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: EVALUATION OF SOIL INTERVENTION VALUES IN MINE TAILINGS IN NORTHERN CHILE
Review round: 2
Reviewer: 1

Output:
Basic reporting: I'm not native english speaker and I have not got an opinion about language quality.
Experimental design: No comment
Validity of the findings: No comment
Additional comments: Although it could be very local, I think that it is very interesting for other researchers.

In addition, this paper was improved since the last submission.

Besides, this paper was improved since the last submission.


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: DYNAMIC PROTEOMIC CHANGES IN SOFT WHEAT SEEDS DURING ACCELERATED AGEING
Review round: 1
Reviewer: 1

Output:
Basic reporting: This paper reports on the proteomics changes during accelerated aging or different grain germination rates. It is an important topic and an interesting article. However, I raised some points that need to be clarified by the authors. This paper has a high similarity with Lv et al., 2016 by the same authors and the originality of the current paper need to be also explained. Also, a discussion between the differential expressed proteins and their respective discussion need to be included, because to better comprehend the effect of different germination rates in the proteomic was the main objective.
The term “storage tolerance” can be very sound for the authors but talking about wheat research, the term storage can mean different things, and I suggest the authors adopt another term, such as “post-harvest storage-tolerance or wheat seed storage tolerance”.
Abstract
Line 20-22. This comparison appears incomplete “more than” what? Please re-write.

Introduction
Line 29-30: Why the wheat seeds storage in China is carried out in horizontal warehouses and why for so long? I think that a simple sentence here could help the reader to better understand the justification and practical application of the work, since in the other countries the way how seeds are storage is completely different.
Line 44-45: In this sentence, it means that germination ratio and physiological enzymatic activities varied with hardness when seeds were submitted to long-term storage or during grain storage?
Line 51-52: The authors cited some previous works of the group, and they justified this current work saying that “the dynamic physiological and quantitative proteomic changes in soft wheat seeds that take place during accelerated aging remain unclear and, need to be elucidated”, but it is not clear in which aspect the previous works needing better comprehension, not only for the authors group, but taking account the literature. For example, in 2016 Lv et al. showed 162 differentially expressed proteins during artificial ageing. Please be clear about the originality of this work.
Experimental design: Line 59: Please provide more information about the classification of “soft cultivars”. Is it Triticum aestivum? Yangmai 15’ represents only one cultivar or variety? Is it a soft common/bread wheat, like soft US wheats?
Line 60: Please explain what “original germination rate” is or how it can be achieved.
Line 61: I think this is the average of seed moisture content.
Line 66: What do the authors want to say with: “seed samples with three biological replicates”? It means three seeds, three lots, please make it clear.
Line 75: Since the main objective of this work is to perform a tandem mass tag-based quantitative proteomic analysis, is essential that the authors explain this methodological step, because it is not clear. For example: the samples were labeled and put all together ?
Moreover the sentence: “Peptides from YM97, YM45, 76 YM28, and YM6 were labelled with 126, 127, 130, and 131, respectively” is not clear. Please specify.

Line 87: Since the MS method used is based on DDA acquisition, please provide methodological details of how the peptides were selected for MS/MS.
SDS-PAGE analysis is presented in the results but not mentioned on the methods. Please include.
Validity of the findings: Results
Line 113: About SDS-PAGE analysis, there is no comment in the results? Why there is a difference of intensity between samples if for all sample 30 ug were loaded?
For  LC-MS/MS analysis the sample were injected in the same concentration?
Line 120: What criteria were used to quantify 4509/6271 proteins?
From the quantified proteins and DEP how many are uncharacterized?
The results section is basically descriptive, I think it will be useful to highlight differences between samples, it means between germination rates, since in the discussion section the authors did rather a comparison with the literature, and an interesting discussion about the DEP but not explain the difference of the DEP in different germination rates that remains unclear.
For example, in the conclusion it is stated “1010 DEPs were identified, most of which were present when Gr was 6%.” This statement is not clear presented in the results and discussion.

Lines 146-149: For example, in the Figure 2 C the DEP belonging to molecular function of YM97 was extremely different from the others. How can the authors explain this result?
In the same way, in lines 150-153 there are differences between GR28% and GR6%, what is the practical comprehension? How these results can be interpreted concerning storage tolerance? The alpha-amylase is very important for the wheat quality and it is expected that there is an increase when germination occurs.
Line 169-172: This result was found for all samples? Why is inconsistent? The enzymes were up-regulated and their inhibitors were down expressed…
Discussion
Line 194-196: Again there is an incomplete comparison ‘Yangmai 15’ was higher than that of ‘Aikang 58 in which terms? Why these results confirmed the superior tolerance of soft cultivars?

Line 242-243: Why the results involving Glutathione related enzymes were unexpected?

Lines 246-248: It is not clear which traits/features lead to superior storage tolerance.

Figures: Please give a better explanation in the legend about the comparison between the differentially expressed proteins: for instance what does it means YM6/97 in up-regulated, means that proteins are up regulated in Gr6% compared to Gr97%. It I not clear in the legend neither in the text.
Additional comments: No comment.


