needed to be enhanced for better communication. An overall grammar check might also give the manuscript better readability.
Experimental design: 1.Throughout the manuscript, authors refer to different gene signatures as "gene biomarker", where there are two different sets used. A more specific naming for these gene signatures, for example, Diabetes-PC or non-Diabetes-PC, must be given to maintain the readers' attention and understanding.

2. A heatmap for differential expression between Diabetes and Non-diabetes must be given for proposed gene biomarkers, in addition to LASSO cox regression. The expression levels of proposed genes are equally important for the overall analysis. This must be further added to the discussion.
Validity of the findings: no further comment
Additional comments: No additional comments


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

Input:
Title: AN IN VITRO OVARIAN EXPLANT CULTURE SYSTEM TO EXAMINE SEX CHANGE IN A HERMAPHRODITIC FISH
Review round: 1
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: In this report, authors try to establish an in vitro culture system for ovarian tissue of protogynous wrasse. This culture system (for 21 days) is used to evaluate the effects of different steroids on female-to-male sex change. Unfortunately, only histological data used in this study. It is very difficult to observe the differences on cellular and molecular levels.

1.	For cell proliferating activity, IHC staining with a proliferating marker PCNA or proliferating cells labeling with the BrdU could help authors to evaluate the effect of 11KT and cortisol.
2.	For male differentiation, several sex-dimorphic expression genes (Dmrt1, Amh, Gsdf) could help authors to evaluate the effect of 11KT and cortisol.
3.	How authors to define the difference between the oogonia and spermatogonia in bisexual gonad? Authors have to add the figure to descript the characteristics of oogonia and spermatogonia in the early stage of sex change.
4.	In Figure 2, the letters used to indicate significant difference is very difficult to understand.


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

Input:
Title: AN IN VITRO OVARIAN EXPLANT CULTURE SYSTEM TO EXAMINE SEX CHANGE IN A HERMAPHRODITIC FISH
Review round: 1
Reviewer: 2

Output:
Basic reporting: 1) The reviewer feels that the cited references are slightly less.  Adding more references might be informative for the Journal readers. For example at line 58 and 60.

2) line 74:  The reference 11 is focused on in vitro culture for kidney development.  And the reference 10 does not mention in vitro culture in detail.   The reviewer feels that more suitable reference should be cited if possible.

3) line 83 and line 218:  The reference 12 seems to be doctoral thesis.  The reviewer is not sure that this reference is appropriate.

4) line 104:   Your introduction regarding the roles of cortisol need more detail.  The roles on sex differentiation in gonochoristic species and the roles on sex changing species should be introduced, briefly. This might be informative for the Journal readers.

5) line  212 : Regarding the effect of 11KT, information on the related wrasse (J Exp Zool A Ecol Genet Physiol. 2012 : 317:552-60. ) might be helpful for discussion.
Experimental design: 1)   line 137:  Your organ culture technique in materials and methods need more detailed explanation.　Information on the amount of medium in each well and the frequency of medium exchange would be important information. Those should be mentioned.

2) For image analysis using Ilastik , information on sizes of previtellogenic oocytes would be important.
Usually, ovarian tissue has various sizes of previtellogenic oocytes.  Therefore, the reviewer is wondering whether previtellogenic oocytes at smaller size can be distinguished by Ilastik software?
Validity of the findings: no comment
Additional comments: Some histological photos at high resolution might be helpful to see the size of cells. Those photos are also necessary to understand whether non atretic cells are normal or not.  Photos of D0 tissue might be needed, too.
The reviewer would like to know if the sizes of remaining oocytes differ between each steroid treatment.


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

Input:
Title: AN IN VITRO OVARIAN EXPLANT CULTURE SYSTEM TO EXAMINE SEX CHANGE IN A HERMAPHRODITIC FISH
Review round: 2
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: In this report, authors try to establish a ovarian culture system of wrasse and then use for the future study. However, authors only used image software to validate the ovarian development. It is hard to understand the relation between the oocyte size and cellular situation. Thus, reviewer believes that cell proliferating assay (IHC staining with a proliferating marker PCNA or proliferating cells labeling with the BrdU) and apoptosis assay (TUNEL assay) would help to improve the quality of this study. In general, the commercial anti-PCNA antibody can widely use for many fish species.


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

Input:
Title: AN IN VITRO OVARIAN EXPLANT CULTURE SYSTEM TO EXAMINE SEX CHANGE IN A HERMAPHRODITIC FISH
Review round: 2
Reviewer: 2

Output:
Basic reporting: no comment
Experimental design: The reviewer understands the Ilastik software can be used to distinguish the cells in gonads.  This may be the first trial.  Therefore, the reviewer would like you to recommend to mention about the data settings for Ilastik more carefully.   Does the data vary when the different sizes of previtellogenic oocytes and/or the different stages of atretic cells are labeled to train Ilastik ?   It would be more helpful to understand the availability of Ilastik if you show these kinds of data.
Validity of the findings: no comment
Additional comments: The reviewer would like to express his respect for your efforts to revise the manuscript.
Overall, the manuscript seems to be much improved.
On the other hand, information on the cellular and histological stages of oocytes may be still unclear, although the resolution of pictures has been improved.
As the reviewer might say at the previous comments, the results of the Ilastik analysis for the small sizes of oocytes and the stages of atretic cells has not been clear.  Therefore,  it may be better to use some additional photos to show these histological and/or celllular information.
Moreover, in this manuscript, it might be important to understand the differences of the cellular stages between in vivo and in vitro.  The reader would like to know how different between the initial tissue and the cultured tissue.  In addition, the analyses of histological and/or cellular differences of gonadal change between the present in vitro data and the previous in vivo data during gonadal sex change might be informative.


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

Input:
Title: POPULATION GENETICS OF THE FRESHWATER FISH PROCHILODUS MAGDALENAE (CHARACIFORMES: PROCHILODONTIDAE), USING SPECIES-SPECIFIC MICROSATELLITE LOCI
Review round: 1
Reviewer: 1

Output:
Basic reporting: The study presents population genetics analyses on the migratory <Prochilodus magdalenae> fish species, endemic and highly conspicuous to the Magdalena-Cauca and Caribe Basins in Colombia, while presenting its first set of 11 specific microsatellite DNA markers. The study is notable for its pioneer quality and for a rather fair sample size, over 700 specimens. However, the manuscript emphasizes the use of Next-Generation Sequencing (NGS), whilst not disclosing any NGS data or even reporting on basic NGS results. These would not only be potentially helpful in a lot of predictable and unanticipated ways by the research community, but would also be consonant with the open data ethos. The text needs copy editing and verification. Specific spotted examples are highlighted directly in the annotated manuscript, along with many detailed punctual comments (signed in the PDF as "Vera").
Experimental design: The methods chosen for the development of new species-specific microsatellite markers were spot on, as there is little justification for using more traditional methods aiming this goal, currently. The population genetic analyses are also sound, pertinent and grounded on the precedent literature. Worth praising is the author's use of more current/update estimators for the genetic structure of populations, such as F'st, Jost's D
 and the application of Bayesian approaches. However, Table 1 is presented in the Methods section, while reporting on new results from this original research. It should be moved to the Results section and its findings about expected, observed heterozygosity, exact p and Fis values should be reconsidered, since it has been explained it was composed of a random sample of 88 fish. Given these fish are not from the same sampling effort or location, there is little utility for these results (unless the implications of its nature is stated, accompanied of its respective meaning in discussion, since there will be a tendency towards a deficit of observed heterozygosity and departure of HWE, due to Wahlund effect, as observed in these results). Maybe it could be used to reflect and support the idea the the species does not consist in a single panmitic deme throughout the studied geographic range. Another issue worth raising awareness is the discussion of the inherent flaw in a specific feature of the Micro-Checker (MC) software, used here, presented in Waples, R.S., Journal of Heredity, 2015:106(1):1–19, doi:10.1093/jhered/esu062. The authors should reflect and consider if this could be of compromise to their intended goals ("detect potential genotyping errors") in the present study. I can't seem to locate the reporting of these results below. If it is the case, one could omit the use of this specific software, unless it supports/contradicts directly or indirectly the the study findings. If appropriate, the authors could state that the MC analyses simply did not show evidence of genotyping errors.
Validity of the findings: The paper presents a thorough description of population genetics analyses for this species, the first using species-specific microsatellite markers and will be of great value as a departing port for guiding further studies, proposing testable hypothesis about this species biology and ultimately helping in policy guidance, environmental mitigation and management decisions, in face of the coming wave of population genomic applications, fostered by NGS. This could be made explicit in the discussion (i.e. the results inherent limitations on number of markers sampled in the genome), in the context that the paper forwards 11 useful loci (~11/27 chromosomes (?), or an approximate average of less than one marker per each two different chromossomes in this species complement) and that the current study conclusions or further inquiries would greatly benefit from further future NGS based genotyping such as RAD/GBS and similar approaches, now at hand. A very positive point is that there was a strong selection criteria for loci to be applied in the genetic structure diversity analyses.

