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

Input:
Title: EFFECT OF SEX ON GLUCOSE HANDLING BY ADIPOCYTES ISOLATED FROM RAT SUBCUTANEOUS, MESENTERIC AND PERIGONADAL ADIPOSE TISSUE
Review round: 1
Reviewer: 1

Output:
Basic reporting: General,
The paper reports an experiment done to measure metabolic activity in adipose depots of rats.  English is clearly not the first language of the authors,  but it is basically acceptable.  There are a few rough constructs that I have pointed out.   Unfortunately   the  bigger issues are that the authors clearly do not know the literature,  the truly massive and thorough literature on adipose tissue metabolism.  A review published in 1980 (yes 1980,  38 years ago) in Progess in Lipid Research listed over 400 papers in adipose tissue metabolism,  JUST IN RUMINANTS.  There were hundreds more in rats.  Finally,  the completely incomprehensible statement of objectives precludes any ability to understand what they were doing and it was made even less understandable when the methods were given,  which were absolutely inadequate to review as to what was actually done.
Abstract
Line 15: I don’t know what ‘canonical results’  means here,  please define or reword
Line 57: “During decades’  is likely not the best translation in English.   I might suggest “For most of the earlier research, the….”

Line 62;  similar here,  a segue is needed:  “In the last twenty hears,  many other functions of WAT have been discovered  including  synthesis of hormones and growth factors, insulin resistance in some diabetic states, interactions with other cell types that affect overall health.  Included in these roles are the close control of….”
L 105,  I am not sure of the precise anatomical definition of ‘inguinal cordons’…is there a different word?  Epidydimal fat pads?  Inguinal fat pads?
L 197 to 110  (the objective or hypothesis)  I am very sorry to say but this is totally undefinable…it is impossible to tell what in fact the objective(s) or hypothesis(es) was or were.
‘which was the possible balance between the defining characteristics and relative metabolic uniformity”.
Balance between characteristics?
Balance between a characteristic and a metabolic activity?
What is a ‘relative metabolic uniformity’  and how can it be compared to anything else?
What is a ‘defining characteristic’  (anatomical location,  fat cell size  time of day….)

L 111.    ‘influence of sex was included in the equation”  what equation?  Sex or gender?

This alone makes the work unable to be reviewed. It is impossible to know what you were testing.  I will look over the rest quickly.

L 119  ‘acclimation’  to what?     And did you measure feed intake or body weight?
L 153 to 157.   Glucose, lactate, glycerol and NEFA were measured on what?  With what sampling technique?  On the cells?  In the medium?  Both?
L 175 to 178,  as expected from the nebulous statement of objective, there is no clear statement of experimental design or statistical analysis.  What ‘groups’  were compared and to what?  Once the authors determine what their objectives and hypotheses were, and what treatments were applied and what was measured to test those objectives and hypothesis,  they will need to clearly and explicitly state the statistical model used, including the proper error terms.
L 239-241, this is a massive assumption, as likely as it may be..without actually measuring incorporation.   But release G3p from lipolysis could be reesterified.
L 244 there is absolutely no basis for this statement,  without knowing the physiological and nutritional state of the animals when they were killed.  If they were in energy and carbon balance  it is obviousl that lipogenesis would be minimal, and in short term incubations there would not be sufficient time to induce all the enzymes and pathways to make significant amounts of fatty acids.  You cannot use one set of animals in an undefined state to make such an inference.
L 255  same comment as above,   the change in expression cannot absolutely predict the change in metabolic rate…it can only be supportive or consistent.
L 275 to 282,  all of this is not surprising at all given what has been published many times on the amounts and transcriptional and posttranslational regulation of these enzymes  which precludes a usual tight relationships (high CC or RSQ) between mRNA and activity.

L 305 to 309.  This study does nothing of the sort,  and unfortunately this paragraph simple demonstrates the authors ‘ignorance’ (and that is meant in the strict definition of the term..they clearly are not aware of the body of knowledge on WAT metabolism).   Without sounding like a snide old man,  it is likely that many of the authors were born after most of the work was done.   Much of this would be found in works conducted from the 1960’s (or earlier) through the early 1980s.   But to say ‘studies analyzing metabolic pathways are too few and incomplete to support it.  Sir Hans Krebs, Veech, Per Belfrage, C. Holm, R. Vernon,  D. Bauman, , RL Baldwin,  R. Prior,  Metz and Van den bergh, Abraham, Edwin Krebs,  Katz, McNamara, Loor, etc…….might disagree with you.

I stopped reading here.
There is no way that they can possibly know glucose uptake or lactate uptake or output or NEFA or glycerol uptake given what they stated that they measured,  as they did not state that they measured anything specifically in cells or media.  AND in the absence of using some kind of radio or mass label,  or measuring over time (0 minutes to XX minutes of incubation) how could they possibly know that ‘uptake’ and ‘release’ occurred compared to just recycling?

I think the authors collected much useful data and it would be nice to be published.  But prior to redoing this paper with a new submission to this or any other journal,  that they run a search at www.pubmed.com with the following search terms:
Title:  adipose tissue metabolism
Date:  1950/01/01 to 1989/01/01.
Mine just now yielded 150 papers,  several of which will be reviews and which cover all species including rats.

When they have a better command of the field and can make statements that demonstrate that knowledge,  and when they can clearly describe their treatments, conditions an d
Experimental design: the review is above.
Validity of the findings: the review is above
Additional comments: the review is above


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

Input:
Title: EFFECT OF SEX ON GLUCOSE HANDLING BY ADIPOCYTES ISOLATED FROM RAT SUBCUTANEOUS, MESENTERIC AND PERIGONADAL ADIPOSE TISSUE
Review round: 1
Reviewer: 2

Output:
Basic reporting: English must be improved. Also, the manuscript is lengthy and needs to be dramatically shortened.
Literature is fine.
Figures need to be re-arranged so that they are more understandable, shortened and obvious.
As present, the data might not support hypothesis.
Experimental design: The experimental design needs to be better described and more details are needed.
Validity of the findings: Any culture studies were conducted and thus, data need to be further confirmed.
Data need to be further verified.
Observed changes could also be due to cell damages and stresses experienced during adipocyte preparation, which needs to be better described.
Additional comments: Abstract: the background is not well described and sex difference was not even mentioned.
The language must be improved. In many cases, it is difficult to understand what were described.
Introduction:
It is lengthy and can be dramatically shortened.
Line 111: 3C needs to be defined.
Line 130: Method for mature adipocyte preparation is critical and more details need to be provided, in order for experiments to be replicable.
Line 131: Which type of collagenase was used? Collagenase with trypsin activity could cause significant damage to cell surface proteins, rendering them to behavior abnormally during culture.
Line 139: 30 ml/L , 3% serum, which is quite low. Why was it used?
Line 141: What are the relevant of glucose concentration? Why were 7 and 14 mM chosen?
Line 136: The microscopic views of prepared adipocytes need to be shown.
Line 148: Why chose 24 h or 48h? Wasn’t cells need certain time to recover from damage due to collagen treatment, centrifuge and other stresses during cell preparation? The difference between 24 h and 48 h cells could be due to cell adaptation to the in vitro culture system?
Line 445-449: These sentences are inaccurate. This study was done in cell culture, which does not contain mature adipose structure, and also the cell composition is different. Therefore, it is inaccurate to claim that adipocytes in vivo have similar changes observed in cultured cells.
Figure 2. It will be better to label significant difference in bars. Also, the variations for data of most figures were very small, which appeared unlikely for biological replicates. Do you use cell from individual animals and individual animal was considered as an experimental unit? These need to be added into the statistic section.
Figure 3 to 6. I found that these figures are lengthy, but provide little information. And frankly, difficult to understand. I would suggest to use tables or other ways to make them clear and concise. Also, I would like to see statistic difference to be labeled directly on data points.
Figure 7: Red circles need to be defined, such as representing cell membrane, mitochondria etc.
Metabolic pathways can be grouped into beta-oxidation, lipolysis, lipogenesis, and glycolysis, etc. And also needs to be properly labeled.
Overall, the manuscript is lengthy, and many sentences, words and explanations are unnecessary and then the trimmed off.
Language needs to be improved.


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

Input:
Title: EFFECT OF SEX ON GLUCOSE HANDLING BY ADIPOCYTES ISOLATED FROM RAT SUBCUTANEOUS, MESENTERIC AND PERIGONADAL ADIPOSE TISSUE
Review round: 2
Reviewer: 1

Output:
Basic reporting: First,  thanks for the thorough response from the authors,  it clarifies many things.   I am sorry if I seemed too harsh , but whether we like it or not,  the English language can be difficult and in in scientific writing there is even more difficulty of making things clear.  We really do sometimes need to repeat things or to spell things out specifically, even though we think that the reader can connect one sentence to previous ones.
I also apologize if I criticized too harshly the understanding of the literature by the authors but as the paper was written,  it was not at all clear that the authors had the knowledge of adipocyte metabolism, in any species (I just used ruminants as an example,  there was a lot done in rats and pigs in the 'early days').   I think the new version is much improved.

I don't have any further comments other than the description of statistics is still not specific enough...you need to include the entire model statement(s).