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

Input:
Title: DYNAMIC PROTEOMIC CHANGES IN SOFT WHEAT SEEDS DURING ACCELERATED AGEING
Review round: 1
Reviewer: 2

Output:
Basic reporting: Wheat is one of the major food crops of the world. However, little is known about proteome of different wheat cultivars during different conditions. Here, Lv et al performed a detailed analysis of the proteome of aging wheat of soft wheat cultivar. The quantitative proteomics analysis revealed some significant differences with respect to protein abundance. In addition, the biochemical analysis reveals differences in respiration and germination of the aging soft wheat. This paves the way for further in depth, quantitative (phospho)proteome-wide differential analyses upon aging/stress/pathogen attack or environmental change. The manuscript introduction is clear. Literature is well referenced. Figures are relevant, well-labelled and described.
Experimental design: The authors have done commendable job in defining the research question. It is clear the work by Lv et al fills an important knowledge gap.
Validity of the findings: The methods are sound and details have been provided. Overall investigation performed is technically sound. Conclusion are well stated and limited to supporting results. The major problem is authors only look upto 180 days storage. Wheat is routinely stored for years and it is important to study aging in wheat stored for 1-2 years. The authors should mention limitation of the study in abstract and discussion.
Additional comments: Specific comments:
1.	The authors use inconsistently either ageing/aging. Please use one word consistently throughout the manuscript.
2.	In Discussion Line194 authors suggest “Our current results….” While comparing Yangmai 15 and Aikang 58. These strains are not compared here.
3.	There are problems with references formatting “Lv…Plos One,” and “Pasha….Int.”
4.	In Table 1 the authors should provide values (ratio) of upregulated and downregulated proteins rather than just mentioning regulated type


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: DYNAMIC PROTEOMIC CHANGES IN SOFT WHEAT SEEDS DURING ACCELERATED AGEING
Review round: 1
Reviewer: 3

Output:
Basic reporting: The research is innovative and comprehensively described the proteomic changes in soft wheat seeds during storage through accelerated / artificial aging.  The research will give basics for further investigation of physiological and biochemical changes in potential target proteins.
The results have been described in clear and unambiguous language that is understandable by the readers.
Hypothesis is very clearly explained
Experimental design: The primary research objectives have been clearly addressed in material and methods. The methods have been well designed and justified
Validity of the findings: The research work have some novel findings and the data is pretty useful for others. The manuscript has been well concluded.
Additional comments: 1. Introduction and review have been described in clear and unambiguous language that is understandable by the readers. However, there are chances for improvement at some places like in line number 44-45 (Zhang et al., 2017), the sentence should be re-written for clear perception.
2.	As described in material and methods that authors have conducted first tandem mass tag (TMT) -based dynamic proteomic analysis in lines 53-54, but there is no detail for importance of using this technique in the manuscript. Provide more information regarding the said technique.
3.    In lines 128-131, no reference method is provided for protein annotation, GO, Domain and pathway analysis.
4. 	Under the heading of discussion, the reference used in line number 212 (Aoyama et al., 1994) is old one and also not related to seed storage, research was related to rat hepatoma cell and immunoblot analysis conducted to study the peroxysomal fatty acid β oxidation enzyme. Reference should be reviewed.
5.	In discussion part there is contradiction b/w two statements regarding "up regulated" and "down regulated " enzymes in tricarboxylic acid cycle. line 226-229.
6.	There is ambiguity in word "themselves" in line 231 under discussion. It should be replaced with suitable noun like soft wheat seeds or grains etc. according to the manuscript.
7.	The reference mentioned in line 244 (Wang et al., 2015) is not related to research commodity because it is related to perishable commodity i.e., tomato that is not seems suitable for this work. Ref should be replaced with suitable reference related to seed storage study.
8.	Reference is missing In lines 248-251 where the behavior of histone H2A/B has been described. Add some suitable reference regarding this change that occur during aging.
9.	Overall the work is comprehensively described for proteomic changes in seed of soft wheat variety which will help in further research in deteriorative changes in storage grains.


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: DYNAMIC PROTEOMIC CHANGES IN SOFT WHEAT SEEDS DURING ACCELERATED AGEING
Review round: 2
Reviewer: 1

Output:
Basic reporting: Self-contained with relevant results to hypotheses.
Experimental design: Research question well defined, relevant & meaningful. It is stated how research fills an identified knowledge gap.
Validity of the findings: Conclusion are well stated, linked to original research question & limited to supporting results.
Additional comments: Wheat is one of the major food crops of the world. However, little is known about proteome of  different wheat cultivars during different conditions. Here, Lv et al performed a detailed analysis of the proteome of aging wheat of soft wheat cultivar. The quantitative proteomics analysis revealed some significant differences with respect to protein abundance. In addition, the biochemical analysis reveals differences in respiration and germination of the aging soft wheat. This paves the way for further in depth, quantitative (phospho)proteome-wide differential analyses upon stress/pathogen attack or environmental change. I have some minor comments.