My main points to be discussed are:

1) No NGS results used for generating the data sifted to isolate microsatellites were reported (e.g. assembly results and statistics, depth of sequencing, number of contigs obtained/screened, number of microsatellites found, average PHRED quality, etc). No NGS data (e.g. FASTQ files, assemblies, etc.) was made public through adequate data repositories (e.g. NCBI's SRA). Ideally, each contig used for a validated microsatellite should be publicly revealed. Its average depth of coverage would be also a useful information. Still more preferable, the availability of alignments of short reads onto contigs for each locus should be made available or reported here. If not possible, the authors should disclose/discuss why.

2) What was the test significance (alpha value) after the Sequential Bonferroni procedure? Please, confirm if this was indeed a Holm-Bonferroni type multiple test correction or just a simple Bonferroni correction (less appropriate than the former) and clarify if it was used with all the HWE values from the whole sample, with or without the tests carried in the preliminary testing step, with the 88 fish (i.e. is there a single value for the study, or does the study show different values for Tables 1 and 2)?

3) Are the Fis values found actually accounted by breeding system endogamy? What would be the evidence supporting it, against the other concurrent hypothesis of  sub-structuring and Wahlund effect? Even having the authors put it as non-excludent alternatives, in the light of the study's conclusion, I would tend to interpret these numbers as an effect of mixture of fish from different true stocks, so I advise caution with this conclusion. Same goes, in a less critical way, for disentangling effects from selection, from those of admixture.
Additional comments: The manuscript reports on a notable work in the area of Neotropical fish population genetics and adds to the expanding literature on prochilodontid species, with clear implications for production, management and conservation of an important fisheries resource and describes 11 new valid microsatellite markers isolated from <P. magdalanae>. It could be improved by a detailed copy editing (beyond this reviewer's pointed instances) and by addressing the specific questions raised, including why the NGS results were not presented and made available along the article, since they are an integral part of the works repeatability and could be used in other diverse potential works.


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

Input:
Title: POPULATION GENETICS OF THE FRESHWATER FISH PROCHILODUS MAGDALENAE (CHARACIFORMES: PROCHILODONTIDAE), USING SPECIES-SPECIFIC MICROSATELLITE LOCI
Review round: 1
Reviewer: 2

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: This paper is effective, concise, and makes a compelling case for the study of the populations de Prochilodus magdalenae using species-specific microsatellite loci. I have no major issues with the theory or methods applied but would caution the authors to reconsider some details raised for the manuscript (see my comments below). I think it’s important to adhere to the most standardized, accessible terminology here, especially in the area of the conservation and population genetics (see my comments below). The structure of the paper is generally good, but the paragraph and sentence structure (particularly in the Introduction) needs to be better organized. While I recognize the importance of the data provided in the present study, I have detected a number of problems in the manuscript that make it unsuitable for publication the way it is. Therefore, in my opinion, there are some issues which need to be reviewed and/or answered in order for the manuscript to become suitable for publication. In addition, a review of English is required. I encourage the authors to review these issues and resubmit the manuscript for appreciation.

Line-by-line revisions:

Line 18: Change “The genetic structuring patterns...” to “The genetic structure patterns of populations....”

Line 23: Withdraw “... next-generation sequencing, bioinformatics, and...” to “... next-generation sequencing, and...”

Line 25: Withdraw “... and plausible signs of erosion...” to “... and signs of erosion...”

Line 29: Add “genetics” here: “... the genetics diversity and structure of P. Magdalenae...”

Line 39: Change “... fish species along the main river...” to “... fish species in the main river...”

Line 41-42: Change “... body sizes and high fecundities and abundances, representing around 50–80% of the biomass caught in artisanal and commercial fisheries throughout the distribution area...” to “... body sizes, high fecundities and abundances, representing around 50–80% of the biomass caught by the subsistence and commercial fisheries in some regions of your distribution area...”

Artisanal fisheries is seen as subsistence fishery. However, care should be taken when generalizing a fisheries resource as representative for all your distribuition area. The information has to be very solid.

Line 50: Add “ , ” here: “... permanent resource availability, as well as to guarantee...”

Line 54: Change “... of 2,182.67 metric tonin 2013...” to “...of 2,182.67 metric tons in 2013...”

Line 55: Add “the years of” here: “... between the years of 1978 and 2012...”

Line 56: Change “... population densities, catches (approx. 85%), and mean catch sizes...” to “... populations densities, mean catch size, and catch reduction around 85%...”

Line 61: Change “... counteract its detrimental situation...” to “...counteract this detrimental situation...”

Line 63-64: Change “... was catalogued as under critical threat in 2002 and as vulnerable since 2012 in the Colombian Red List of freshwater fishes (Mojica et al., 2012)...” to “... was catalogued as critically endangered in 2002 and, in 2012 was considered as vulnerable for the Colombian Red List of freshwater fishes (Mojica et al., 2012)...”

Line 65-66: Change “... their efforts on population reinforcements (improperly called restocking) of natural stocks in...” to “... their efforts in the restocking population of natural stocks threatened in...”

Line 69: Change "... regulation of fish farming (Povh et al...” to “... regulation of the fish farm (Povh et al...”

Line 75: Change “... for population reinforcements of natural stocks...” to “... for population restocking of natural stocks...”

Line 75: Change “Hence, natural stocks...” to “Thus, natural stocks...”

Line 81-82: Withdraw and Chance “... the observation that Prochilodus lineatus (Godoy, 1959) and Prochilodus argenteus (Godinho & Kynard, 2006) show fidelity to spawning sites (“homing”) suggests that P. magdalenae may exhibit...” to “... observations performed in the species Prochilodus lineatus (Godoy, 1959) and P. argenteus (Godinho & Kynard, 2006) show that these species present fidelity to the spawning sites (“homing”) suggesting that the species P. magdalenae can exhibit...”

Line 84-85: Withdraw and Change “... Indeed, previous genetic studies have found the population structure and/or coexistence of multiples stocks along the Magdalena River and several tributaries...” to “... Previous genetic studies have found population structure and/or coexistence of multiples stocks along the Magdalena River and of several your tributaries...”

Line 84-92: Withdraw and Change “Although this structure may result from the unregulated population reinforcements of the natural stocks, it may also reflect a natural behavior of P. magdalenae since similar patterns of genetic population structure have been found in other congeners such as Prochilodus reticulatus (López-Macías et al., 2009), P. argenteus (Hatanaka & Galetti Jr., 2003; Hatanaka, Henrique-Silva, & Galetti Jr., 2006; Barroca et al., 2012a), P. lineatus (Ramella et al., 2006; Rueda et al., 2013; Gomes et al., 2017), and Prochilodus costatus (Barroca et al., 2012a,b)” to “Although the cause this structure can be the result of the restocking of populations unregulated for the natural stocks, it can also reflect in the natural behavior of P. magdalenae since similar patterns of genetic structure have been found in other congeners such as P. reticulatus (López-Macías et al., 2009), P. argenteus (Hatanaka & Galetti Jr., 2003; Hatanaka, Henrique-Silva, & Galetti Jr., 2006; Barroca et al., 2012a), P. lineatus (Ramella et al., 2006; Rueda et al., 2013; Gomes et al., 2017), and P. costatus (Barroca et al., 2012a,b), respectively”

Line 95-97: Withdraw and Change “Likewise, we compare the genetic diversity and structure with those of five sites (Pijiño, Llanito, Mompox, Palomino, and San Marcos) previously studied by Orozco Berdugo & Narváez Barandica (2014)” to “Thus, we compare the genetic diversity and structure with the sites (Pijiño, Llanito, Mompox, Palomino, and San Marcos) previously studied by Orozco Berdugo & Narváez Barandica (2014)”

Line 98: Add “study of” here: “... their advantages in study of population genetics...”

Line 104: Change “... the river mainstream and floodplain...” to “... the river main stream and floodplain...”

Line 104-106: Add “samples, (river name?), of, farm and (fish famr name?)” here: “... a total of 725 muscle tissues samples of P. magdalenae from the river main stream (river name?) and floodplain lakes along of the different Colombian hydrographic areas of the Magdalena-Cauca and Caribe (Fig. 1; Supplementary Information) and, 40 juveniles from a local fish farm hatchery (fish farm name?).

Line 109-110: Change “... Territorial de Colombia #0155 on January 30, 2009 for Ituango hydropower...” to “... Territorial de Colombia #0155 (January 30, 2009) for Ituango hydropower...”

Line 112: Change and Add “respectively” here: “... permit #1293 of 2013 of the Universidad del Magdalena.” to “... permit #1293/2013 of the Universidad del Magdalena, respectively.”