I have no further comments and do not need to see any further revisions, I'll leave the final decisions to the Editor.
Experimental design: see above
Validity of the findings: see above
Additional comments: see above


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

Input:
Title: EFFECT OF SEX ON GLUCOSE HANDLING BY ADIPOCYTES ISOLATED FROM RAT SUBCUTANEOUS, MESENTERIC AND PERIGONADAL ADIPOSE TISSUE
Review round: 2
Reviewer: 2

Output:
Basic reporting: In this revision, authors only addressed a portion of reviewers' comments. The manuscript remains long and lack of contents.
Figure 1 and 2 can be combined into one figure.
For other figures, the statistic tables below the figure panels are not informative and suggest to remove. Instead, it will be much better and clearer to label significant difference directly above bars. This comment has been proposed previously, but authors chose to ignore.

In addition, only in vitro data were conducted. Because adipocytes are severely stress (if they survive), the difference both 24 and 28 h data might just represent cell adaption to the new in vitro environment. As such, I think that presenting both 24 and 48 h data may not be meaningful.
Experimental design: Only in vitro data were present, which may not represent difference in vivo.
Validity of the findings: The validity of findings remain questionable.
Additional comments: Authors only address my comments partially.


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

Input:
Title: EFFECT OF SEX ON GLUCOSE HANDLING BY ADIPOCYTES ISOLATED FROM RAT SUBCUTANEOUS, MESENTERIC AND PERIGONADAL ADIPOSE TISSUE
Review round: 3
Reviewer: 1

Output:
Basic reporting: This is the second revision, and I do not want to be too critical. In my opinion, the presentation of figures remains confusing.

Also, the English must be improved.
Experimental design: Only in vitro, which is a concern.
Validity of the findings: In my opinion, validity of the findings may be questionable, due to the lengthy adipocyte separation which likely altered cell behavior, among other problems.

I agree with authors that the manuscript will be difficult to further improve and, thus, I will leave it to the editor to make a decision.
Additional comments: The reviewer appreciate the effort of authors to improve the manuscript. However, there is only limited improvement.
The English must be improved.


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

Input:
Title: GENOME-WIDE IDENTIFICATION AND CHARACTERIZATION OF NON-SPECIFIC LIPID TRANSFER PROTEINS IN CABBAGE
Review round: 1
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: The manuscript” Genome-wide identification and characterization of non-specific lipid transfer proteins in cabbage” by Ji et al detailed analyzed non-specific lipid transfer proteins (nsLtps) in cabbage based on the public genome and transcriptome data and experimental analysis. This manuscript generally fit the criteria of PeerJ journal, clearly descript the work of nsLtps identification and related study. The experimental design was good. Research question well defined, relevant and meaningful. All the data the authors got were relatively reliable. However, there are still some problems need to be concerned and corrected:
1.	In the part of characterization on 62 BonsLtps are summarized, authors didn’t explain why they chose BonsLtpI.7 and BonsLtpII.3 to perform the subcellular localization. The subcellular localization of All BonsLtps should be predicted by using bioinformatic tools (i.e. Cell-PLoc) and then select some interesting BonsLtps to do the subcellular localization.
2.	Phylogenetic analysis of the nsLtp family revealed sLtps in Arabidopsis and cabbage are closer to each other. As we know, both Arabidopsis and cabbage are belonged to dicot, while rice is belonged to monocot. Is this difference due to the difference between dicot and monocot? Authors should combine more organisms nsLtp in phylogenetic analysis to get more biological meaning.
3.	In the part of the expression analysis of nsLtp genes, authors combined public RNA-seq date to analysis the expression profiling of BonsLtps at the transcriptional level. Did they validate the RNA-seq data before analysis? That’s essential to make data more reliable.
Based on these concerns, I think this manuscript should be resubmitted with minor revisions.


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

Input:
Title: GENOME-WIDE IDENTIFICATION AND CHARACTERIZATION OF NON-SPECIFIC LIPID TRANSFER PROTEINS IN CABBAGE
Review round: 1
Reviewer: 2

Output:
Basic reporting: In the introduction there are some errors or misunderstandings regarding the evolution, expression, function and lipid-binding of LTPs that needs correction.

•	Firstly, for an update on plant LTPs I suggest that authors read and include a citation to Salminen et al 2016 (Planta. 244(5):971-997) which is a recent and very informative review on the plant LTPs.
•	On line 2, when the authors write that the LTPs exists widely in the plant kingdom. The sentence should be complemented with a citation to Edstam et al 2011 (Mol Plant. 4(6):947-64). In this paper it is shown that LTPs are present in bryophytes, ferns, conifers and flowering plants.
•	On line 33 the authors state that LTPs are involved in transfer of lipids between membranes. This is not correct since the LTPs are generally secreted and located on the outside of the plasma membrane.
•	Also on line 33, the authors state that LTPs are transporting glycolipids. To my knowledge there is no publication supporting this statement. I suggest that authors provide a citation or rewrite the sentence.
•	On lines 43-44, the authors mention that LTPs are involved in cell wall extension. This should be complemented with citation to Nieuwland et al (Plant Cell. 2005, 17(7):2009-19).
•	On lines 43-45, it should be added that LTPs are suggested to be involved in transport of cuticular lipids. (Since this is proposed in DeBono et al 2009, which is cited.)
•	On lines 49-50 the authors should mention that LTPs also are expressed in roots. (See for instance, Edstam et al. 2013 Plant Mol Biol. 83(6):625-49.)
•	Lines 50-52, the authors should add some more recent citations regarding the stess-induced expression of LTPs.
•	Line 56, the authors write that cabbage is cultivated due to its excellent quality. Here I suggest that the authors are more specific and describe which qualities they are referring to.

Comments on the reporting of the results
•	Lines 243-250: Here the authors describe the expression pattern of Arabidopsis LTPs presented in other reports. This information should be moved to introduction or discussion.

Comments on discussion
•	Lines 270-278 are irrelevant and should be omitted.
•	Lines 291-292; include a citation to Edstam and Edqvist 2014 (Physiol Plant. 152(1):32-42) in which it is shown that several LTP genes are involved in maturation of seed and pollen.
•	The discussion includes rather large segments where the results are repeated, for instance on lines 280-287, 314-319. I would suggest that these parts are omitted or minimized, and that that the discussion is used to put the obtained data in a larger context.
Experimental design: Line 82: Why were the sequences with GPI-anchor removed? Provide arguments:
Lines 111-113: The localization study should be performed also with 1-2 control proteins with known localization patterns, for instance in plasma membrane or cell wall. As it is the localization study seems rather meaningless. Also add information about the microscope used.
Lines 121: Why was the expression of LTPs during response to black rot disease investigated? Are there some specific arguments that support a role for LTPs during the response?
Line 148: I would recommend using the more recent LTP classification system described in Edstam et al 2011 (Mol Plant. 4(6):947-64) and Salminen et al 2016 (Planta. 244(5):971-997). This classification system is useful since it is possible to apply also on LTPs from bryophytes and lycopods, as well as on the LTPs from flowering plants. It is also less complicated as it divides the LTPs in fewer major types, and keeps the “traditional” types LTP1 and LTP2.
Validity of the findings: Lines 332-334: The authors should avoid listing experimental results in the conclusion. I would suggesting rewriting the conclusions with more focus on extensions of the work. But without using standard clichés (such as now on lines 337-338).
Additional comments: This manuscript is basically just an identification of the members of the LTP gene family. It is not providing us with any new clues to help us understand these enigmatic proteins. The information on the expression of LTPs in the male sterile cabbage is interesting and should be investigate further.

Unfortunately, the text in the introduction of the manuscript  suggests that the authors lack detailed and updated knowledge of the plant LTPs.


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

Input:
Title: GENOME-WIDE IDENTIFICATION AND CHARACTERIZATION OF NON-SPECIFIC LIPID TRANSFER PROTEINS IN CABBAGE
Review round: 2
Reviewer: 1

Output:
Basic reporting: 1. Work description is Clear and unambiguous, professional English used throughout;
2. The introduction is sufficient, Authors detailed introduced the background and the work fits into the broader field of knowledge. Literatures referenced appropriately;
3. The structure of this article is acceptable with well-designed figures and tables;
Experimental design: Well designed experiments. Research question well defined, relevant and meaningful. All the data the authors got were relatively reliable.
Validity of the findings: no comment
Additional comments: The second version of the manuscript “Genome-wide identification and characterization of non-specific lipid transfer proteins in cabbage”, authors have made some revisions to respond the reviewers’ comments and upgraded figures to make data more robust. Based on this revised version, the manuscript meets the PeerJ criteria and should be accepted as is.