1.	The title is somewhat vague and does not justify the study. Since in this study the authors have compared just two wheat strains and a more rigorous analysis is needed to verify these results with other wheat varieties. I believe limitation of the study should be mentioned.

2.	Improve English usage in manuscript. I will cite few examples
a.	Line 140 “indicating that the protein was not degraded and hence” It should be “indicating that the proteins were not degraded and hence”
b.	Abstract : “…..response to stimulus functions, consistent with processes occurring in hard”


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: ONTOGENETIC CHANGES IN SWIMMING SPEED OF SILVER CARP, BIGHEAD CARP, AND GRASS CARP LARVAE: IMPLICATIONS FOR LARVAL DISPERSAL
Review round: 1
Reviewer: 1

Output:
Basic reporting: This manuscript makes a useful contribution to the study of fish early life stages behaviour. It provides data on the ontogeny of swimming abilities of 3 species of invasive carps, which is crucial for developing accurate bio-physical models of dispersal and connectivity. The whole manuscript is very well written and nicely structured, with well referenced, relevant and up to date references. Introduction provides the appropriate background the reader needs, and addresses the knowledge gaps that the study will help filling. However, I have some doubts regarding the swimming terminology, which I would like to see addressed (see Experimental Design). Methods are well described, although I have some specific comments on this section (see Experimental Design). Results are, for the most part, straightforward and interpreted correctly, figures are clear and self explanatory, and the raw data has been made available and is nicely structured. However, I have some suggestions on this section, which I consider will help improving the manuscript (see Validity of Findings). Discussion wraps things up nicely and places the study in an appropriately broad setting. However, I would like the authors to further discuss some of the findings (see Validity of Findings).
Overall, I think this manuscript is an important contribution that certainly deserves publication in PeerJ, but I am recommending major revisions based on the specific comments below.
Experimental design: The research question of the current manuscript is very clear and nicely introduced in the Introduction. My main concern in the Introduction and Methods has to do with swimming terminology and methodology.
Swimming ability in fishes is generally classed into three types, including: 1) burst, which uses exclusively anaerobically powered muscle and lasts for less than 20 seconds; 2) prolonged, which may include both aerobic and anaerobic muscle activity lasting from 20 seconds to 200 minutes (Critical speed is a measure of prolonged speed); or 3) sustained, consisting of aerobically powered muscle activity lasting for longer than 200 min (review by Fisher & Leis 2009, which is included in the reference list of the manuscript). These types of swimming abilities differ in their ecological function, as well as in the methodology. However, there seems to be some confusion regarding concepts and methods.
The authors state that measurements of critical and burst (defined by the authors as maximum speed) speed use the same techniques and equations, being different only in step duration. I would like the authors to provide a reference for this statement, as according to the literature I am aware of, this is not the case. Measures of critical speed involves placing a fish in a swimming flume and increasing the speed incrementally over time until it can no longer maintain position. U-crit is then calculated as: U-crit = U + (t / ti * Ui), where U is the penultimate speed, Ui is the velocity increment, t is the time swum in the final velocity increment and ti is the set time interval for each velocity increment. This is exactly what the authors have calculated, but have named it as Maximum swimming speed, and according to the definition used in the introduction, this would be equivalent to the burst speed.
Burst speed is the fastest speed of which an individual is capable and because the speeds involved are so great and take place over very short periods of time, high-speed photography is required. Most commonly, burst speeds are measured as a startle response - a fast start behaviour that most larvae exhibit when startled by a perceived threat, and typically reflect the “escape” speed of larvae (review by Fisher & Leis 2009). To my knowledge, burst speeds are usually not measured in swimming flumes.
In summary, it seems to me there is some confusion regarding terminology and methodology, which I would like to see better explained by the authors, because as it stands, the results may confuse ecological interpretation of research findings.
Moreover, the time interval for each velocity increment the authors use when measuring swimming speed in the flume is 1 second. I would like the authors to explain why they chose to use such a short step duration. I am aware that both time and speed increments used to calculate critical speed are frequently species-specific, and stage-specific (e.g. Farlinger, S., and F. W. Beamish. 1977; Hogan et al. 2007). However, I have never seen a study using such small-time increments, and I would like the authors to justify their choice.
Other than these issues, the Methods are robust and well explained.

References:
Farlinger, S., and F. W. Beamish. 1977. Effects of time and velocity increments on the critical swimming speed of largemouth bass (Micropterus salmoides). Trans Am Fish Soc 106:436-439.
Hogan, J. D., R. Fisher, and C. Nolan. 2007. Critical swimming abilities of late-stage coral reef fish larvae from the Caribbean: A methodological and intra-specific comparison. Bulletin of Marine Science 80:219-232.
Validity of the findings: One of the greatest input of this work is the ontogenetic description of swimming abilities, in a multi-species approach. However, and as clearly stated by the authors, developmental time varied among the species, meaning that species differ in size of their larvae at comparable stages of development, hindering the opportunity to better compare swimming performance among the 3 studied species. In this sense, I suggest the authors to use the Ontogenetic index (by Fuiman 1994, 1997) to characterize the different ontogenetic states of the three carp species. It is a quantitative scale of ontogeny and has proven very useful when applied.
Also, it would be visually interesting to see in the graphs some of the ontogenetic landmarks described in the result section, such as the development of the gas bladder, fins, and others. If the authors include a graph with the Ontogenetic index, this could be easily visualized in a three-panel graph (1 panel per species) with arrows pointing to the landmarks shared by the 3 species.
Other than these suggestions, Results are well described, straightforward and statistically sound (large number of tested individuals, highly significant regressions).
Discussion wraps things up nicely, is well linked to the original research question and places the study in an appropriately broad setting. However, I would like the authors to address some of the suggestions I’ve commented on above.