Line 114-116: Withdraw “... P. magdalenae from the middle section of the Magdalena River was performed using the Illumina MiSeq v.2 instrument using the "whole genome shotgun" strategy and the Nextera library preparation kits for the sequence...” to “... P. magdalenae for the region of middle Magdalena River was performed using the Illumina MiSeq v.2 platform (manufacturer, city, country). An alternative approach to shotgun whole genome sequencing (WGS) was applied using the Nextera Library preparation kit (specify the Nextera kit) (manufacturer, city, country) for the sequence...”

Line 117: Change “... steps concerning the read cleaning, contig assemblage, identification...” to “... steps regarding the reads quality and filtering, contig assemblage, identification...”

Line 120-126: Redo all this passage (see my comments on this topic – Major (1)).

Line 137-139: Withdraw and Change “... using LIZ500 (Applied Biosystems) as the internal molecular size. Allelic fragments were denoted according to their molecular size and scored using GeneMapper v.4.0 (Applied Biosystems)...” to “... using the GeneScan LIZ-500 standad size (Applied Biosystems, city, country) to determine fragment length. The alleles were scored based on the consistent pattern of their stutter peaks, and on the peak intensity corresponding to each individual at each locus using GeneMapper v4.0 (Applied Biosystems, city, country)...”

Line 142-144: Withdraw and Change “... for departures from Hardy–Weinberg linkage equilibria as well as the observed (HO) and expected (HE) heterozygosity and the inbreeding coefficient (FIS) were estimated using Arlequin v.3.5.2.2 (Excoffier, Laval, & Schneider...” to “... for Hardy–Weinberg equilibrium (HWE), observed (HO) and expected heterozygosity (HE) and, the inbreeding coefficient (FIS) were estimated using Arlequin v3.5.2.2 software (Excoffier, Laval, & Schneider...”

Line 146-147: Withdraw and Change “... for each marker were calculated with GenAlEx v.6.503 (Peakall & Smouse, 2006) and Cervus v.3.0.7 (Marshall et al., 1998), respectively” to ... for each SSR were calculated with GenAlEx v6.503 (Peakall & Smouse, 2006) and Cervus v3.0.7 software (Marshall et al., 1998), as well as to estimate the genetic diversity of P. magdalenae”.

Line 148-150: Withdraw “The average number of alleles per locus, observed and expected average heterozygosities, and fixation index (Hartl & Clark, 1997) were calculated with GenAlex v.6.503 (Peakall & Smouse, 2006) to estimate the genetic diversity of P. magdalenae”.

Line 150-153: Withdraw and Change “... geographical samples was calculated using the standardized statistics F ́ST (Meirmans, 2006) and Jost ́s Dest (Meirmans & Hedrick, 2011) and analysis of molecular variance (AMOVA) (Meirmans, 2006) with 10,000 permutations and bootstraps included in GenAlex v.6.503...” to “... geographical samples were calculated using the standardized statistics F ́ST (Meirmans, 2006) and Jost ́s Dest (Meirmans & Hedrick, 2011) and, analysis of molecular variance (AMOVA) (Meirmans, 2006) with 10,000 permutations and bootstraps included in GenAlex v6.503 software...”.

Line 156-167: Add "software" and Withdraw “ . ” for all programs used. Example: Structure v.2.3.4 to Structure v2.3.4 software
CLUMPP v.1.1.2b to CLUMPP v1.1.2b software
Distruct v.1.1 to Distruct v1.1 software

Line 176: Withdraw and Change “... using the software BayeScan v.2.1 (Foll & Gaggiotti...” to “... using the BayeScan v2.1 software (Foll & Gaggiotti...”

Line 181-192: Add "software" and Withdraw “ . ” for all programs used. Example:
software jModelTest to jModelTest software
software MrBayes v.3.2.6 to MrBayes v3.2.6 software
Figtree v.1.4.3 to Figtree v1.4.3 software

Line 208-209: Change “... revealed that 8 of 11 loci exhibit allelic frequencies concordant with Hardy-Weinberg equilibrium expectations in at least one case (Table...” to ... revealed that 7 of 11 loci exhibit allelic frequencies concordant with Hardy-Weinberg equilibrium expectations in at least one case (see Table...”.

Line 225: Withdraw “... magnitude, heterozygosity deficits and inbreeding coefficients...” to “... magnitude and inbreeding coefficients...”

Line 228-230: Change “... of the tests performed using Bottleneck (Table 4) were significant for all populations under the infinite alleles model (IAM) and for most populations under the two-phase model (TPM), whereas they were generally non-significant under the stepwise mutation model (SMM)” to “... of the genetic bottleneck were significant for all populations under the infinite alleles model (IAM) and for most populations under the two-phase model (TPM), however, was non-significant for the stepwise mutation model (SMM) (Table 4)”.

Line 241: Change “... and AMOVA (F ́ST(7, 1407) = 0.009...” to “...and AMOVA (F ́ST(7, 1407) = 0.009...”

Line 242-244: Change “... statistics F ́ST (Meirmans, 2006) and Jost ́s Dest (Meirmans, & Hedrick, 2011) showed additional genetic differences among Atrato, the fish hatchery, Sinú, and the remaining rivers (Table 6) as well as...” to “... statistics F ́ST (Meirmans, 2006) and Jost ́s Dest (Meirmans, & Hedrick, 2011) showed additional genetic differences among Atrato, the fish farm hatchery, Sinú, and the remaining rivers (Table 6), as well as...”

Major:
(1) The authors discuss the parameters required to validate new microsatellite (SSR) primers, but it is not clear what these parameters. The criteria adopted by the authors to choose the 11 microsatellite loci, only two I consider important for validation of new microsatellite loci: (i) value of F or FIS (however, this depends directly on the pValue for the deviation of the Hardy-Weinberg equilibrium - HWE ) and (ii) the Polymorphic Information Content (PIC) - loci should be polymorphic. The authors speak of low levels of heterozygosity deficit, but when I see F or FIS values (see Table 1, HO < HE), I observe high levels of heterozygosity deficiency for all loci, as well as 16 loci with deviation HWE after Bonferroni correction (5%, p <= 0.05 / 21 = 0.00238). In addition, it is important to perform the linkage disequilibrium (LD) test for the validation of the 21 SSR loci. I suggest you take the LD test and add the information in the manuscript.

1.1. What are the criteria actually used for the choice of the 11 microsatellite loci for the present study? “I am not against the choice of 11 loci for this study, however, I would like this to be clear to me”.

1.2. What did mean by low levels of heterozygosity deficit?

1.3. Why did not check the private alleles in the populations studied? I suggest adding the information from the private alleles in the manuscript.

(2) Why was not an analysis for isolation by distance (IBD) in the distribution area of the species studied? This would be interesting and would greatly contribute to the manuscript data. I suggest that this analysis be carried out.

(3) I suggest the author better explain of the data information found in Figs. 2, 3 and 4 (structure), as well as the DAPC data. The current form as presented is very fragmented and can generate misinterpretation.

What I observed in this part of the manuscript related to the structure and DAPC data:
Figure 2A, two populations on the Magdalena River, and Fig. 2C two populations - Sinú and Atrato Rivers (Population 1) and the Cauca, Magdalena, Cesar, San Jorge and Nare Rivers (Population 2), respectively. The Fig. 3A shows that Magdalena (MG), Cesar (CS), San Jorge (SJ) and Nare (NA) rivers populations are more related than wtih the Cauca (CA) river populations. This is clearer with Fig. 3B and 3C, when we observed the Cauca (CA) and Magdalena (MG) rivers populations separately. Individuals from the fish farming hatchery, the author suggests that they originate from several rivers. It would be important for the authors to have this information about the origin of the individuals fish farm. Data on distance isolation (IBD) and a UPGMA tree would help to better understand Figs. 2, 3 and 4.

NOTE: I suggest the authors make a new run in the strucuture software up to a K = 8 (seven rivers + fish farm), adding each result of K to a single figure. Present the data for gene flow between the studied rivers. This information will be important to verify the level of reproductive isolation that these populations present.

Even with the existence of structure, the DAPC data show a genetic mix between the studied rivers, but this would be justified by the repopulation carried out in the region with individuals of the species Prochilodus magdalenae. The authors constructed a Bayesian tree (Fig. 5) using the cox1 gene. I suggest building a new tree containing the origin of each sample to understand the DAPC data.

(4) When checking Tables 2 and 3, it is observed that the majority of the populations of the target species are with values above 10% for FIS values. The FST values (Table 6) suggest that there is a good gene flow among the populations, however, the FIS values indicate the occurrence of mating with related individuals, what can be caused by the genetics bottleneck and/or restocking causing by the mixture of the population (the parents used in the restocking program probably originate from the same area of study). In addition, possible signs of local adaptation could have been verified (from high FST values, number of private and low alleles or absence of genetic flow).