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

Input:
Title: GENOME-WIDE IDENTIFICATION AND CHARACTERIZATION OF NON-SPECIFIC LIPID TRANSFER PROTEINS IN CABBAGE
Review round: 2
Reviewer: 2

Output:
Basic reporting: The manuscript is clear and easy to follow. However, the spelling should be checked once more.
Experimental design: No comments
Validity of the findings: No comments
Additional comments: The revised manuscript is very much improved compared to the original version. My previous remarks were carefully considered by the authors. I have no further 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: RELATIVE TELOMERE LENGTH AND OXIDATIVE DNA DAMAGE IN HYPERTROPHIC LIGAMENTUM FLAVUM OF LUMBAR SPINAL STENOSIS
Review round: 1
Reviewer: 1

Output:
Basic reporting: No comments
Experimental design: No comments
Validity of the findings: No comments
Additional comments: Telomeres are very important for chromosome maintenance and contain highly conserved repetitive DNA sequence 5’TTAGGG 3’. It forms the ends of linear eukaryotic chromosomes and protects it from damage and all. The authors have studied the relative telomere length and oxidative DNA damage in ligamentum flavum tissue collected from 48 adult patients suffering from lumbar spinal stenosis. The authors have compared the relative telomere length from genomic DNA of non-hypertropic and hypertropic LF tissues using RT-PCR and also analyzed 8-OHdG levels, the marker for oxidative DNA damage. The authors also observed the positive senescence signal in pathologic LF fibroblast cells. The current study clearly demonstrates that the patients suffering from lumbar spinal stenosis has shorter RTL and higher oxidative DNA damage in hypertropic ligamentum flavum. This is a very novel finding and manuscript is suitable for PeerJ with minor changes.

Minor comments:

•	Please reframe this statement: “Telomeres are nucleoprotein TTAGGG DNA sequences at the end of chromosomes.”

•	Please add these reference regarding telomeres:
1)	Lingner, J. and Cech, T.R. (1998) Telomerase and chromosome end maintenance. Curr. Opin. Genet. Dev. 8, 226–232.
2) McEachern, M.J., Krauskopf, A. and Blackburn, E.H. (200

•	Please add the abbreviation of Reactive Oxygen Species (ROS) in the background section.

•	Line # 70: Please elaborate the end replication problem of telomere.

•	Line # 126: The concentration of DNA samples was measured…

•	Line 129: Cawthon et al: reference is missing

•	Please cite: Roderick J. O’Sullivan and Jan Karlseder, Nat Rev Mol Cel Biol. (2010). 11(3): 171-181: “Reference: Hypertrophy of Ligamentum Flavum in Lumbar Spine Stenosis Is Associated with Increased miR-155 Level.”

•	The authors should also include the increased expression and activities of various matrix metalloproteases, connective tissue growth factors, inflammatory cytokines etc. with proper references.


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: RELATIVE TELOMERE LENGTH AND OXIDATIVE DNA DAMAGE IN HYPERTROPHIC LIGAMENTUM FLAVUM OF LUMBAR SPINAL STENOSIS
Review round: 1
Reviewer: 2

Output:
Basic reporting: Minor suggestions:
1.	Briefly explain what LSS is, in the introduction.
2.	Define T/S ratio briefly in the materials and methods section.
3.	Mention lysis buffer constituents in the materials and methods section.
4.	Include the citation for Cawthon et al. (for the technique followed to determine RTL) in the references section.
Experimental design: In this manuscript titled “Relative telomere length and oxidative DNA damage in hypertrophic ligamentum flavum of lumbar spinal stenosis” the authors examine the role of telomere shortening and oxidative stress as possible mechanisms underlying lumbar spinal stenosis pathogenesis. The authors present data to claim that hypertrophic ligamentum flavum (LF) obtained from patients with lumbar spinal stenosis (LSS) has lower relative telomere length (RTL) and higher oxidative stress compared to non-hypertrophic LF samples obtained from the same patients, using whole DNA extracts. Furthermore, the authors present data showing lower RTL and higher senescence associated β-galactosidase in fibroblasts isolated from hypertrophic LF tissues of specific patients.

This manuscript provides some evidence of accelerated senescence and oxidative stress induced DNA damage in hypertrophic LF tissues of patients with LSS compared to normal LF tissues of the same patients.

Some noteworthy aspects of the manuscript:

1.	The authors have provided detailed descriptions of the experimental methods and provided sufficient background information relating to the study.
2.	Use of non-hypertrophic LF tissues from the same patients as negative controls.
3.	Acknowledging the limitations of the study in an honest manner.
Validity of the findings: 1.	From Figure 1, the RTL of non-hypertrophic LF tissues is marginally higher than hypertrophic LF tissues and from Figure 2, the 8-OHdG levels is marginally lower in non-hypertrophic LF tissues than the hypertrophic counterparts. This phenomenon is constant in all age group of LSS patients used in the study (i.e. from age 45-87- data on supplemental S1-sheet 1).

Although use of non-hypertrophic LF tissue samples from the same patient is a good negative control to show that telomere shortening and oxidative damage is accelerated during hypertrophic conditions, it is possible that these non-hypertrophic tissues (especially if obtained from the lumbar area) are showing early signs of telomere shortening/oxidative damage that can result in low statistical significance. This can be seen from the fact that the RTL of non-hypertrophic and hypertrophic LF fibroblasts is comparable after passage 3 (Figure 3).

If the authors have access to normal LF tissues or LF fibroblasts from normal age-matched people with no LSS, or any other normal comparable tissues, that would serve as a good control to determine normal RTL and 8-OHdG levels.

2.	The authors have used fibroblasts from patients aged 61, 66, and 77 years to examine RTL changes during passaging. The RTL represented by T/S ratio in the first passage of hypertrophic cells from the above patients are 3.36, 4.32, and 4.82 respectively (Supplemental S1- sheet2). However, the RTL (T/S ratio) of the hypertrophic LF tissue from the same patients is 1.61, 1.43, and 1.19 respectively (Supplemental S1-sheet1). Can the authors explain why LF cells in culture have different RTL compared to the parent tissues from which they have been isolated?

3.	In the discussion, paragraph 2, the authors state “We showed that hypertrophic LF tissue from LSS patients presented shorter RTL than non-hypertrophic LF tissue, suggesting that the hypertrophic LF tissue may exhibit high turnover rate of cell division.”

The authors are encouraged to present data comparing the growth rate of hypertrophic and non-hypertrophic LF fibroblasts.

4.	In the Discussion, paragraph 3, the authors state “Cells from elderly patients might have lower levels of telomerase activity and senescent cells accumulate with age.” In the absence of data about telomerase levels, can the authors include β-galactosidase data for the patients in the 45-55 age group? Are they different from the older patients i.e. patients >60 years?
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: RELATIVE TELOMERE LENGTH AND OXIDATIVE DNA DAMAGE IN HYPERTROPHIC LIGAMENTUM FLAVUM OF LUMBAR SPINAL STENOSIS
Review round: 2
Reviewer: 1

Output:
Basic reporting: 1. I recommend the authors to refer to the Supplementary data on the growth rates in the Results, and Discussion sections (e.g. line 227, 232) and also include the experimental method in the Materials and Methods section.

2. In the discussion, line 228, the statement ".... suggesting that the hypertrophic LF tissue may exhibit high turnover rate of cell division" needs to be revised to change the word "tissue" to "cells" to reflect the supplementary growth data.
Experimental design: The authors have included additional data on the growth rates to add validity to the statement that hypertrophic LF cells exhibits higher rate of cell division compared to non-hypertrophic LF cells.
Validity of the findings: No comments
Additional comments: The authors have sufficiently addressed the reviewers' concerns. The manuscript is suitable for publishing. However, I recommend the authors to refer to the Supplementary data on the growth rates in the Results, and Discussion sections and include the experimental method in the Materials and Methods 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: COLONIZATION AND USAGE OF AN ARTIFICIAL URBAN WETLAND COMPLEX BY FRESHWATER TURTLES
Review round: 1
Reviewer: 1

Output:
Basic reporting: This is a very descriptive survey of turtles found in Tommy Thompson Park, a large created urban wetland complex in Toronto. It does not have any experimental design, no hypotheses to be tested, and no theories to explain the observations.  The data provided are useful for further studies/investigations or for others to use in meta-analyses. There is no figure other than the maps showing location of the wetlands and where turtles overwintered.  At least some of the information buried in tables should be made into figures. GIS maps to show the distribution of individual and population home ranges for the Painted and Snapping turtles would have been informative.  The way in which they reported mean and SE needs to be streamlined  (e.g. line 185)--perhaps journal has a required style. Figure 2 needs to be revised so that all of the labels do not obscure the information.  Perhaps try to make inserts with an expanded view of those ponds and have lines pointing to the location of the overwintering sites.
Experimental design: There is no experimental design.  The goal of this project is mainly to describe where and how many turtle species and individuals used the created wetland complexes. The CMR approach used in both years to determine population size of the two dominant species, as well as the tracking program they carried out were all appropriate and the data from these were very useful and credible.  Nevertheless, they were not used to their fullest extent, and no spatial analysis was performed to determine how the turtles used their habitats.  I also disagree with their analyses on a pond-by-pond basis rather than treating the population as a whole for the entire complex.
Validity of the findings: I have no concerns with the validity of what they have presented. Unfortunately, the authors did not ask more from their data.  In particular, the movements and home ranges of the turtles would have added greatly to this study. I had many questions while reading their manuscript.  Why did turtles only use some of the sites, and not others.  What are the predators and how were they surveyed?  Where is the reference wetland that was mentioned in the Discussion (line 346).  How do these turtles behave relative to Snapping turtle populations in non-urbanized settings--provincial parks and other protected areas?
Additional comments: There is a lot of great information in this study that should be communicated to the conservation audience. For instance, the fact that Blanding's turtles can exist there at all is very surprising and interesting.  The relatively large population size of the two dominant species in such a setting is similarly important to note. That said, there is missing information that could make this manuscript more valuable to scientists because as it reads currently, it is a technical report that would form the basis of a published paper.  Missing things include home-range analyses, possible vehicular traffic estimates, location of turtles relative to the type of habitat features within the ponds, embayments, etc., presenting movement and use information according to the behavioural seasons (pre-nesting, nesting and post-nesting/overwintering), etc.