References:
Fuiman L.A. 1997. What can flatfish ontogeny tell us about pelagic and benthic lifestyles? Journal of Sea Research 37: 257–267.
Fuiman, L. A. (1994). The interplay of ontogeny and scaling in the interactions of fish larvae and their predators. Journal of Fish Biology 45(Suppl. A), 55–79.
Additional comments: Overall, I think this manuscript is an important contribution that certainly deserves publication in PeerJ, but I am recommending major revisions based on the specific 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: ONTOGENETIC CHANGES IN SWIMMING SPEED OF SILVER CARP, BIGHEAD CARP, AND GRASS CARP LARVAE: IMPLICATIONS FOR LARVAL DISPERSAL
Review round: 1
Reviewer: 2

Output:
Basic reporting: ### Clear and unambiguous, professional English used throughout.

The english is good overall. A few comments:
- I noticed several instances of double spaces after full stops. It should either be 1 space or 2 spaces throughout. I suggest the former, the final editing being done by the journal.
- l. 51 to 60 could be clearer. I suggest "The eggs of these fishes are semi-buoyant and their survival is considered to require a length of river for them to drift downstream before hatching and a minimum turbulence or velocity to keep them from settling and being covered by sediment (Kolar et al. 2007, George et al. 2015). Previous works have often assumed the requisite river length to be approximately 100 km and the minimum velocity to be around 0.7 m/s (Soin and Sukhanova 1972; Krykhtin and Gorbach 1981; Kolar et al. 2007; Kocovsky et al. 2012). However, because several interacting variables control the development and settling of eggs, determining the adequacy of a river for egg survival requires a model such as FluEgg ..."
- "parameter" is often used to mean "variable". Prefer "variable" or "factor". NB: a "parameter" is a fixed value that determines the dynamic behaviour of a system: a growth rate, a metabolic conversion factor, etc.; a "variable" varies and its variations may influence the system: temperature, light intensity, etc.
- The phrase "leaving the drift" sounds strange but my English knowledge is not good enough to be sure what to recommend; maybe "and leave the drift in search of nursery" -> "and stop drifting to search for nursery"? Check throughout.
- l 96: BL/s is not defined at this point. Define it.
- "maximum speed" is a speed measurement, like "critical speed", "routine speed", all of which can be "absolute" (in cm/s) or "relative" (in BL/s). Therefore use "absolute maximum speed" (or simply "maximum speed") and "relative maximum speed", not "maximum relative speed". I suppose you would not write "routine relative speed" but rather "relative routine speed", the same goes for "maximum speed".
- l 100: 0.35 m/s = is that in burst? Overall, it could be made clearer what speeds are reported throughout this paragraph. It is often clear but sometimes not.
- l 232-246 and 248-263: the text is heavy and would greatly benefit from synthesising the numerical results in a table. Text should then only comment on the increases, decreases, etc. relevant for the biological discussion. Also, for maximum speed, the various numbers are given at different times post hatch and, apparently, different developmental stages too. This makes it impossible to compare and having the numbers is therefore a bit pointless, they should be made comparable and be displayed in a clear table.
- swimming speed is sometimes referred to as "swimming rate"; it sounds strange. A rate is indeed "some quantity per unit of time" but I would stick to speed in this particular case.
- l 248: "actual" -> "absolute"

### Literature references, sufficient field background/context provided.

The reference:
Leis, Jeffrey M. "Are larvae of demersal fishes plankton or nekton?." Advances in marine biology 51 (2006): 57-141
is a bit old now but is probably the most complete reference regarding behaviour of marine fish larvae, including the various measurements of swimming speeds. It could be added or replace several others.

### Professional article structure, figs, tables. Raw data shared.

The structure of the article is adequate.

The figures are really subpar:
- Fig 1 is dominated by extreme values and "outliers" (w/r to how they are defined in boxplots) and makes the general pattern very difficult to see.
- The info for Fig 2 could easily be included in Fig 1 and suffers for summarising the data too much (the mean only, with no indication on the variation)
- Fig 3 tries to convey too much information at the same time: growth, absolute swimming speed, and relative swimming speed all on the same plot! Multiple y axes on the same figure are usually a very bad idea, because they make the different variables seem to be numerically related to each other when this is just an artefact of the scales (this is very much the case for length and speed here). Also it does not depict the linear regression which is computed on absolute speed, while it would have the opportunity to.

I strongly recommend to rethink the figures and present growth, then routine speed, then maximum speed. I attach code and figure examples to convey how I would recommend doing so.