The Wahlund effect on large rivers with the fragmentation of populations may be due to the construction of hydroelectric power plants (barrier to gene flow) e/or restocking program consequence performed in the rivers (different populations coexisting). To confirm the Wahlund effect due to the coexistence of genetic stocks in the study area, it will be important for the author to see the allelic frequencies that are different between populations and which has caused the heterozygosity deficit.

“Thus, it is lacking in the discussion a greater exploration of the consequences that this can bring if the management for species is not applied in the studied área”.

Minor:
(1) In the methodology nothing was found about of the DNA extraction step in the P. magdalenae samples. It is important that this information is contained in the material and methods. Please, add in the body of the text the method applied for DNA extraction.

(2) “The extension step and a final elongation were absent in this thermal profile”. Justify why the absence of this step in the PCR? Did you use any method on this? If yes, it should be cited in the text.

(3) Caution: all chemicals and equipment must bear the following information - manufacturer, city and country. Example: GeneScan Liz-500 (-250) standard size (Applied Biosystems, Waltham, USA) or 96-well Veriti™ Thermal Cycler (Applied Biosystems, Waltham, USA)

(4) I am does not understand what the author meant by this passage  - Line 197-198:  "A total of 21 of the 50 loci microsatellite evaluated were polymorphic and showed allelic frequencies that departed from Hardy-Weinberg equilibrium".

How do you know that the allelic frequency departed from Hardy-Weinberg equilibrium? I suggest that add a supplementary table with the data of the allelic frequency.

(5) Standardize in the manuscript: “Ho” to “HO”, “He” to “HE”, “F’ST” to “F’ST” and “F” to “FIS”, respectively.

(6) The fixation index (F) and inbreeding coefficient (FIS) they are not the same thing? Review table 3.

(7) The genetic structure tends to decrease when populations are mixed, increasing or restoring the gene flow among individuals of different populations. Thus, I did not understand what the author wanted to say in the line 275-276: "... the genetic structure of the samples shaped by the mixture of two genetic stocks..."


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

Input:
Title: POPULATION GENETICS OF THE FRESHWATER FISH PROCHILODUS MAGDALENAE (CHARACIFORMES: PROCHILODONTIDAE), USING SPECIES-SPECIFIC MICROSATELLITE LOCI
Review round: 2
Reviewer: 1

Output:
Basic reporting: The authors have full, direct and satisfactorily addressed all remarks, questioning and suggestions I have made in the first round of revision. They have added a profuse and detailed suite of supplemental data that will certainly enrich the paper’s open virtual environment presentation. I also take the opportunity to thank back the authors for their acknowledgement and I hope they will forgive me, though, and cope with new little remarks and details I have freshly raised in this second round or review, of elements already present in the original manuscript, along one main important point:
-Although there are no rules of name translation, throughout the text, tables and supplemental material, do prefer the use of the English term “the Caribbean” instead of “Caribe” (e.g. L18, L94 and Supplemental Table 1);
-  Line 92  Since the objective deals with an atemporal aspect of the study (what it does) prefer the used of the present tense instead the past tense: "this study tests the hypothesis";
Experimental design: No new comments;
Validity of the findings: No new comments;
Additional comments: My only point of concern is that the paper devalues itself while not making clear in the discussion and in the conclusion that these are indeed the FIRST species-specific microsatellite DNA markers published for this fish species (see instances on the annotated review manuscript), at least to the best of my knowledge. A search of GenBank revealed a dubious claim of 17 P. magdalenae species-specific microsatellites (implicit in the title of an unpublished manuscript – e.g. entries MH586829 through MH586842) for what actually seem to be other already published microsatellite loci for P. lineatus, P. argenteus and P. costatus. If the authors arrive to the conclusion this here is the first set of microsatellite markers for P. magdalenae, it certainly needs to be brought to light in the final work.


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

Input:
Title: POPULATION GENETICS OF THE FRESHWATER FISH PROCHILODUS MAGDALENAE (CHARACIFORMES: PROCHILODONTIDAE), USING SPECIES-SPECIFIC MICROSATELLITE LOCI
Review round: 2
Reviewer: 2

Output:
Basic reporting: The present study shows great improvements after the suggested revisions and has relevant information for the conservation of the species Prochilodus magdalenae. The information presented here is clear and the article presents an adequate structure. Figures and tables (supplementary material) bring information that strengthens the content presented in the paper. The references are adequate for the discussion of the results, however, I felt a lack of more current references from the last 3 years (2017-2020). The paper presents only 7.8% of references for the years 2017-2020.
Experimental design: The research presented here is adequate to the scope and objective of the journal. The paper's objective is well defined and relevant to the study area. The methodology applied is adequate and describes in detail the study.
Validity of the findings: The underlying data has been provided (supplementary material) and they are robust for a better understanding of the results. The conclusions are very clear from the study proposal and linked to the research question.
Additional comments: I congratulate the authors for the importance of the study and its contribution to the management and conservation of the species Prochilodus magdalenae. It would be interesting if the authors had applied a greater number of microsatellite loci, as well as some mitochondrial genes for further investigation. However, the work brings relevant information and contribution to the study area. I highlight here the sample size to the number of microsatellite markers used in the study. The review of the paper brought improvements to the study.


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

Input:
Title: POPULATION GENETICS OF THE FRESHWATER FISH PROCHILODUS MAGDALENAE (CHARACIFORMES: PROCHILODONTIDAE), USING SPECIES-SPECIFIC MICROSATELLITE LOCI
Review round: 3
Reviewer: 1

Output:
Basic reporting: The authors have, once more, addressed all suggestions and points raised. In particular, regarding one of my inquiries, they have argued for their position on not emphasizing the novelty aspect of the developed microsatellite markers set. They have opted for a more humble stance, conceding to the benefit of the doubt, relating to a previous 2018 GenBank deposit of microsatellite clones for P. magdalenae, by a third party. While, as the own efforts of the present study might testify, there is a huge gap between isolating/characterizing clones and empirically validating it for practical use, and while a peer-review publication should take precedence over isolated GenBank accessions as a primary source, given the nature of the study, which focus on its genetic diversity structure description goal, I am ready to acknowledge this position, knowing the molecular markers developed by the paper’s team will certainly be readily available to other research initiatives aiming this species, all the same. It is an important fisheries resource and, from a biological perspective, it clearly needs more study and an integrated taxonomic revision, mainly in regard to its status along with P. reticulans, as hinted in this report and on previous literature and also discussed during this peer-review process. I think the paper is suitable for publication in its current state and I congratulate its authors and research institutions for their efforts.
Experimental design: No new comments.
Validity of the findings: No new comments.
Additional comments: No new comments.


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

Input:
Title: POPULATION GENETICS OF THE FRESHWATER FISH PROCHILODUS MAGDALENAE (CHARACIFORMES: PROCHILODONTIDAE), USING SPECIES-SPECIFIC MICROSATELLITE LOCI
Review round: 3
Reviewer: 2

Output:
Basic reporting: All questions were resolved and improved in the body of the paper.
Experimental design: All questions were resolved and improved in the body of the paper.
Validity of the findings: All questions were resolved and improved in the body of the paper.
Additional comments: The authors made the improvements suggested in the final version, and justified all the points covered in the review. I consider the paper adequate and ready to be published in Peerj.


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

Input:
Title: A FORECAST MODEL FOR PREVENTION OF FOODBORNE OUTBREAKS OF NON-TYPHOIDAL SALMONELLOSIS
Review round: 1
Reviewer: 1

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


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

Input:
Title: A FORECAST MODEL FOR PREVENTION OF FOODBORNE OUTBREAKS OF NON-TYPHOIDAL SALMONELLOSIS
Review round: 1
Reviewer: 2

Output:
Basic reporting: The paper is difficult to read due to the dense technical language and equations used to describe the models.

The figures a small.  They are not well labeled.
Experimental design: I am not a statistician, and cannot comment on the extensive discussion of the development of the model.
Validity of the findings: The source of the data are described, but the data themselves are not. For example, is Salmonella enteritidis intended to refer to the serotype enteritidis, or to Salmonella enterica, generally?  If the first, why is pork demand being used to predict the occurrence of a serotype associated primarily with chickens? If the second, the data need to be properly described.