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

Input:
Title: COLONIZATION AND USAGE OF AN ARTIFICIAL URBAN WETLAND COMPLEX BY FRESHWATER TURTLES
Review round: 1
Reviewer: 2

Output:
Basic reporting: In general, the article is quite interesting, with a well-defined objective. The authors contextualized the question satisfactorily, and the practical implications is very clear.

- Background/context

Line 47-51: The territorial proportion and conservation status of the wetlands are relevant information, but insufficient to highlight their importance. The authors could discuss more about the ecosystem services that the wetlands provide.

- Structure, figs, tables

Figure 1: I believe the line between the image and the "Embayment B" label has been misconfigured. For a standardization criterion, I suggest to include a line between the image and the "Embayment D" label.

Figure 2: Too much information in the figure, which makes it confusing for the reader. It does not seem to facilitate the understanding the information that was presented in the text.

Table 2: Standardized the abbreviations in table and title.

Table 4: I suggest that the authors report the density and biomass for all the wetlands. It is only three more!

Table 5: The table title is uninformative. Formatting (font type and font size) need to be standardized.

Table 6: What is “na”? – Make an observation or explain in the title.
Experimental design: - Some points of methods need a more detailed description.

Line 84-90: A more detailed characterization of water bodies is necessary. Do they differ only in size and connectivity with Lake Ontario?

Line 97-99: The authors could provide some more relevant information, such as mesh size and size of the traps.

Line 110-112: Why basking traps and seine net were trapped only in 2016? Could this have affected the success of the recaptures in 2017?

Line 113 -114: Need a more detailed explanation of CRM protocol, or at least a reference for consult.

Line 119-121: Why some wetlands were sampled only in one year? Does this have implications for population estimates?

Line 127: A character (multiplication) is missing in the formula.

Line 145-147: It was not clear why new radios were added to females, while others radios was removed from males.
Validity of the findings: No comment
Additional comments: The modification of aquatic and semi-aquatic environments because of urbanization is a global problem that affects different populations of a wide variety of organisms that depend in some way on these environments. In the specific case of turtles, which have certain intrinsic biological characteristics, such as low recruitment rates in their populations, low rates of body growth and late sexual maturity, these modifications can be critical. On the other hand, some turtle species have the ability to adapt to non-optimal conditions and therefore to establish themselves with some success in environments where natural conditions have changed. Thus, the study presents a very relevant issue from the point of view of conservation and restoration of both wetlands and native turtle populations in the Lake Ontario region.

Specific comments:

Line 168-169: Why differentiate between new individuals and recaptures in the year of 2016? This information is not used for the estimates of population size, and the study is limited to data from 2016/2017. By highlighting the recaptures in 2016 the authors can confusing the reader about how the population estimates were calculated.

Line 175-176: The definition of “species assemblage” is not related to the proportion between different species as presented in the manuscript. I suggest to the authors to see Fauth et al. (Am. Nat., 1996, V. 147 pp. 282-286) and Stroud et al. (Ecology and Evolution, 2015, V. 5(21), pp.
4757-4765) for a discussion about the terminology.

- It is unnecessary to present both proportions! I suggest keeping only the most direct one (second proportion).

Line 185-188: Data presentation need to be standardized. For one species size data is presented with standard deviation and for another it is not. In addition, size data are presented with minimum and maximum, while mass data is not.

Line 189-191: Replace "weight" with "mass". Standardize the unit presentation.

Line 220-221: Consider substitute “visiting other wetlands” for “visiting more than one wetland".

Line 232-233: Consider substitute “explored at least two other wetlands” for ““explored at least three wetlands”.

Line 288-291: The authors use the sentence “species assemblage ratio”. See the suggestions for the lines 175-176.

-The ratio between species is always skewed towards Painted turtles, therefore the sentence “The TTP Painted to Snapping turtle species assemblage ratio of 3.5:1 was skewed towards Snapping turtle compare to…” is incorrect. Although the intention was compare this result to the ratios found in other areas, the only difference between areas is the force of skew and not the direction of skew. I suggest the authors rewriting the phrase.

Line 292-294: In the text is not clear if the ratio presented is between Painted turtle and Snapping turtle. I suggest substitute “… found an overall 1.3:1 ratio” for “… found an overall 1.3:1 ratio between this 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: COLONIZATION AND USAGE OF AN ARTIFICIAL URBAN WETLAND COMPLEX BY FRESHWATER TURTLES
Review round: 2
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: - Original primary research within Scope of the journal.

 Yes

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

The paper brings an interesting study with relevant implications for conservation and ecological restoration. However, the new objective presented is less clear and concise than the previous one. The distinction and the hierarchical relationship between the main objective and the specific objectives should be clearer. For example, in my opinion, investigating the colonization of a complex of artificial wetlands by turtle species is the main objective, while determining the populations of origin of Blanding's turtles, and the distribution of species along the wetlands complex are specific objectives.

- Rigorous investigation performed to a high technical & ethical standard.

Yes, all the technical and ethical standards were performed.

- Methods described with sufficient detail & information to replicate.

Some details of the sampling design still need to be better explained in the manuscript. Although the authors have justified some choices in the response letter, without these justifications readers may be confused (For more details see specific comments section).
Validity of the findings: no comment
Additional comments: In general, the authors answered almost all my questions in a satisfactory manner. Although I lacked a more standardized sampling design, especially with regards to the sampling of different wetlands and different years, this lack did not affect the results and conclusions presented in the manuscript. Anyway, I suggest that the authors include in the manuscript some of the answers presented in the response letter that have not yet been added, as this will facilitate readers comprehension and help to resolve doubts that may arise during the reading. I point out the answers that I believe should be included in the manuscript in the specific comments section.

Specific Comments

- Answers that should be included in the manuscript:

Line 110-112: Why basking traps and seine net were trapped only in 2016? Could this have affected the success of the recaptures in 2017?
The seine net was only used one day in 2016 (one snapping turtle captured) and was unavailable in 2017 and the basking traps had little success in 2016 and were not used in 2017. Unlikely, given how little success they had in 2016.

Line 119-121: Why some wetlands were sampled only in one year? Does this have implications for population estimates?
Wetlands that had very few turtles captured in 2016 were not re-sampled the following year.

Line 145-147: It was not clear why new radios were added to females, while other radios was removed from males.
In 2016 we tried to get an even number of males and females.  After an initial data investigation, we noticed that the females were moving more than the males.  In 2017 we were more interested in the movement of females, so added transmitters only to females.  When we recaptured males in 2017, we removed the old VHF transmitters (most had stopped working anyway).  We added a line of text to better describe our motivation.

- Table 4: Replace "weight" with "mass".

- The presentation of the results and discussion about the genetic data of Blanding's turtle is confusing. In the passage "Microsatellite genotypes for the two Blanding's turtles were not strongly assigned to any previously genotyped populations (Davy et al., 2014) but were most strongly assigned to the previously profiled populations in the Kawartha Highlands and the east shore of Georgian Bay.", it is unclear whether the two individuals sampled in the TTP are associated with any population cited in Davy et al. 2014. Do the populations of Kawartha Highlands and the east shore of Georgian Bay refer to other paper?


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

Input:
Title: INDOLE AND 2,4-THIAZOLIDINEDIONE CONJUGATES AS POTENTIAL ANTICANCER MODULATORS
Review round: 1
Reviewer: 1

Output:
Basic reporting: 1.	Repeated mislabeling and misreferring of figures in both figure legend and result sections. For example, figure legend-4 refers to the figure-1 for the MTT assay scheme but figure-1 is the synthesis strategy of the TZD compounds. Line-222; Figure 4A and 4B are referred for the cell migration assay but the figure-4 data shows cell viability.
2.	Figure-4 and Figure-5 (MTT assay) are the subset of data in figure-3. There is no reason to project the same data repeatedly in different figures.
3.	Line 189-192; the table 1 shows IC50 of all the three compounds (18, 20, and 22) are 5μM at 48h. However, the statement made in this line is not supported by the data in table 1. Moreover, the figure referred for the statement (Figure2) has no relevance with the statement. Please explain.
Experimental design: 4.	How do you assay the IC50? Is it different from MTT assay? If not, I suggest to combined in a single paragraph under the heading “Inhibition of cell viability in MCF-7 and PC3 cells” without “IC50” or “MTT” as heading.
5.	Though the treatment of the cells with the TZD compounds increases BCL-xL in temporal manner (gradually increased from 24h to 72h) but the authors claims other way around. The increase BCL-xL is thought to act as anti-apoptotic. Please compare 24h, 48h and 72h of each compound, the BCL-xL is either increases or unaltered. Please explain.
6.	I suggest assaying for other proapoptotic marker in the treated samples, for example Caspase 3 or PARP cleavage.
7.	The endogenous loading control is missing in the BCL-xL western blots.
8.	I suggest the authors to assay the binding affinity of compounds 18, 20, and 22 for the PPARs in comparison to Rosiglitazone.
Validity of the findings: 9.	Line 188; the statement claims IC50 5μM at 72h whereas data provided in table-1 shows IC50 5μM at 48h. Please clarify.
10.	Introduction should include more details background on different genera of TZD and their specific drawbacks and the knowledge gap should be established clearly.
11.	Please explain why RPMI media was used for the wound healing assay (line 160) while cells were maintained in DMEM media.
Additional comments: 12.	Please provide literature support for concept why indolyl and indolinone sidechain may decrease side effect of TZD (line-72-74).
13.	Please provide the buffer composition used for total cell extract preparation and catalog number of the antibodies used.
14.	Move down line 97-101 to the end of the “Synthesis of TZD analogues” section.