Most of the raw data is shared (which is very useful for the review, see attached file). The development stage values are not shared with the rest of the data, which prevents replicating those results. Given that a conclusion is that development stage may be a better predictor of swimming speed than size, it would be important to have those.

### Self-contained with relevant results to hypotheses.

Not sure what that sections means... Anyhow, the topic of the paper is well defined and the text addresses it correctly.
Experimental design: ### Original primary research within Aims and Scope of the journal.

Yes.

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

The point of the paper is well made but, to make it really meaningful, I would have liked to see an expanded discussion of:
- the usual flow speeds in rivers where those species live (including their variability); a bit of information is included near l 337-346 but it is mixed with technical information regarding other speed measurement experiments. It needs to be extended if possible and made into a standalone paragraph with the clear point of comparing the speed measured here to the speed of flow in rivers.
- the relevance of maximum speed for navigation in rivers. One sentence is present at l 88-91 but this is not enough. In the marine environment, half the critical speed is often considered to be relevant for navigation because it measures intense swimming yet that can be sustained for some period of time. I understand that, in a very turbulent environment such as a small river, burst swimming may also be relevant; but I suppose that some sort of sustained swimming is also necessary to achieve significant displacement, in particular in larger rivers. This needs to be discussed.

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

How were the fish euthanised after the experiments? I do not know the details of the regulation in the US but for vertebrates, the euthanasia procedures are usually regulated.

### Methods described with sufficient detail & information to replicate.

l 78-92: The initial description of the various measures of swimming speed in the introduction is not clear/detailed enough. A "step" is mentioned on l 83 but the stepwise increase in speed has not been described at this point. "Routine speed" is not defined, it is therefore difficult for a reader to understand how it relates to "vulnerability to predation" (in addition, I would have supposed that escaping predators is more related to burst speed). "Burst speed" is used in the following paragraph but not defined in this one. I think this paragraph should be a concise but complete description of the various measures of swimming performance.

l 162: How where the fish collected from the pond for the experiments? 890 m2 is large! Collecting them with a net may have damaged them.

l 167: "high" -> "long"?

l 167: Was there flow in the tank? If not, why make it rectangular (100 x 23cm) rather than square or circular? The rectangle has a smaller surface / perimeter ratio and the larvae may have interacted with the borders more, hence degrading the quality of the measurements. I suppose it has something to do with the capture of pictures; please explain it.

l 168: 15 cm deep and 23 cm wide seems quite limiting to measure speeds of up to cm long larvae that often swim at ~ 2.5 cm/s, frequently up to 5 cm/s. Any evidence of frequent interactions with the surface/bottom and sides? Were these data points removed or kept in the dataset?

l 177-181: The capture of pictures is not explained clearly enough. I gather that there were 2 cameras, taking pictures from the top and from the side, but this is never really stated. Maybe a small schematic drawing could simplify the text and describe the experimental setting (with measurements of the tank, distance of the cameras, etc.).

l 178, Table 1: the table of camera settings is useless without some quantitative information on the lighting intensity. I suggest to remove it (or place it in supp. mat.).

l 182-194: The tracking of larvae would require a more thorough description. It is nice to have the code of the LARVEL program but you cannot expect all readers to go through it. Because there is no technical paper describing LARVEL, please explain briefly here how the thresholding, measurement of particles, and association of particle position into a track is done (references to the algorithms mentioned in the code etc.). One thing I was not sure about is whether you have a continuous trajectory for each individual larva through the observation period or just associations or positions in two successive images. I suppose the former but it needs to be clearer.

l 182-187: Related to the above: you state that the relatively low image capture frequency "did not affect conclusions" or "provide[d] accurate data". Without more details on the tracking these statements are impossible to judge; in particular, assembling a continuous track for an individual from low frequency captures in a setting where individuals may cross paths is very difficult. Also, did you quantitatively assess those statements (by degrading the signal from higher frequency trials for example)?

l 208: It is not clear that Hz is the measurement units of the speed controller in the Loligo tank. Make it explicit.

l 209: How was the speed increase controlled? Manually? It seems difficult to reliably turn a control knob by 0.1 unit every second, on the second. Have you tested the replicability of this technical aspect?

l 212 and 214: I supposed that the Hz values in the data table provided are computed with formula at l 214 and that computing speed in cm/s would just be a matter of applying the formula at l 212. But this does not work, even after trying to scale the values in case there was a mistake in the units (see attached code). As such, the results are therefore not replicable. This *absolutely* needs to be investigated and fixed.

l 215: How was the time swum in the last speed increment measured (given that it needs to be sub-second accurate)?

l 217: Make it clearer that it is the fish that swam that were measured (which is how I understand the data table provided). It is important that you have the individual size of the fishes that swam.

l 223 (related to the above): Why compute the relative maximum speed from the *mean* of the body length when you have individual length measurements? Speed and length are not independent and mean(speed/mean(length)) is different from mean(speed/length) (I checked with data restricted to lines where both are available). Computing the individual ratio yields better estimates (for the mean but most importantly for the variance) and it is therefore preferable even if some measurements of length are missing (hence some speeds have to be discarded).