How is predicting the occurrence of an outbreak based on generic distribution of a commodity useful for prevention?
Additional comments: The authors have developed a statistical model to help predict the occurrence of non-typhoidal Salmonella outbreaks. Much of the paper lays out the theoretical basis and historical development for the modeling approach. This is rich in technical terms and equations that are frankly beyond my expertise. The paper needs to be reviewed by a statistician to vet the language, equations and approach.
My own expertise as an epidemiologist relates to the identification, use and interpretation of the data used to validate the model.  The authors present a case study for predicting outbreaks of Salmonella in Sydney, Australia based on estimates of mean maximum temperature, mean 3 pm relative humidity, and weekly demand for pork.  They apply their algorithms to the data and forecast the occurrence of Salmonella based on forecasts of the occurrence of the covariates.
There is very little information presented about the data used in these models. The first question relates to the Salmonella counts. The title of the paper discusses nontyphoidal Salmonella, which encompasses a wide range of different Salmonella serotypes and likely reservoir sources.  However, the paper discusses obtaining counts of Salmonella enteritidis. Salmonella enterica serotype Enteritidis is a specific serotype of Salmonella that has been primarily associated with chickens.  The organism colonizes chickens asymptomatically and can infect the hen’s ovaries.  This led to global problems with S. Enteritidis transmission as a result of egg production. More recently, S. Enteritidis has become a recognized problem with chicken meat production.  While the serotype is not biologically restricted to chickens, it is not clear why its occurrence would be predicted by consumption of pork.  If the authors are using the “Salmonella enteritidis” to refer to Salmonella enterica, generally, they need to revise their description of the data. With the three years of data on case counts obtained, how many outbreak-associated cases were included?  Distinguishing between sporadic cases and outbreak-associated cases could influence the development and use of the data. In the US, 5-10% of Salmonella cases are associated with an outbreak. Large outbreaks embedded in the data could affect the modeling, and any adjustments made to account for the possible presence of outbreaks in the data should be described.
The authors should also discuss the rationale for the use of temperature and humidity in the models.  Temperature is certainly known to affect the growth of Salmonella.  However, what data support the affect of humidity?  Certainly, temperature and humidity are seasonally associated environmental factors, but it is not clear what the independent contribution of humidity may be.
The final point I would raise is on the usefulness of predicting the occurrence of outbreaks.  It is true that Salmonella has a strong seasonality with increased occurrence of cases and outbreak potential during the summer months.  However, outbreaks occur within defined settings, or associated with specific food exposures.  A predicted increase in pork consumption during a hot, humid stretch, could lead to public health warnings. Is this what the authors envision?  Every year in the US such warnings are issued with respect to the advent of outdoor cooking of hamburger to prevent E.coli O157:H7 infections. This type of general advice does not require the use of elegant models.  Beyond this, what specificity of prediction is possible.  On a global basis, the incidence of Salmonella infections appears to be slowly increasing. However, one of the reasons for this is the broad host range of nontyphoidal Salmonella. Thus, focusing on a single commodity, would not address the actual population exposure risk.  If the models could be broadened to accommodate the full range of Salmonella attribution, it would be interesting.  Perhaps this initial model could serve as a test of concept for a broader approach.
In terms of the construction of the manuscript, the technical language and volume of equations makes reading the paper difficult. There are numerous figures present that are reproduced at such a small scale as to make them difficult to interpret.  Also, the labels on the figures do not adequately describe the data.  For example, in figure 5, it is not clear what the various scatterplots are showing.


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

Input:
Title: A FORECAST MODEL FOR PREVENTION OF FOODBORNE OUTBREAKS OF NON-TYPHOIDAL SALMONELLOSIS
Review round: 1
Reviewer: 3

Output:
Basic reporting: The manuscript is overall well presented, however the English language in most parts is grammatically incorrect, unclear and very difficult to interpret. For instance, l. 11-15 (convoluted and overlong sentence with grammatical errors), l. 18-19 (‘health public’ is a wrong term) – and generally throughout the manuscript. I suggest that the authors pay very close attention to writing clear, concise sentences – particularly as this is quite a technical manuscript which otherwise can become too hard to read.
The figures presented need more information:
•	What exactly is presented in Figure 1? Are these predicted Salmonella cases? What is the time line (it does not correlate with either 156 weeks or 3 years).
•	The axes on Figure 2 are hard to read.
•	Figure 4 needs more elaboration: again what is the variable under study? What are the three different lines in part (c) – they are hard to read.
•	Figure 5 lacks a clear explanation; without reading the main text it is almost impossible to determine what this figure depicts and even then it is hard to interpret. Consider presenting these data in a different way.
•	Figure 7 is possibly the most interesting figure in the manuscript but again hard to interpret. Does ‘upperbound’ indicate the upper threshold? What does ‘infected’ mean – predicted or observed numbers? I can’t see any green crosses in this figure although the legend indicates them – and what do they mean, an observed outbreak? Please also indicate that the forecast is per month (and provide a scientific rationale for this choice).
Experimental design: Reading through the manuscript, it is hard to understand what parameters specifically relating to Salmonella the forecast model is based on. Or does the model estimate general parameters for infectious/foodborne diseases which are then used for the forecasting? Please describe exactly the background knowledge, data (and not just the mathematical assumptions) that went into developing the model. For an epidemiologist and any public health official working with disease surveillance, this is crucial knowledge.
In the case study, the authors use relative humidity, temperature and weekly demand for pork to predict the future number of Salmonella cases. Where is the scientific basis for using exactly these variables? Salmonellosis is often a highly seasonal disease and so any variable that fluctuates with season could technically be used in a forecasting model, but without much epidemiological sense. Also, the forecast does not account for either serovariant, subtype or geographical variation which is a serious omission as almost all Salmonellosis outbreaks are firstly determined by serovar or (genetic)subtype and often geographically limited. The authors state that their model covers Salmonella enteritidis infections, however, the raw dataset includes many non-enteritidis cases. Were these excluded from the forecast? And what is the purpose of focusing solely on enteritidis when Salmonella Typhimurium is the most common serovar in Australia?
Lastly, I am concerned about the forecasting in general which is not well described - how long ahead does the model validly forecast the number of cases? Is it on a weekly basis or monthly basis as indicated in Figure 7? What is the scientific rationale behind this selection? How does the forecast deal with weeks/months where zero cases are observed, and was the forecasting adjusted to deal with a zero inflation?
Validity of the findings: Please refer to my comments above.
Additional comments: The authors present a potentially useful tool for predicting outbreaks of a common foodborne disease. However, the manuscript lacks important information which is needed for a proper epidemiological evaluation of the usefulness of the predictions. An infectious disease epidemiologist would need to see more disease-specific knowledge used in such a study in order for the results to be of public health importance. In general, the manuscript is highly technical and suitable for a specialized audience, and I think that the public health relevance is not particularly evident as it is presented now. The English language concerns also poses a hindrance for fully understanding the methods employed.


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

Input:
Title: A FORECAST MODEL FOR PREVENTION OF FOODBORNE OUTBREAKS OF NON-TYPHOIDAL SALMONELLOSIS
Review round: 2
Reviewer: 1

Output:
Basic reporting: There are numerous small grammatical errors throughout the manuscript.  For example, lines 12,13 "for the an early alert...pathogen, whose are related...", line 18 "is a foodborne illness considered is a..".
Experimental design: As previously noted, the paper is dense with technical biostatistical language I am not competent to evaluate.  The authors do not really address the issue of why better prediction is needed to improve source control (lines 23-24).  There is another issue in that the ability of the model to predict outbreaks is dependent on the interplay of recognized hazards and associated conditions.  It does not fundamentally allow for assessment of the emergence of novel food safety hazards. One application of such a model could be to identify when the known effects fail to predict the occurrence of disease, which could be a flag for the emergence of a new food safety hazard.  Some consideration of this idea is warranted.
Validity of the findings: In the previous version of this paper, the authors included consumption of pork as a predictor. It was pointed out that pork is not a recognized vehicle for Salmonella Enteritidis.  In this version of the paper, the authors have included consumption of eggs as a predictor.  This is good.  However, in comparing the two versions of the paper, it appears that Figure 7 is identical.  The labels have been changed but the underlying figure and the three alarms generated appear to be the same.  How is it that changing a major input, such as consumption of eggs for pork, has no impact on the predictive value of the model?
Additional comments: No additional comments


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

Input:
Title: A FORECAST MODEL FOR PREVENTION OF FOODBORNE OUTBREAKS OF NON-TYPHOIDAL SALMONELLOSIS
Review round: 3
Reviewer: 1

Output:
Basic reporting: OK, technical writing is dense.
Experimental design: OK.
Validity of the findings: OK, but the model seems insensitive to the inclusion of specific exposure variables.
Additional comments: The authors have made suitable modifications to prior comments, and appropriately qualified the use of their predictive model.


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

Input:
Title: A NEW GENUS OF ORYZOMYINE RODENTS (CRICETIDAE, SIGMODONTINAE) WITH THREE NEW SPECIES FROM MONTANE CLOUD FORESTS, WESTERN ANDEAN CORDILLERA OF COLOMBIA AND ECUADOR
Review round: 1
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: The article reports the discovery of a new oryzomyine genus obtained from field surveys in the Chocó ecoregion of Ecuador and Colombia. Brito et al. present an exhaustive and detailed anatomical description of the new genus and comparison with two closely related and a sympatric genus together with high-quality illustrations. From my point of view, the generic status is largely justified by phylogenetic analysis (molecular and morphological characters) and quantitative anatomical traits and dental morphology. I included minor suggestions in a marked pdf.