The manuscript entitled “Indole and 2,4-thiazolidinedione conjugates as potential modulators of cellular apoptosis and proliferation” by Messineo et. al. has tried to address an important clinical problem by a synthetic chemistry approach. I commend the authors for their extensive effort for the synthesis of TZD derivatives. I found the conjugation of heterocyclic moieties to the TZD derivatives synthesis are compelling. Though, several efforts have been reported in the literatures (reviewed by M.J.Nanjan et.al. Bioinorganic Chemistry,2018) for the synthesis of TZD derivatives in the past to reduce side effect and increase specificity for specific PPARs. I think the authors fail to build a strong rationale in support to their specific synthesis approach why they specifically synthesize indolyl/indolinone TZDs, thought indolyl/indolinone conjugation thought to reduce pharmacological side effects. Overall manuscript is poorly written with redundant experimental data and several mislabeling. The authors repeatedly presented same data at multiple figures. At the present stage of the manuscript, I don’t recommend considering for the publication in PeerJ unless a major revision address all the concerns.


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

Input:
Title: INDOLE AND 2,4-THIAZOLIDINEDIONE CONJUGATES AS POTENTIAL ANTICANCER MODULATORS
Review round: 1
Reviewer: 2

Output:
Basic reporting: The manuscript #24396, entitled “Indole and 2,4-thiazolidinedione conjugates as potential modulators of cellular apoptosis and proliferation” by Messineo et al. describes the anti-proliferative and anti-migratory capacity of three novel TZD derivates, suggesting their putative utility as anti-tumor compounds.

This is an interesting study expanding the putative utility of TZD-derived compounds and providing relevant information regarding novel anti-tumor candidates. Only few comments are raised:

1)	The title should be adapted in that it does not accurately reflect the results presented herein. No direct assessment of apoptosis capacity is demonstrated and this should be eliminated from the tittle.
2)	Abstract, line 36: Should it say migration instead of proliferation?
3)	The discussion section can be expanded substantially, incorporating the discussion of additional previous results.
Experimental design: 1)	In the western-blot section of the material and methods, more extended information should be provided in terms of methods used for protein extraction, blotting procedures and data analyses.
2)	The statistical analysis description is missing in the material and methods section. It should be provided.
3)	It is odds that the authors used apoptosis markers to demonstrated anti-proliferative effects, in that these effects are not necessarily mediated by apoptosis induction. The authors should want to separate both sections by 1) adding some proliferation marker and 2) demonstrating induction of apoptosis. This could greatly increase the impact and significance of the study
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: INDOLE AND 2,4-THIAZOLIDINEDIONE CONJUGATES AS POTENTIAL ANTICANCER MODULATORS
Review round: 1
Reviewer: 3

Output:
Basic reporting: No comment
Experimental design: In the manuscript by Messineo et al, the authors detail the rationale for designing analogues of Thiozolidinediones (TZD). They have done a commendable job in designing 26 derivative and testing them for anti-proliferative effects in human breast and prostate cancer cell lines by MTT assay. However, several key controls were missing in the subsequent evaluation of apoptosis-western blots were missing loading controls and migration assays were missing zero hour controls.
Validity of the findings: The data lacks important controls (see below). Based on this the conclusions are over stated claiming them to have promising potential in treating breast and prostate cancer.
Additional comments: In the manuscript by Messineo et al, the authors detail the rationale for designing analogues of Thiozolidinediones (TZD). They have done a commendable job in designing 26 derivative and testing them for anti-proliferative effects in human breast and prostate cancer cell lines by MTT assay. Based on the IC50 curves, the authors prioritized AC18, AC20 and AC22  to carry out cell migration and evaluation of anti-apoptotic markers. Listed below are some of the major concerns regarding this manuscript:
1.	The MMT assays show that AC 19, 25 ,26 show significant anti proliferative effects with effects as early as 24h but these did not make the cutoff based on IC50 values and these discrepancies were not addressed.
2.	For the blots demonstrating changes in Bcl-xL, the western blots lacked loading controls and therefore the results described by the authors could not be validated. Further, for apoptosis induction, the direct analysis of cleaved PARP and cleaved caspase 3 are considered gold standard assays, which were not evaluated in this manuscript. Alternatively, the authors could potentially use propidium iodide based staining to confirm cell death by apoptosis.
3.	For the analysis on cell migration by the wound healing assay, the critical 0 hour control images were missing. This result is necessary for the evaluation of width of the scratch at time 0 and consistency of this width between the different treatment conditions. In the absence of this control, it is not a well-controlled experiment to draw conclusions about the different responses.


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: INDOLE AND 2,4-THIAZOLIDINEDIONE CONJUGATES AS POTENTIAL ANTICANCER MODULATORS
Review round: 2
Reviewer: 1

Output:
Basic reporting: The authors have improved overall quality of the manuscript significantly. I would recommend the paper to be accepted for publication in PeerJ.
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: INDOLE AND 2,4-THIAZOLIDINEDIONE CONJUGATES AS POTENTIAL ANTICANCER MODULATORS
Review round: 2
Reviewer: 2

Output:
Basic reporting: No comment
Experimental design: No comment
Validity of the findings: No comment
Additional comments: The authors have appropriatelly addressed all my 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: INDOLE AND 2,4-THIAZOLIDINEDIONE CONJUGATES AS POTENTIAL ANTICANCER MODULATORS
Review round: 2
Reviewer: 3

Output:
Basic reporting: The manuscript is clearly written and easy to follow. The figure labeling has been corrected.
Experimental design: The authors have addressed the concern of loading controls for western blots. However,
for the key comparison of Bcl-xl levels between control and test samples (AC18,20,22), the blots should not be separated and be analysed in the same exposure conditions in Fig 4A and B.

In the PARP blot, the trend in both C-PARP and b-Actin is the same, suggesting no increase in the cleaved form of PARP with Rosi or AC22 in Fig 4A.

The b-actin blot in Fig 4B is over exposed and therefore making the results hard to interpret.
Validity of the findings: The authors have addressed the other concerns made in
1.  rationale for picking AC 18, AC20 and AC22

3. addition of 0h controls in Figure 5.
Additional comments: The western blot data for cell death is not convincingly supporting the claims made in the paper.


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

Input:
Title: SEXUAL DIMORPHISM IN ADULT LITTLE STINTS (CALIDRIS MINUTA) REVEALED BY DNA SEXING AND DISCRIMINANT ANALYSIS
Review round: 1
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: This paper is clear advancement in sexing of an abundant and widely distributed wader species, the Little Stint, which is inadequately studied in many respects. The developed discriminant functions for sexing will be of great help for further studies of this species. The paper is clearly and well written. It does not evolve any criticism. But I have several small comments/suggestions.
Line 82: It is worth adding that it is not only long-distance species, but also abundant one.
Lines 83-84: The described non-breeding distribution reflects areas where most Little Stints are wintering, but in small numbers they can be found farther east (similarly with low densities in the east of their breeding range). See: Round P.D. et al. (Stilt, 2012, V61, Pp55-56) and Tomkovich et al. (Wader Study Group Bulletin, 2009, V116, No3, Pp199-201).
Lines 229-230: There is no need to repeat here that females have longer wings. This is said in the previous paragraph.
Lines 245-250: This speculation about reasons of the revealed sexual size dimorphism does not sound convincing to me, although I have nothing to suggest instead (this issue needs study). Little Stint males differ from most other sandpipers by reduced breeding territoriality and related displays. Females of this species are bigamous indeed, but they are bigger than males similarly with monogamous Calidris sandpipers. This means that probably not bigamy, but something else instead is a reason for females to be bigger.