l 223: TL stands for Total Length, which is different from Body Length (BL) (also called Standard Length); https://en.wikipedia.org/wiki/Fish_measurement. You measured Total Length apparently (based on the legend of the data table); please make the text clear in that respect (i.e. replace BL by TL everywhere).

l 228: Why compute the regressions with the daily mean rather than the full data? If I understand correctly all measurements are taken from different individual fish, they are therefore independent and can all be used in the regression, no need to average. The distribution of variance will be better represented with individual measurements.
It seems the assumptions for linear regression (normality of residuals, homoscedasticity) were not checked; it would have been useful to be able to compute, and report, p-values for the regressions. My tests suggest they were not met. Was this the reason to not report p-values? If so, please state it. In that particular case, I think R2 is indeed the most interesting statistic, but reporting p-values is usual and may be required by other reviewers.
Finally, I recomputed the regressions with individual values of swimming speed. I have problems replicating the values of speed displayed in Fig 3 (as explained above) but the overall aspect of the plot is similar, so the results of the regression should be comparable. Yet, I do not get the same qualitative results in terms of fit. For the regression of speed on DPH, the order in terms of quality of the fit (i.e. R^2) is SVC > GSCP > BHC when you have GSCP > BHC ~ SVC. For the regression of speed on length, my results are SVC > GSCP ~ BHC, you have SVC > GSCP > BHC. Again, here the results do not seem replicable. This needs to be investigated and checked.

l 236: what is n? Define it clearly. I suppose it was number of couples of positions between which speed could be measured. That means measurements for several fishes were pooled together, indiscriminative of the fish they were measured on. Have you examined variability in routine speed between individual fishes (this is why having individual tracks would be important)? Are the variations at the population level (what you report on here) much larger than the variation at individual level? If not then maybe the statistics should be computed per individual and then summarised at population level, to not mix the two sources of variability (individual and age) in the results.

l 235 and following: the time of "gas bladder inflation" is taken as a reference to discuss swimming speed, which is relevant, but we need to know at which approximate age this occurs in each species, which is not stated. It would also be useful to have this displayed (as a vertical line) on plots.
Validity of the findings: ### Impact and novelty not assessed. Negative/inconclusive results accepted. Meaningful replication encouraged where rationale & benefit to literature is clearly stated.

No comment, content of the paper is appropriate.

### Data is robust, statistically sound, & controlled.

See remarks pertaining to the methods above.

### Conclusion are well stated, linked to original research question & limited to supporting results.

l 364-373: I am not sure I completely understood this paragraph on entrainment by barges. Barges would be able to create a current fast enough to counter the effect of the river flow? Larvae could ride it? Please make it a bit clearer for non-specialist (maybe a few introductory sentences with some size order of the flow speeds involved would be enough).

l 374-382: This paragraph has two points: (i) relative swimming speed decreases after the larval stage, (ii) flow conditions at that moment in the life of the species considered can still be intense. The overall point seem to be that settling larvae have to find paths of low flow to be able to settle. I am not sure the point is well made because the *relative* speed decreases, indeed, but the *absolute* speed *increases*; it is the absolute speed (and how it compares to current speeds) which is relevant for settlement. This paragraph needs a bit of rethinking and some numbers (see my point above about discussing speed of larvae vs speed of flow).

### Speculation is welcome, but should be identified as such.

No comment, content of the paper is appropriate.
Additional comments: The transition between vertical and horizontal swimming in these species surprised me. In other species, the separation between vertical and horizontal swimming is rather done in terms of swimming *efficiency*: when larvae begin to swim, they swim slowly and that makes swimming significant only in the vertical direction (because vertical currents are often much slower than horizontal ones), then, after swimming abilities have developed, horizontal swimming may become efficient. In the species studied here, it seems there is a real behavioural transition between vertical and horizontal swimming which I had not heard about before. I think it would deserve a bit more explanation around lines 69-70.

I attach some code and its output, which I used to try to replicate the findings and show how I would plot the data. The code is in the R language, while the authors seem to be more familiar with MATLAB, but the general idea is likely transferable. I tried to add many comments to make it clear what I was doing. I would be happy to interact with the authors further on this.


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: ONTOGENETIC CHANGES IN SWIMMING SPEED OF SILVER CARP, BIGHEAD CARP, AND GRASS CARP LARVAE: IMPLICATIONS FOR LARVAL DISPERSAL
Review round: 2
Reviewer: 1

Output:
Basic reporting: First, I thank the authors for their careful considerations of my remarks and those of the author reviewer. They addressed most points to my satisfaction. Below I highlight a few minor additional improvements.