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

Input:
Title: A NEW GENUS OF ORYZOMYINE RODENTS (CRICETIDAE, SIGMODONTINAE) WITH THREE NEW SPECIES FROM MONTANE CLOUD FORESTS, WESTERN ANDEAN CORDILLERA OF COLOMBIA AND ECUADOR
Review round: 1
Reviewer: 2

Output:
Basic reporting: In this study the authors employ an integrative systematic approach and described a new genus within Oryzomyini, which has at least three new species, two of them are also named and diagnosed. For these descriptions and analyses, the authors perform an integrated approach from phylogenetic analyses, morphological comparisons and detailed anatomical evidence partially based on micro-computed tomography, on specimens colected in Ecuador and Colombia.

I think that the phylogenetic and descriptive approaches are very consistent and adequate to test the hypotheses about the differentiation and presence of novel clades.

English is clear, but I suggest to improve it in some parts of the manuscript.

Bibliographic references are appropriate.

Of course I have really enjoyed reading this study on Orizomini taxonomy and Neotropical biogeography.
Experimental design: Experimental design are relevant and meaninful.

In summary, the experimental design and conclusions are very robust. I only suggest a few questions from the statistical point of view, in relation to phylogenetic inference analyzes (all details are provided to the authors).
Validity of the findings: Findings of this manuscript are very interesting. I think that the conclusions of this article can have a great impact on what is the taxonomy and biogeography of these clades of small rodents. The approach is quite original. The discussion is very well structured and argued, with an easy and interesting reading.
Additional comments: Comments to the authors:

Phylogenetic and descriptive approaches in this study are very consistent and adequate to test the hypotheses about the differentiation and the presence of a new clade in this region.
The authors have made a very good comparison with the two closest Orizomini clades (Nephelomys and Mindomys), and in turn with the clade that geographically has the greatest phylogenetic similarities (Tanyuromys). I think the authors have correctly integrated both molecular data and descriptions of body and cranio-dental morphology, in order to make a good description of a new genus within Orizomini. Both phylogenetic analyzes and descriptions of external and cranio-dental morphology support the presence of a new genera within Orizomini. Also, the exclusive molar morphology of Pattonimus among oryzomyines provides the best evidence of the ecological uniqueness of this taxon.
The study is extensive and the descriptions of the study material are correct and very thorough. I consider it to be a very interesting contribution to the biogeography and taxonomy of rodents in one of the most diverse areas in the Neotropical region. The study contributes to expanding the taxonomy and knowledge of this large rodent clade. The manuscript has a very good balance between Tables and Figures on the main text, and those materials reported as Supplemental Information.

Of course I have really enjoyed reading this study on Orizomini taxonomy and Neotropical biogeography. One of the recommendations for future studies would be to explore the ecological spaces occupied by these new species based on ecological niche models.

In order to improve this manuscript, I have some major and minor recommendations to the authors. For instance, I think grammar and spelling need to be improved in many sections of this manuscript.

Major comments:

1) The authors performed PCAs in order to explore some differences in the putative species described here. In this sense, PCAs included six external, 19 cranial, and six dental morphological variables. Beyond that the logarithmized data tends to homogenize the variances in the data, I suggest (at least) to perform independent PCAs using variables corresponding to each category of data (e.g. PCA with only cranial measurements, or a OCA with only external measurements). By adjusting variables with such different magnitude values, the relative weight of a particular cranial-dental variable could be clouded. I think that this approach should be attempted, thinking about the significance of each variable in each main component.

2) I think that in many cases there are species with very incomplete sequences, such as Nephelomys devius, Pattonimus ecominga, Nephelomys keaysi, Pattonimus musseri (sequence data from S4). I think the authors should evaluate how this missing data within sequences might affect the phylogenetic inferences.

3) From lines 301 to 318, about the phylogenetic inference using the supermatrix. The authors use parsimony and argue that cytb mitochondrial DNA could be the reason for saturation at higher taxonomic level evaluated here.

I think there are many ways to assess the saturation level of a particular molecular marker, such as the use of the DAMBE program, which I recommend to be used by the authors. In this sense, and according to my proper experience in other rodent groups, the citb would not have to show high saturation at this taxonomic level ("tribe" level). On the contrary, I think that high levels of homoplasy could come from the morphological variation (e.g. how morphological variation has been treated in this study). In fact, it is very difficult to assign saturation problems to molecular markers when working together with sources of morphological variation. For this reason, I suggest to the authors to explain the possible limitations when both type of data sets (morphological variation vs. molecular variation) are taking into account in order to infer phylogenetic relationships.
Beyond these questions, it is clear from the phylogenetic analyzes (using both data sets and using only molecular data) that the clade proposed as a novel genus is recovered as monophyletic, reinforcing the authors' idea regarding the proposal of at least two new species.

4) Considering the phylogenetic inferences, did the authors use any substitution model for each independent molecular partition? Authors should estimate the particular mutation rates for each DNA fragment (nuclear and mitochondrial), and incorporate this information in the phylogenetic analyses (BI analysis, for instance). The steps would be to calculate the best nucleotide substitution model for each particular DNA fragment (e.g. using Jmodeltest), then concatenate the genes and check the level of saturation (e.g. DAMBE software), after that include this information into the Bayesian inference (e.g. BEAST program).

Minor corrections:

In this section I summarize minor concerns, which in many cases are style or writing suggestions.
Abstract:

1) About “Extensive field work in two protected areas enclosing outstanding remnants of Chocó montane forest retrieved a variety of small rodents”; this sentence sounds a bit ambiguous, I suggest explaining how the rodent diversity was found in the two protected areas (e.g. recovered a high diversity of small mammals).

2) I suggest to connect better this sentence “Colombian and Ecuadorian Pacific cloud forests are under rapid anthropic transformation”.

Introduction:

3) I suggest to replace this sentence “The Oryzomyini, measured by number of genera and species, is the largest tribe of the 56 sigmodontine radiation, and according to current counts it comprises about 152 living (including 57 those historical extinct) species distributed in 33 genera (Weksler, 2015;
Pardiñas et al., 2017)” by “The Oryzomyini is the largest tribe resulting from the large radiation of the 56 sigmodontine rodent clades, and according to current counts it comprises about 152 living (including 57 those historical extinct) species distributed in 33 genera (Weksler, 2015; Pardiñas et al., 2017)”.
I also suggest putting this tribe in a broader geographic context, and adding something like "It is also the tribe with the widest geographic distribution, extending from the southeastern United States of America to Tierra del Fuego and the Cape Horn islands, plus some oceanic islands and the Antillean region”.

4) I suggest to replace “For this reason, and because of its high degree of threats,…” by “For this reason, and because of its high degree of threats to biodiversity,…”.

Material and Methods:

5) This study only use pitfall traps… I ask the authors (and only as a particular concern) why sherman traps have not been used, which in addition to live captures of small cursorial mammals allow to have (to a much greater extent) captures of scansorial species (or inclusive arboreal rodents).

6) In the text, the section “Anatomy, age criteria and measurements” does not lead to its corresponding figure (can be?).

7) Line 181, about “Females and males were combined in all analyses, following Voss (1991) and Abreu-Jr. et al. 182 (2012), who concluded that sexual dimorphism was not an important source of morphometric variation in oryzomyine rodents”. I suppose this will depend on the type of specimens and their particular body weights. The most correct procedure in this case is to do an allometric analysis to evaluate possible sex differences in adults. I suggest justifying why the latter was not carried out.

8) In some parts of the text, the authors use the passive voice, such as “For the fresh tissue samples (90% ethanol), DNeasy (Qiagen) or Puregene 204 (Gentra) extraction kits were used. For museum samples the protocol of Bilton & Jaarola (1996) was used”. In this cases, I suggest to use the first person.

9) Line 201. I suggest to replace “We amplified two genes: the mitochondrial gene cytochrome-b (Cytb) using…” by “We amplified two genes: the mitochondrial cytochrome b (Cytb) gene using…”.

10) Line 207. Modify “gene, amplified using…” by “gene using…”.

11) Replaced “The amplicons were sequenced by Macrogen (South Korea, Inc)”.

12) Line 216. This sentence should be referred to the corresponding Table or Appendix: The taxonomic sampling of the used morphological matrix corresponds to that of Pine et al. (2012) with the additions of the new material described here.

13) Between lines 209 to 222. The authors refer to the morphological matrix in Supplemental Information S3, but this file corresponds to a nexus file from DNA sequences. Please, check carefully.

14) From lines 227 to 229. The authors should include de original number of sequences that correspond to this study and to these molecular partitions.

15) Line 236. Replace “…while the molecular only datasets were…” by “…while the molecular datasets were…”.