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: SEXUAL DIMORPHISM IN ADULT LITTLE STINTS (CALIDRIS MINUTA) REVEALED BY DNA SEXING AND DISCRIMINANT ANALYSIS
Review round: 1
Reviewer: 2

Output:
Basic reporting: .
Experimental design: .
Validity of the findings: .
Additional comments: This paper describes a solid piece of work, which should be published after revision.  I have the following opinions and comments, which will need some thought, and further analyses if my comments are acceptable.
1.	Abstract line 35.  Change “plumage” to “primaries”.
2.	There is both English and American spelling.  See lines 52 “behavior” and 53 “moult”.
3.	Line 86. “gap year” is slang. Remove.
4.	Why were first-year birds ignored?  Seems a shame just to do adults. Either explain why first-years were ignored (small sample), or include basic morphometrics to show that they are the same as adults, or carry out discriminant functions for them too.
5.	Line 109.  “chord” is wrong.  This is the distance between the leading and trailing edge of a wing.
6.	Line 109. The tarsus + toe measure is first described by Anderson (c. 1975) in Wildfowl on moorhens.
7.	Line 142. Give months covering “before and after primary moult”.  Further, if wing length decreases during the inter-moult period, this will affect both the discriminant functions, and their application.  You need to show how and when wing length reduction occurs.  For some species (red knot), it is a steady reduction – this would mean that each month needs to be adjusted to a given standard month.  However, if there is no measurable wear between arrival and the start of mouth, and between the end of moult and northward migration, then there is no need for adjustment.  Therefore, wing length change (accounting for sex) needs to be shown for the before and after moult periods.  Note too, that application of your functions cannot apply to breeding birds.  You need to say this.
8.	Total head length is not as frequently measured as bill length.  Therefore, your functions are unlikely to be used by others.  Further, I would disagree that bill length has more error than total head length.  Can you show that you are correct?
9.	Likewise, tarsus length is a poorly defined parameter by most (between observer variation is large).  Tarsus + toe is less problematic in this respect.
10.	Following on from 8 and 9, you could provide functions that are more likely to be used by others by using the parameters that are most likely to be used by others, even although the power of discrimination is poorer (I would guess this would be minor).  Otherwise, your study becomes one of limited application.
11.	Line 192.  What does D actually mean?  What does it say for an individual.  Ideally, one want’s to know the probability of a bird being male or female.  One can then make a judgement on the value of that probability.  I would accept a 0.9, but not 0.6.
12.	Line 248.  I don’t think it is called “successive bigamy”.
13.	There is not consistency in the references.  See line 372.
14.	Line 254. The statement about which parameter is most dimorphic is incorrect – see table 1 - (4.48).
15.	Table 1 needs to show wing length before and after moult.
16.	Figures.  What are the boxes, vertical lines?  What do the +ve and –ve scores mean?  I would want to see the 95% ranges (not CLs) for both 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: SEXUAL DIMORPHISM IN ADULT LITTLE STINTS (CALIDRIS MINUTA) REVEALED BY DNA SEXING AND DISCRIMINANT ANALYSIS
Review round: 1
Reviewer: 3

Output:
Basic reporting: In general the article is well written, concise and easy to read. Is in accordance with the norms of scientific writing.

The literature references were appropriately chosen for the theme. They can be complemented as indicated throughout the manuscript.
In the background, the authors introduce the text with the sexual selection (line 15, pg 1) which leads to understand in the context that is the one responsible for the RSSD observed in Little Stint (Calidris minuta). Additionally, at least the three largest measures of sexual dimorphism should be indicated in the abstract.
The authors should make it clear that the results should be used carefully because of the misclassification of sex (21.2 to 15.3%), according to the used FDA.

The structure of the article is in conform the suggested format and raw data are available.
Table 1 shows a mistake. Storer’s index is not calculated according de formula of the Methods section (all measurements).
Example: Wing SI on table 1 = 3,80
Wing = (101,19 – 97,42)/ (101,19 + 97,42) *100           Wing = 1,89
DIstorer^' s=  (mean ♀-mean ♂)/(mean ♂+mean ♀)*100
Figures 1 and 2 are not required
Include a figure in Species and Study area section (pg 4, line 100) – The described area would be better represented on a map of the country with the study area.

The manuscript presents clear hypotheses and has been satisfactorily addressed in an objective and concise manner.
Experimental design: The study is original, examines an alternative of identifying the sex of monomorphic shorebird and fills a gap in Calidris minuta sexing that has not yet been studied.
I emphasize that the authors performed a great fieldwork with a large number of captured shorebirds, and a fine lab work with a great number of samples, which gives good credibility to the results.

The study was well conducted with a good sample size, careful collection of birds morphometric measurements, advanced techniques of DNA extraction and amplification of the gene of interest for the birds sexing.
International standards for capture, handling, sample collection and laboratory work were observed.

The methodology has been described in details and can be replicated easily elsewhere.
Pg 5, line 100 - It would be better represented on a map of the country with a study area;
Pg 5, line 115 – “and body mass to 1 g or 0.1 g in different years (weighed with an electronic scale)” – the body mass weighed to 1g has a low accuracy in measurement
Validity of the findings: The study is original and employs modern techniques for sex determination of birds, in the present case in Calidris minuta. It has application in every wintering area of the species in West Africa. In this sense, it has an important impact for other studies to be carried out with the species, as well as significantly reducing the effort and costs to carry out fieldwork that intend to determine the sex of the bird to identify specific patterns in these birds.

The large number of samples is a strong point of the manuscript, since it allowed the performance of statistical analyzes with high reliability and robustness. The sample collections and analyzes were obtained with care and standard procedures of accomplishment and control.

The conclusions are clearly presented and are in line with the proposed objectives. They also present the limitations of the presented results, which contributes to avoid future errors in the use and application of the same in other field studies. They could address and discuss a little more about the Storer’s Index that indicates RSSD in the species under study.

Unidentified speculation in the text
Additional comments: Please see further review comments throughout the manuscript in word document.
I congratulate the authors for the great effort in carrying out this work. It was eight years of catches of the species (probably many more species) with mist nets and walk-in traps, sample collection, DNA extraction, gene amplification and analyzes. All this carried out with high quality and use of procedures standardized and used internationally. Congratulations.


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: DENOISING THE DENOISERS: AN INDEPENDENT EVALUATION OF MICROBIOME SEQUENCE ERROR-CORRECTION APPROACHES
Review round: 1
Reviewer: 1

Output:
Basic reporting: This manuscript uses clear and professional English throughout. They also cite appropriate references from the literature and provide sufficient background. Tables and figures are well organized and informative. They hypotheses presented were supported with relevant results.
Experimental design: The research question is well defined. The authors' primary goal is to assess the computational time and accuracy of different ASV calling methods.  This question is highly relevant to the field.

Please clarify why an abundance filter of .1% was selected, and why the rarefaction levels presented were selected. Was there an obvious cut point?

QIIME1 is no longer supported, so I wonder why the authors chose to use this method instead of several other publicly available packages to calculate diversity and generate principle components.

I would be interested in seeing comparisons with Deblur without the positive filtering method. It seems likely that this positive filtering may bias findings towards sequences present in greengages.
Validity of the findings: The conclusion of the paper seems rather weak. From reading this paper, I did not come to the same conclusions as the authors; it seemed to me that based on this data, the three methods were not comparable. The authors make this statement based on weighted Unifrac differences, though this alone cannot justify this statement as different experimental questions will justify the use of different packages.

The authors often state that DADA2 calls more ASVs than other methods, but does not venture into whether these are spurious calls or likely to be true. This can be best evaluated with the mock communities where the "answer" is known. Table S2 appears to be the only mock community with discordant identification of ASVs between methods, and  DADA2 identifies more ASVs correctly, though it is unclear whether this is just a function of identifying more total ASVs.

The authors should clarify the implications of using a pooled verses sample-by-sample workflow. While the sample-by-sample approach may be more computationally efficient, it looses the ability to correct errors within batches. For multi-run experiments, this could lead to even greater batch effects.

Is it a fair comparison to use dada2 filtering scripts before inputting data into all three pipelines. Would the typically user of these pipelines do this filtering since other parameters were left at default for this comparison?
Additional comments: The authors evaluate three ASV calling methods on multiple mock communities and datasets. Overall, this is a solid manuscript that makes a significant contribution to the literature.


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: DENOISING THE DENOISERS: AN INDEPENDENT EVALUATION OF MICROBIOME SEQUENCE ERROR-CORRECTION APPROACHES
Review round: 1
Reviewer: 2

Output:
Basic reporting: No comment
Experimental design: No comment
Validity of the findings: No comment
Additional comments: Identification of ASVs or zOTUs from microbe amplicon sequencing is a raising area. Most of the proposed methods are not well benchmarked yet and a fair comparison between these methods is in great need. Hence the efforts and findings of the paper should be encouraged. However, the following issues in the experiment design should be further considered in order to achieve a solid benchmark:
1)  It should be noted that the methods tested in the paper used different chimera-checking pipelines (and possibly also other pre-processing steps), this would affect the initial point of the evaluations. Please check the consistency of chimera-checked sequences generated from different pipelines.
2) For denoised sequences, a 97% similarity is quite a loose match which is at least unintended for the denoising algorithms. I suggest to draw a complete similarity-proportion curve for each method (at least on one example) instead of using the 97% threshold alone.
3) For the claim "DADA2 tended to find more ASVs than the other two methods", it should be noted that each algorithm provided parameter settings that can trade-off between the recall and the quality of ASVs (e.g., for unoise you can tune the lower limit of sequence count for detecting an ASV) . It is fair to plot the complete trade-off curve using different parameter settings before making the claim.
4) The proportion of unexpected but matched ASVs is large, this should be explained.