l 84 : "The swimming capabilities of larvae change during ontogeny at gas bladder emergence from vertical swimming to horizontal swimming, in a transition marked by both behavioral and anatomical changes" -> "The swimming capabilities of larvae change during ontogeny, in a transition marked by both behavioral (from vertical swimming to horizontal swimming) and anatomical (gas bladder emergence) changes"

paragraph starting at l 96: The text is improved but could still be clearer. Start by presenting the various speeds measures and what they quantify:
- routine = free swimming; aerobic;
- critical = sustained forced swimming; aerobic + anaerobic,
- maximum = short term forced swimming; anaerobic
- burst = nearly instantaneous escape reaction; anaerobic.
The remarks of reviewer 1 in the first round of review summarise this even better.
Qualify maximum speed as "intense short term swimming" and not "burst", otherwise it is confusing relative to what is usually called burst speed. Then use the sentence that was added to justify why maximum speed is the most appropriate to measure to then model advection and navigation in rivers.

l 252: "1m tall, 0.23 m wide, 0.15m deep". I am still not sure what is what; both tall and deep evoke the vertical direction to me. If the vertical dimension was 1m then there is no problem to measure vertical speeds; but the longest horizontal dimension would be 23cm, which is short to measure horizontal swimming. Otherwise, 15cm is the vertical dimension (which is what I thought initially) and I was wondering whether that would be enough to measure vertical speeds and avoid too frequent interactions with the bottom (but the authors responded on that point already). Maybe use Length (= longer horizontal direction), Width (shorter horizontal direction), Height (= vertical direction)?
Also, be consistent with spaces before units.

l 303: "Each path was visually examined" -> "Each *individual larval* path was visually examined"

l 356: "Daily measurements of total length for the 10-20 larval fish that swam in the swim chamber  Developmental" too much has been deleted; a portion of the sentence is missing.

l 363: "mean total body length"; should be either total or body but not both. Total includes fins (the caudal fin in particular here); body is juste the *body*, down to the peduncle of the caudal fin.
Experimental design: ### Methods described with sufficient detail & information to replicate.

"Fish were euthanized with an overdose of MS-222 (standard methodology approved by the IACUC)."
This should probably be in the paper.

"Fish were collected with light traps and nets (as necessary) and acclimated overnight in a tank. Care was taken to ensure that fish were in good condition and undamaged before they were used in the swim chambers."
Should be in the paper

"The photo frame did not include the sides, surface, or bottom of the tank, so there were no recorded data on interactions.  But in general, we do not see larval carp interacting with the sides of any tank.  They often lay on the bottom, and we do see them at the surface, but in both cases, they tend to not be moving."
Add a note about this in the methods, in the section about how the pictures were taken, e.g. "The photo frame excluded the sides and bottom of the tank to avoid measuring speeds biased by the interaction with a substrate."

"The means were used because we could not in all cases match the length data to the swimming speed of each individual larvae"
This is now clearer in the text of the paper (and the response to reviewers). But I was actually misled by the accompanying data which gives, on the same line, a speed and a length for a species at a certain age. I assumed those were measured on the same fish. If this is not systematically the case, please put these two values in two different tables. If this was the case, then it seems there would be plenty of data to perform the statistical analysis at individual level.
Validity of the findings: ### Data is robust, statistically sound, & controlled.

"P-values were reported in the caption to the table, all were significant, and are now added to the table,"
I don't see them, but it is fine like this. Sorry I missed it in the caption.
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: HOME SICK: IMPACTS OF MIGRATORY BEEKEEPING ON HONEY BEE (APIS MELLIFERA) PESTS, PATHOGENS, AND COLONY SIZE
Review round: 1
Reviewer: 1

Output:
Basic reporting: The authors have developed a commendable approach in an attempt to explain the effects of modern apiculture practices on honey bee colony health. Specifically, the authors attempt to explain the effects of a.) migration on colony population size and pathogen profiles, and b.) the effects of exposing stationary colonies to migratory colonies returning from California on pathogen profiles. The experimental approach developed in this study has the potential to resolve the complexity associated with modern apiculture on colony population health.

The introduction is clearly worded, and expertly organized. There is a clear conclusion paragraph which describes the goal of the study, and the main hypotheses being tested. However, the intro would benefit by elaborating on some mechanisms of colony health decline, and including relevant citations. For example, the species of Nosema responsible for colony health decline should be elaborated-on, considering evidence exists that Nosema ceranae has been linked with colony death in Spain (Higes et al. 2008).
Experimental design: The experimental approach is the main strength of this study. The authors are correct that many studies monitoring colony health lack an appropriate control group, and therefore I think this study will progress the field. However, I would like the authors to acknowledge a few experimental shortcomings:

a.) after returning from California, migratory colonies were placed next to stationary colonies in the same bee yard, but no additional colonies were placed next to the stationary colony. How can the authors conclude that pathogen exchange is as a result of migratory colonies, and not simply from increasing densities of bees in the yard? This relates to discussion lines 354-356

b.) if colonies are blocked spatially for the duration of the study, is it still appropriate to treat each colony as an independent individual? Are there block effects on colony condition?

c.) The analysis was performed on a limited set of bee pathogens. What might be the case if the authors were to survey for some more common bee pathogens (Lotmaria passim, LSV complex, KBV, PCR amplified Nosema ceranae, etc...)?

d.) The temporal scope of their study is somewhat short.