16) Lines 284-288. In relation to Figure 1, the authors report that Bayesian trees and maximum likelihood trees were similar in terms of clade conformation. In order to avoid the polytomies observed in the Bayesian tree, I suggest evaluating the possibility of reporting the maximum likelihood tree topology. In which case, reports the reason why the authors have not proceeded in this way.

17) Line 329. I suggest to replace “The reduced molecular only analysis (Fig. 2B) also recovered the new taxa as a monophyletic group, but sister to Mindomys hammondi” by “The reduced phylogenetic tree using only molecular data (Fig. 2B) also recovered the new taxa as a monophyletic clade, but sister to Mindomys hammondi”.

18) Fig. 2. The topology of the phylogenetic trees (A and B) corresponds to de BI or ML inference?

19) In order to improve this manuscript, suggest to the authors check carefully the grammar and spelling. English need to be improved in many sections of this manuscript.

20) Fig. 5. It is very confusing which letter corresponds to each specimen. Maybe there is something here that I did not understand very well. It is not understood to which occlusal series each taxon belongs. In addition to this, italicize the scientific names of the species.

21) Lines 404-407. This information seems somewhat redundant, consider whether to leave it or delete it.

22) Line 408-418. About the pelage description of each entity, authors say that specimens from Drácula and Río Manduriacu reserves are externally very similar, although the fur of latter is less dorso-ventrally counter shaded as they exhibit greyer bellies. These chromatic differences are also displayed in the tails, which are more dark above and below in animals from Río Manduriacu.
Is there any quantification regarding the population variability of these traits at the phenotype level? How many individuals have been compared in relation to these results? The authors should indicate in the text the number of individuals that have been taken into account to make these descriptions.

23) Lines 414-416. I suggest to rewrite this sentence as follows: In contrast, the antorbital bridge from specimens of Río Manduriacu is dorsally narrow, the alar fissure has a marked basal notch, and the lateral expansions of the parietals are absent.

24) Lines 447-450. Consider dividing this sentence into two parts.

Discussion:

25) From lines 880 to 882. Here, the authors say that this genus could represents a novel transition to a dental morphospace within the tribe, combining lamination, increased crown height (i.e., relatively more hypsodont), occlusal simplification, and a mesiodistally compressed m3 (Fig. 13).
I think it is difficult from these photographs to observe the hypsodoncia patterns. I suggest, if were possible, add some kind of scale.

26) Lines 922-924. Where the authros say “although an important degree of transverse lamination (i.e., confluence of directly opposing folds; Hershkovitz 1962: 92) is observed in a few taxa, such as the sigmodontine Irenomys”. There is any citation for the lamination pattern observed in Irenomys?

Legend of Tables and Figures:

Line 1444. I suggest to express in the following form: Clades are indicated by letters (A to D).
Line 1448. I suggest modify by “…from 45 terminals.”. It is already clear in the text the number of total base pairs in the concatenated DNA fragment.


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

Input:
Title: A NEW GENUS OF ORYZOMYINE RODENTS (CRICETIDAE, SIGMODONTINAE) WITH THREE NEW SPECIES FROM MONTANE CLOUD FORESTS, WESTERN ANDEAN CORDILLERA OF COLOMBIA AND ECUADOR
Review round: 1
Reviewer: 3

Output:
Basic reporting: Field sampling and curatorial efforts leading to these new specimens are commendable, as are the comparisons with relevant museum specimens across several collections and the careful morphological descriptions.  Unfortunately, the molecular efforts are far more limited and essentially add new cyt b and IRBP data to the broader, existing datasets for these loci.  The resulting support for the key clades (new genus, distinct species) is very limited.  Statistical support is only moderate and ultimately based on these two loci.

The language and style of the manuscript are professional and only minor tweaking would be required from that standpoint.
Experimental design: The new molecular data are limited: 8 new IRBP sequences and 16 new cyt b sequences.  Recognizing that these are the two most commonly used loci for phylogenetic analysis in Sigmodontinae, the resulting database is limited to support a new genus and three new species. It would seem that several additional loci would be required to secure such support.

In terms of taxonomic representation, the molecular data used to represent the genus Nephelomys are also very limited and make it difficult to assess whether these two genera are indeed reciprocally monophyletic or the new genus is embedded within Nephelomys.
Validity of the findings: The presentation of levels of divergence between the relevant taxa is very limited, both in the text and in figures and tables.  Table 1 shows overall (presumably combined cytb-IRBP) divergence among genera, but the details are too limited to evaluate the meaning of these values.  How does divergence in cyt b and IRBP compare to each other across species and genera?  The pairwise comparisons among putative new species, provided in the text, are also sketchy and do not allow clear assessment of divergence and congruence across these two loci.  The possibility that the proposed new genus is but part of Nephelomys, and the possibility that a single species is represented in the data cannot be critically assessed with the information provided by the authors.
Additional comments: I commend the field, curatorial, and museum research component of this project.  However, I would encourage further efforts in securing additional representation of the relevant taxa (more specimens and taxa) and sequencing additional loci to further clarify the phylogenetic relations and distinctiveness of the taxa proposed here.


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

Input:
Title: GENETIC LOCI DETERMINING POTATO STARCH YIELD AND GRANULE MORPHOLOGY REVEALED BY GENOME-WIDE ASSOCIATION STUDY (GWAS)
Review round: 1
Reviewer: 1

Output:
Basic reporting: See General comments
Experimental design: See General comments
Validity of the findings: See General comments
Additional comments: The paper is on the genetic loci determination related to starch granules in potato. A total of 90 potato samples were used. GWD gene is related to the results. I believe that the paper is interesting and showed new insights in to starch biosynthesis in potatoes. i only have few comments and rather suggest the paper to be acceptance after making comments to mine.
There are ;already papers on the genes responsible for the granule morphology in different crops such as arabidopsis. I hope the authors may comment on how the findings from other crops can be related to the potatoes.
Figure 1, the captions should be translated as most of the readers don't learn Russian. also, which year is this drawn?
GWAS in the title, this may be spelled out in full.
How about the number of starch granules in the amyloplasts?
There are still some errors in your writings, Please double check your paper again to make sure not many mistakes are there.


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

Input:
Title: GENETIC LOCI DETERMINING POTATO STARCH YIELD AND GRANULE MORPHOLOGY REVEALED BY GENOME-WIDE ASSOCIATION STUDY (GWAS)
Review round: 1
Reviewer: 2

Output:
Basic reporting: 1. As I am from a non-English speaking country, it is difficult to judge the English of the manuscript.
2. I feel that in the section Results and Discussion, the literature referencing is insufficient. For instance, the authors discuss the connection of identified SNPs to certain proteins associated with studied traits. However, no references were provided neither for proteins itself nor for the hypothesis behind putative associations.
3. In the section Materials,  it is not clear why the authors did not provide a full list of accessions in the study? A simple reference to another published work is not sufficient. Please provide a separate file with the list of accessions, their origin, and other details.
4. It is not clear why the MLM method was not applied in the GWAS, although the method was stated in the section “Materials and Methods”? A small-sized population and the GLM (alone or with a combination of matrices) are often lead to the detection of false-positive signals due to inflated p-value.
5. I think that Table S4 should be moved to the text itself. The Table itself should have more information, such as QTL effect and trait heritability values. In figures with Manhattan plots, I think that all figs should be given with side-by-side QQ plots.
Experimental design: I have no question to this section, except the fact that the studied population is rather small-sized, which may lead to potential false statements in QTN identification for studied traits.
Validity of the findings: The results of the study are fairly novel. However, I recommend the authors use not only GLM but also the MLM method, which is a more reliable approach to avoid the detection of possible false-positive associations.
Conclusions should be reassessed after the application of MLM methods in the GWAS.
Additional comments: Table S4 is missing information for the QTL effects of the associations.
Also, please provide heritability values for analyzed traits in the study?


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

Input:
Title: GENETIC LOCI DETERMINING POTATO STARCH YIELD AND GRANULE MORPHOLOGY REVEALED BY GENOME-WIDE ASSOCIATION STUDY (GWAS)
Review round: 1
Reviewer: 3

Output:
Basic reporting: Ok
Experimental design: OK
Validity of the findings: OK
Additional comments: 1. Fig.1 should have english note beside the Russian;
2. Only one environment for the trait phenotyping, can this make the significant SNP reliable;
3. Move Some  figures and tables to main text from the supplimentary file;
4. Is there a candidate gene on Chr.2H for the siginficant SNP or a published QTL for this region;


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

Input:
Title: DIFFERENTIAL EFFECTS OF COTREATMENT OF THE ANTIBIOTIC RIFAMPIN WITH HOST-DIRECTED THERAPEUTICS IN REDUCING INTRACELLULAR STAPHYLOCOCCUS AUREUS INFECTION
Review round: 1
Reviewer: 1

Output:
Basic reporting: In the research article titled "Differential effects of cotreatment of the antibiotic rifampin with host-directed therapeutics in reducing intracellular staphylococcus aureus infection", the authors have tried to study the impact of cotreatment of ML141/simvastatin with rifampin against S. aureus infections. Rifampin is a front line drug for treating S. aureus infections but is associated with bacterial resistance. the authors have explored a possibility of combinatorial therapy of using an anti-virulent molecule along with antibiotic rifampin to check for treating S. aureus. This concept is fast gaining attention for various infectious diseases.
Experimental design: No comment
Validity of the findings: No comment
Additional comments: The manuscript is well written with supporting evidence, however the following points needs to be addressed.