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: DENOISING THE DENOISERS: AN INDEPENDENT EVALUATION OF MICROBIOME SEQUENCE ERROR-CORRECTION APPROACHES
Review round: 1
Reviewer: 3

Output:
Basic reporting: This study, presented by Nearing et al., is an important addition to the scientific literature. With the plethora of tools and software becoming available for 16S rRNA gene sequence processing, an unbiased assestment of ASV processing tools is imperative to the microbiome field. This reviewer is also encouraged to see that testing communities were pulled from human and soil microbiomes as it is important to test the performance of these software on a variety of bacterial communities containing many different types of species and levels of diversity.
In multiple places thoroughout the manuscript, this reviewer required more detail as to methods, results, and interpretation, and in some places found differing accounts of the results between the manuscript and figure legends. Specific lines with suggestions are outlined below. However, a lot of these questions and concerns could have been mitigated by including the specific scripts/code for the processing and analysis presented here. Because this is a manuscript which focusses on the comparison of various software, the exact commands that have been used to generate the BIOM tables should be provided. I do appreciate that most of the processing has been completed with Microbiome Helper (which is open source and public); however, steps to reproduce, for example, the UNOISE3 Pipeline (lines 118-126) would be difficult given the current information. Further, reproducing the analyses within would be extremely difficult without the appropriate code to do so. This supplemental material would be advantageous to the community as more tools become available, or if readers are interested in testing these 3 tools against their own mock communities.
Experimental design: In general, the experimental design is sound. As mentioned above, having the processing pipeline available in the supplemental material would aid in the reproducibly and visibility of this work.
Through the Materials and Methods the authors refer to "soil", "mouse", and "human" datasets; it would be advantageous to orient the reader in the Sequence Acquisition section as to which of these (HMP mock community, Zymomock, Extreme, fungal) are from which environments, and to use consistent naming throughout the text.
line 85: The HMP produced 2 mock communities, both with the same species composition but with different relative abundances. It would be advantageous to indicate which of these is used here without having to refer to Corneau, Douglas, & Langille 2017.
line 102: Why were cutoffs of 270 and 210 base pairs employed? Please detail here.
line 105: This sentence is repetitive of that above it.
line 148: Which database was BLASTN used against?
line 154: Is this 0.1% filter in addition to analyses done with a 0% filter? Wording is not clear here. (This does become apparent in the Results but I would still request that it is clarified here.)
line 173: It is not clear here what is meant by "medium stringencies" here.
Validity of the findings: I disagree with the authors indication that the biological results from these methods are unaffected by the chosen ASV processing tool. Having significantly different levels of ASVs assigned from each method (as shown in Fig1) indicates that these tools are identifying a different number of amplicon sequences in these samples. Although the genus-level assignments are very similar, differing numbers of ASVs are likely to severely impact alpha and beta-diversity metrics when performed at the ASV-level; the implications of these differences have been previously shown at the OTU level (for e.g. DOI 10.1186/s40168-017-0314-2). Further, it is more common to calculate these metrics at the OTU/ASV-level than at the genus-level (for e.g. as done in these recent publications doi: 10.1128/AEM.02335-17,https://doi.org/10.1073/pnas.1717617115), and is actually suggested in the DADA2 Pipeline Tutorial (1.6). I do understand that calculating beta-diversity at the genus-level is useful here in order to identity that these 3 methods all produce similar genus-level identifications of this community. However, it is imperative to point out that most investigations of the microbiome calculate beta diversity at the OTU or ASV level, not based on genus-level assignments. A supplemental to Fig3 should indicate whether similar analyses at the ASV-level produce similar results, and a point in the Discussion be made as to why this is important.
As a follow up to this point, it would be interesting to note the differential in the number of ASVs called by each method in the processing of the real soil dataset.
Fig1: How was the number of expected ASVs per community identified? Details on how this was determined should be provided in the Materials and Methods. This is a cruical detail which determines how the results of this Figure are interpreted.
Fig1: Due to a lack of SupFig legends (see below) I can't be sure which is missing, but why is one dataset missing from SupFig1?
SupTable1-4: It is not clear to me whether the "Percent Abundance" column is the Expected or Observed relative abundance of the organism in the mock community. Regardless, it would be advantageous to include both the Expected and Observed abundances of each organism here in order to make an informed descion as to tool performance. The text also indicates that information as to percent identity (100% match, >97% match) will be found in these Tables; however, they are not present.
line 206: Sequencing depths of the mock (and real) communities should be included in the Materials and Methods in order for the reader to estimate whether low abundance taxa were missed due to low sequencing depth.
Fig2: Unique colours should be used to distinguish taxa.
-lines288-295: These results should be included in the manuscript.
I really appreciate the inclusion of Supp Fig 6; the distribution of samples is often ignored and this reviewer appreciated being able to take a peak at them via this Figure.
Additional comments: I was unable to find the Figure legends for any of the Supplemental Figures. This may be an issue with file upload, or my own naivity, but it still should be noted.
line 50: Clarity is needed here; 16S rRNA gene sequencing is so widely adopted because its sequence tends to be similar among members of a species but differ across species. Also, because of its structure it uniquely allows areas of high conservation to be used in the creation of (near) universal primers which can be used to target areas of high variation. The way in which the authors currently introduce 16S rRNA gene sequencing here doesn't give the methodology justice.
line 54: Many research groups and projects still rely on OTUs, they are not yet "in the past". For example, the following studies were published in 2018: https://doi.org/10.1186/s40168-017-0389-9, doi:10.1038/s41598-018-23261-1, doi: 10.1128/AEM.02335-17.
line 61: There seem to be missing symbols following many references within the test which are appearing as empty boxes.
SupTable1-4: Spelling error in Table headers.
-The text indicates that Fig3a is based on genus-level distances; the Figure legend indicates that they are based on ASV-level distances.
-SupFig7: need y-axis labels.
-Table 1 is only mentioned in the Discussion; the information contained within would be more beneficial if it was brought to the readers attention at the beginning of the Results section.
-line335: This should be mentioned in the Materials and Methods.
-line357: What is the sequence bleed-through rate of an Illumina HiSeq? This information would be helpful here to make the comparison clearer.


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: DENOISING THE DENOISERS: AN INDEPENDENT EVALUATION OF MICROBIOME SEQUENCE ERROR-CORRECTION APPROACHES
Review round: 2
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: All of my comments have been addressed.


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: DENOISING THE DENOISERS: AN INDEPENDENT EVALUATION OF MICROBIOME SEQUENCE ERROR-CORRECTION APPROACHES
Review round: 2
Reviewer: 2

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: The revision is completely unsatisfactory in my point of view. See below.
1) ”but the chimera-checking methods employed by each method are tightly integrated within the documented use of each denoising method and it is unlikely that a typical user would choose a different chimera checking method from a different bioinformatic pipeline" -- This is not a reason for not carrying out additional experiments. A capable evaluation paper is not just about running code and collecting results. The authors should try to find the cause that make the discrepancy and suggest potential directions for improving. For the chimera-checking issue, what the authors should do is :(1) Create a pre-processed, chimera-free data set that would not be further filtered by the tested algorithms (or by-pass additional fitering steps of the tested algorithms if possible), and compare the observed outcome across algorithms with the one using raw data. (2) If chimera-checking does affect the ASV results, try to explain the cause.
2) "however, the majority of users will most likely use the default values and for this reason we tested these default parameters only"  -- Again, this not a reason for not taking comparisons. Parameter varation analyses and trade-off analyses are basic requirements for an evalutaion paper.
3) “We also found a high proportion of matches to a database but not the expected sequences on multiple occasions. We believe this is most likely due to the introduction of contaminate DNA sequences during the preparation of the mock communities for sequencing.” -- This is a completely baseless guess, because inproper choice of database query assignment criteria or representative sequences would as well lead to the case. If should be further noted that different methods report quite different portions of unexpected sequences, this cannot be explained by contamination. Moreover, large-scale contamination is not expected to happen for a mock community study. If unfortunately (though dubiously) this happened to be true, the authors would have to use other less-contaminated data for test instead.