1. One of my biggest concern is missing control for all the experiments. Neither positive nor negative controls were used in the experiments. using these controls can help us to understand how efficiently the experiment worked.

2. There is inconsistency in the number of cells used for various experiments which the authors have not reasoned. proper justification is required for why for Figure 1, HEK293-A cells were infected at a MOI of 2 for figure 1A and MOI of 100 for figure 1B.
Similarly, the authors have used 3 X 105 cells/ml for all the experiments where 5 X 104 cells were used for Cell viability experiment in Figure 2A. It will be better if the authors reasons why different amount of cells were used for these experiments.

3. The results could have been explained better. Details regarding  the experiments/assay performed and what observations were made were missing in the Results section.


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

Input:
Title: DIFFERENTIAL EFFECTS OF COTREATMENT OF THE ANTIBIOTIC RIFAMPIN WITH HOST-DIRECTED THERAPEUTICS IN REDUCING INTRACELLULAR STAPHYLOCOCCUS AUREUS INFECTION
Review round: 1
Reviewer: 2

Output:
Basic reporting: 1)	Provide the structure of rifampin, simvastatin, and ML141 as a supplementary file.
2)	Describe what is ML141 and its mechanism of action in detail.
3)	Flow cytometer graphs should be provided and discussed.
4)    Add literature regarding the use of these drugs against S. infections.
Experimental design: 1)	The experiments were performed only with one strain of S. aureus; please include a minimum 3 strains of S. aureus in the study to validate the findings. If possible, antibiotic rifampin-resistant strain should be included as a control.
2)	What are the minimum inhibitory values of the rifampin, simvastatin, and ML141 against S. aureus in vitro? Please provide in vitro synergistic study data by incubating the bacteria with R, S, ML alone, and in combination against the S. aureus strains (minimum 3 strains should be included.).
3)	For the co-treatment assay, what is the basis to select the following concentrations? ML141 (10 μM), simvastatin (1 μM), rifampin (0.01 mg/L), ML141 (10 μM) with rifampin (0.01 mg/L), or simvastatin (1 μM) with rifampin (0.01 mg/L) ?
4)	HEK Cells should be stained with fluorescent dyes and imaged and the live dead cells images should be provided in the support of the experimental claims.
5)	In Rifampin bactericidal activity assay, different concentrations of antibiotics and the ML141 should be used to confirm that the bactericidal activity is not enhanced.
Validity of the findings: 1)	Why simvastatin and rifampin yield differential results and rifampin-ML141 not? The authors should discuss this point in detail and provide multiple hypothesis against these results. These hypotheses should include literature citations.
Additional comments: No additional comments


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

Input:
Title: DIFFERENTIAL EFFECTS OF COTREATMENT OF THE ANTIBIOTIC RIFAMPIN WITH HOST-DIRECTED THERAPEUTICS IN REDUCING INTRACELLULAR STAPHYLOCOCCUS AUREUS INFECTION
Review round: 1
Reviewer: 3

Output:
Basic reporting: The manuscript is simple and well written.
Experimental design: Research question addressed in this manuscript is provides new knowledge on host directed therapies.
Validity of the findings: Overall the data is well represented.
Additional comments: The manuscript by Evans et al., discusses the effect of rifampin therapy in combination with host directed ML141 therapy in S. aureus infection treatment. Overall the topic is interesting and the results are useful for further research. My specific comments are:
1.	In the abstract, results should be more clearly written with exact changes observed with cotreatment as compared to mono-therapy or no treatment.
2.	In methods, the co-treatment method is not clear. The authors have discussed both pretreatment and cotreatment in this section, which is getting a bit confusing. Please modify.
3.	The authors have performed pretreatment assays with ML141. How is it relevant in real infection cases, would the patients will keep taking ML141 as precautionary measure?
4.	Results, line 163-165, “We next assessed whether the reduction in the number of intracellular bacteria was due to…” The authors need to explain the results in more detail instead of directly writing the final results, how they address this question and what experiments were performed.


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

Input:
Title: DIFFERENTIAL EFFECTS OF COTREATMENT OF THE ANTIBIOTIC RIFAMPIN WITH HOST-DIRECTED THERAPEUTICS IN REDUCING INTRACELLULAR STAPHYLOCOCCUS AUREUS INFECTION
Review round: 2
Reviewer: 1

Output:
Basic reporting: The authors have addressed the reviewers comments positively. The current version of the manuscript looks more convincing.
Experimental design: None
Validity of the findings: None
Additional comments: No additional comments


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

Input:
Title: DIFFERENTIAL EFFECTS OF COTREATMENT OF THE ANTIBIOTIC RIFAMPIN WITH HOST-DIRECTED THERAPEUTICS IN REDUCING INTRACELLULAR STAPHYLOCOCCUS AUREUS INFECTION
Review round: 2
Reviewer: 2

Output:
Basic reporting: The authors have responded to all the comments.
Experimental design: The authors have responded to all the comments.
Validity of the findings: The authors have responded to all the comments.
Additional comments: The authors have responded well to the previous critiques.


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

Input:
Title: DIFFERENTIAL EFFECTS OF COTREATMENT OF THE ANTIBIOTIC RIFAMPIN WITH HOST-DIRECTED THERAPEUTICS IN REDUCING INTRACELLULAR STAPHYLOCOCCUS AUREUS INFECTION
Review round: 2
Reviewer: 3

Output:
Basic reporting: The manuscript is overall simple and provides enough background.
Experimental design: Overall experimental design is sufficient.
Validity of the findings: Conclusions are well stated and linked to manuscriot.
Additional comments: Overall I am satisfied with author response and I do not have any major concern on the manuscript. I have one minor comment.
1) In Figure 3c and %c authors should try to label X axis properly, It is unclear how authors did not use log axis beyond 104. This analysis is ok but a more clear representation will help this study further.


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

Input:
Title: THE LAND FLATWORM AMAGA EXPATRIA (GEOPLANIDAE) IN GUADELOUPE AND MARTINIQUE: NEW REPORTS AND MOLECULAR CHARACTERIZATION INCLUDING COMPLETE MITOGENOME
Review round: 1
Reviewer: 1

Output:
Basic reporting: A useful contribution to a growing field cataloguing, describing and developing molecular tools towards understanding land planarians and especially those becoming important invasive pests (for which many have the propensity of becoming). The serendipitous reach of a 'national' citizen science program provided access to various specimens of Amaga expatria for which a mitogenome has been characterized using short molecular barcodes to help verify species identifications.

The writing is clear, professional and high quality. The authors have provided a comprehensive background to the underlying issues and relevant history underpinning the publication. All figures are clear and informative and the article is well executed, with all sections complementing one another in terms of content, interpretation and clarity. I have no concerns with the analysis or interpretation of the data.
Experimental design: The article is novel as the species in question has not been characterised using molecular tools since discovery and description. The delivery of a full mitogenome contributes useful data fro phylogenetics, molecular systematics and potentially diagnostic markers should they be needed. Morphological examination, next generation sequencing, mitogenome mapping for gene boundaries and analysis of NGS data to identify/verify possible food sources are all useful and complementary.
Validity of the findings: All underlying data have been provided; they are robust. Conclusions are well stated, linked to original research investigation and useful for future studies. The TTG/TTA possible start codons are interesting - I do hope GenBank don't have a problem with this (they can be annoying when data don't fit their expectations; good luck)
Additional comments: A few suggested changes in the text:

l.27 we DNA barcoded [to differentiate from 'traditional' barcoding]
l.28 more usually 'next generation sequencing'
l.37 by standard molecular barcoding techniques [as above]
l.173 For 4 specimens, the amplified COI sequences [since the full gene is much bigger than 903 and to differentiate these from the full cox1 gene in the mitogenome]
l.233 BLAST (acronym needs to be capitalised)

Figure 1 and Figure 2 ... indicate annual rainfall [not 'raindrop falls']
Figure 1 and Figure 2 ... where rainfall is high [not 'where raindrops are high']
Figure 7 Amaga expatria, map of the mitochondrial genome [remove 'genomic']
Figure 8 ... I may have missed this; is the phylogeny rooted or unrooted?