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: DENOISING THE DENOISERS: AN INDEPENDENT EVALUATION OF MICROBIOME SEQUENCE ERROR-CORRECTION APPROACHES
Review round: 2
Reviewer: 3

Output:
Basic reporting: All previous comments have been adequately addressed.
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: THE ROLE OF BIOTIC FACTORS DURING PLANT ESTABLISHMENT IN NOVEL COMMUNITIES ASSESSED WITH AN AGENT-BASED SIMULATION MODEL
Review round: 1
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: Dear Authors,
With pleasure I have read your interesting and very well-written manuscript. I like your work, your embedding in the literature and the conclusions you draw from your computational experiments. I have some questions and comments, and a, what I think, important suggestion that I’d like to hear your thoughts on.
To start with the latter:
I think the strong difference between survival rates of competitors and defenders could be due to an ‘imbalance’ in the model: plant growth (equation 2) depends on biomass, both through the zone of influence and the ratio with maximum biomass. Biomass does have a contrasting effect on plant growth through both parts of the equation. It would be nice to see the graphs for some different values of biomass for overall plant growth in the appendix, to get more feeling for what the equation implies. In addition, herbivores damage plants by eating a certain amount of biomass, equal for big and small plants, and don’t spend more time on one than the other. This means that big plants suffer relatively less from the same number of herbivores. In all, biomass is what drives the plant’s survival probability.
(btw: I seem to miss the values for rgr in the paper, are they different for different species?)
And related to this a question: during a run, no new plants appear in the plot, right? So survival rates are a maximum of 1? This means I guess that if a good competitor outcompetes its neighbour (which would usually be replaced by a new competitor), it’s after that care-free, with a bigger zone of influence than before.
If all this means that large biomass is relatively important in the model, it also makes sense that defence values don’t matter: if herbivores are not important then defending against them doesn’t either. We do see trade-offs in the native plant traits, but under model simplification one benefit can overwhelm the other.
(Speaking of which: it seems that 3 native plants have lower seed weight than what is called ‘weak’ for non-natives?)
Other comments:
I think the study (apart from some minor issues) is very well-written and explained. Sometimes even a bit apologetic, as if using a model needs special conditions or warrantees. In line with this:
-ln. 64-67: turn around: non-successful expansions cannot be tracked in the field, and we need full control to test certain hypotheses here, so we use a model.
- ln. 68: a greenhouse is not reality either, you do not need to apologise. rephrase please.
- 74: this should be the view of any person interested in range expansions, leave out the modeller’s view.
- 101: you can make this statement in the discussion, no need to apologise here.
- 111: first mention of dispersal. I value that you show the bigger picture and maybe some reviewer asked for including all this. But I find it confusing to talk about dispersal when it is actually not included in the model..
-130 vs 138: 3-dimensional trait vector vs 4 plant traits?
-139: plants always hatch from seeds :)
-176: so 1 cm2?
-185: what is the purpose of stochastic density of herbivores: is this also not a reason that biomass is valued over other traits? If a herbivore remains in a spot, it would do so on a large plant I guess..?
-220: I guess the same comment as for dispersal: I value that you explain how you actually had herbivores move during a simulation, but for this purpose I would just leave out how you solved this technically and just say they chose a new random position each day.
-233: another one: if you remove herbivores that cannot eat on defended plants, without them bothering other undefended plants with it, this gives a relative advantage for undefended plants. In real life I guess the herbivore would continue searching for a plant that it can eat.
- I don’t understand how you get from eq 3 to eq 4. Maybe something for the appendix (‘appendix for the stupid’ :)
- 271: but theta was between 0 and infinity? (ln 261)
- 289: note that = so
- 292-294: I don’t understand why you do this: is this a trade-off: strong competitors likely maintain more biomass but set less seed per amount biomass?
-304: parameterization?
-334: omit ‘from reality’
-344: so no new plants during the simulation? Which would make survival rates >1
-365: to catch up
-388: because strong competitors end up competing with each other?


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 ROLE OF BIOTIC FACTORS DURING PLANT ESTABLISHMENT IN NOVEL COMMUNITIES ASSESSED WITH AN AGENT-BASED SIMULATION MODEL
Review round: 1
Reviewer: 2

Output:
Basic reporting: The article is generally well-written and follows the ODD protocol structure. This is slightly different to a traditional research article layout but it is commonly used for individual based models and is appropriate here. The authors provide a good background for the study in the introduction and the references cited are appropriate.

However, on occasion the language used is a little hard to understand. Some sentences are quite long and convoluted, and other times the grammar is not quite right, common mistakes include e.g.“are interacting with” should be “interact with” (line 175), “is representing” should be “represents” (line 146). I provide some further examples at the end of this section. In general, I would recommend that a native English speaker provide feedback on the draft before publication.

I also think it would be good to make clear early on that this model has been parameterized using data from a greenhouse experiment, along with some basic information about the experiment – even though the authors provide a copy of this manuscript (thank you!), just some general details e.g. when the experiments took place, the basic design, what species, etc. would be useful to know early on rather than mid way through the paper. At the moment this information is drip-fed throughout the paper, which makes it a little hard for the reader to keep track of.

The raw data has been shared (the netlogo model), however I think that the article could be improved by sharing the R code and model outputs as described in lines 352-358. I talk a bit more about this in section 3 of the review.

Some examples of where language & grammar could be improved:
Line 27-28 and 30-31 - sentences are quite hard to follow, can you replace “as compared to” with “than” perhaps?
Line 43 – it is not clear who “they” refers to here
Line 46 – you use both the terms “range shift” and “border shift” – I suggest for consistency only using the first of these terms as multiple terms can become confusing
Line 48 – suggest “include, for example, ..”
Line 130-131 – Can a vector be 3D? Should this be matrix instead?
Line 162 – Wording is a bit confusing. Recommend “resident herbivores and competitors” instead
Line 128,188 – “Repulsion” instead of “repulse”?
Line 240-241 – this seems like repetition of earlier “interactions” section – probably doesn’t need to be cited twice and don’t need to re-define acronym
Experimental design: The research fits well within the aims and scope of PeerJ, and the research question is well defined and is of great relevance in invasion biology. The introduction is well laid out and explains the state of knowledge well, as well as placing the study in a wider context. However, I think it would benefit from explicit hypotheses or aims at the end, as the current sentence at the end is a little vague and does not strongly describe what knowledge gaps the research is filling.

The methods are extremely detailed and I commend the authors on their use of the ODD protocol and for providing their model. I can confirm that the model works and that the code is well-commented and laid out so that a third party can understand it easily. The technical quality of the work is high and I cannot find fault with it.

My only suggested improvement here is that the binomial GLM models could be more fully described in the text (i.e. replace “and all two-way interactions” with the variable names), as well as how they were assessed (e.g. were data normalized/standardized, were residuals checked, how was the model assessed for degree of fit etc). This, along with providing the R code for these models, would greatly help improve reproducibility.

Another minor comment is that the info tab of the netlogo model is blank when I open it on my computer – I’m not sure if this is because I’m using a newer version of netlogo or because the info tab hasn’t been filled out. If the latter, I recommend doing so to make the model more user-friendly.
Validity of the findings: The data appear to be robust, and the conclusions are well linked to the introduction and research question. However, it is difficult to fully assess the robustness of the results as there is no sensitivity analysis of the model. I would recommend running a full sensitivity analysis for all parameters and presenting these results in a table, as it is difficult to assess the validity of the model without this information.

I think that before publication it would be good to expand the results section – personally I would like to see the results of a sensitivity analysis, the results of the scenarios, as well as the results of the binomial GLMs described in the text. Currently, the results text is a bit vague and lacking in detail, and as such it is hard to assess the robustness of the conclusions even though the authors provide some of this information in figures and tables.
Additional comments: I very much enjoyed reading and reviewing this paper and have no doubt that it will be up to the standards of PeerJ after some minor editing as described in the previous sections. Thanks to the authors for providing clearly-commented code and for following the ODD protocol, this is something I really think we need more of in the modelling literature!

My main suggestions for improvement (in order of importance) are:
1. Provide sensitivity analysis results for model parameters
2. Expand upon results section text and description of GLMs in methods
3. Improve grammar and reduce wordiness throughout

My recommendation (minor revisions) is based on the assumption that the authors have already done a sensitivity analysis during model construction. If this is not the case it could be quite a significant undertaking, if that is the case and if the authors decide not to include a sensitivity analysis, I think this needs to be addressed and justified in the methods as it is fairly standard practice.

My other comments are just minor suggestions. Thanks again to the authors for their well-laid out paper, I had a lot of fun playing with your model, it's very nicely done!


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: HERBIVORE BIOCONTROL AND MANUAL REMOVAL SUCCESSFULLY REDUCE INVASIVE MACROALGAE ON CORAL REEFS
Review round: 1
Reviewer: 1

Output:
Basic reporting: Reporting is clear with sufficient background and references with good statical design, analysis and figures.
Experimental design: Good experimental design
Validity of the findings: As authors themselves write in the discussion, the method is not effective to be recommend for a wide scale management of macro algae problems in the reefs.
Research is good, method is sound, but restricted in its applicability
Additional comments: Authors Neilson et al work related to the control of macroalgae using biological and physical methods is an interesting research carried out in the filed in Coconut Island.
I agree with the concept of the authors that there needs to be some method to control algal outbreak in reefs to help keep balance between algal and coral growths.

Removal or addition of biological organisms to control or prevent something is not new. It all comes to how effective it is in terms of time, manpower, amount spent and final results.
I feel that authors have contrasting ideas when you look at the title-abstract and later part of discussion. It seems to be an effective method when you read only title and abstract, however discussion tells a different story. This is kind of misleading.

Yes, there was a substantial change in the algal cover over time compared to control, but that did not change anything in terms of coral cover? Also on an ecological time scale to see substantial shift, this work is not long-term enough.
As a technique combining physical and biological controls simultaneously, this work is a good example.
Having said that, as authors themselves point out, there are many problems.
The reduction in the algal cover you see may be short-term, they could come back, once physical removal and urchins are stopped.
I do not see any substantial change in coral cover between 2 treatments. So was the presence of macro algae really effecting the coral in treatment area?

It is nice this worked for Coconut Island reef, but it might not be the same if some one try to use this method elsewhere.
There could easily be sea urchin outbreaks. High costs for a long-term period might not be feasible for many management people. Since, this has to be continued as long as macro algae exist in the area.

I think this the scope of this work in terms of its use is not much and limited.
I am not convinced how this can be an effective management tool and can be recommended to managers?