The statistical analysis is creative. Their use of multivariate statistics is important, and is a powerful tool to explain a complex system. However, I feel as if the authors can include more detail into the section regarding the building of linear mixed effects models, and the specific question they intended to answer through this analysis.

e.) was BQCV excluded from the discriminant analysis because it was prevalent in 100% of colonies? I would imagine this makes colonies more similar, and more difficult for the classification tree to discriminate treatment groups.

f.) There was no mention of including interactions in the models. Why include interactions?

g.) How did the authors deal with potential multicolinearity among fixed effects in their models? I would imagine there is temporal autocorrelation among FOB and mite loads.
Validity of the findings: The conclusions of the results are supported by current research in the field. The authors do an excellent job of stating the limitations of their limited temporal study in the conclusion paragraph. However, the authors sometimes do not explain results using honey bee biology.

a.) could the simplest explanation for FOB, mite, and pathogen increase in the third sampling event be because the third sampling event took place during the colony buildup phase? We would expect colony populations to grow to prepare for summer foraging. This could be why the authors find a lot of significant interactions between time of sampling and other fixed effects in the models.

b.) Lines 381-391, while interesting, seems beyond the scope of this study.
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: HOME SICK: IMPACTS OF MIGRATORY BEEKEEPING ON HONEY BEE (APIS MELLIFERA) PESTS, PATHOGENS, AND COLONY SIZE
Review round: 1
Reviewer: 2

Output:
Basic reporting: Background Research in general ok, but referencing could be improved in some places.
Experimental design: The scope of the experiment appears quite limited in terms of number of colonies, exposure time, and observation time.
Validity of the findings: The discussion is not adequate in several places and in need of major improvement. Speculation is abundant.
Additional comments: L52 - "for over a decade honey bees have experienced catastrophic colony failure " is exaggerating and refers, if anything, to the US situation, but is not valid on a worldwide scale.
L 54 - the Zhu et al. 2014 paper deals with honey bee larvae in individual assays and is not a suitable reference for pesticide effects on the colony Level
L55 Varroa mites should be mentioned together with pathogens. I'm also not sure that Evans and Schwarz 2011 is the best refrence in this context; vanEngelsdorp and Meixner 2010 could be a better reference
L 57 - here, a more recent reference might be more suitable (p.ex. Traynor et al. 2016)
What about economy as driver for pollination or other apicultural practices?

The scope of the experiment appears quite limited in terms of number of colonies, exposure time, and observation time.
Sampling - at least for targeting Nosema infection levels, sampling away from the broodnest would have been more advisable (see Fries et al. 2013 JAR 52(1) (BEEBOOK)

Why was the number of brood cells assessed only once at the start of the experiment, and not again in the course of the experiment? Especially in the context with development of the Varroa mite population it would have been helpful to also obtain data on broodnest size.

Discussion:
L312 ff - why is it likely that migratory conditions exacerbated BQCV infection? The evidence is correlative, not causative. Is there any experimental evidence that migration indeed increases BQCV replication?

L317 ff, 320 fff – this reasoning is highly speculative, following from the correlative evidence above. You have no data that would show exposure of your hives to OSS, or allow the conclusion that this (putative) exposure would have resulted in higher virus replication. In addition, you would also have to show that the stationary colonies were not exposed to OSS.
Also - in line of this reasoning - how would you explain that one month later, the BQCV levels of all three groups (including the isolated and the exposed colonies) converged at an even higher Level?
In addition - BQCV infection levels in all three groups appear absurdly high (Fig. 3), compared to the published literature (where levels beyond 10^8 or 10^10 are considered as "high"

L337 ff - as there are no data on the amount of bee brood present, it is difficult to compare the mite infestation levels on adult bees of the three groups. Mite population dynamics and the ratio of mites in brood to mites on bees is highly dependent on the amount of brood present, but can also be influenced by contact with highly infested neighboring colonies (drifting, robbing etc.)

L360 ff - I'm not sure the higher DWV loads in the exposed colonies compared to migratory and isolated colonies can be explained from your data at all .
If I were to speculate, I might say that it looks like these colonies grew faster (they had more bees), but probably they also had a higher brood infestation level (because they probably also had more brood, but this wasn't measured). More mites in the brood usually also  results in higher DWV replication in the brood, and consequently in higher DWV levels


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: HOME SICK: IMPACTS OF MIGRATORY BEEKEEPING ON HONEY BEE (APIS MELLIFERA) PESTS, PATHOGENS, AND COLONY SIZE
Review round: 1
Reviewer: 3

Output:
Basic reporting: The paper is generally clearly written and nicely organized. Although some sentences could benefit of clarity by being much concise or rephrase, the manuscript was easy to read. The introduction and background are highlighting the need to better understand the link between parasitism / pathogens risk associated with migration in honey bees. The work offered to test and quantify the effect of migration on colony size, pathogens prevalence and loads among 48 colonies submitted to different risk exposure despite originated from the same apiary. The results of different tests converged toward a link between beekeeping migration for pollination and negative impact on colony health in honey bees.

I would suggest a few modifications/check in the introduction part to ease the reader in the topic:
-	L49: Please check the value you are stating from Gallai et al. 2009. I remember reading in this reference that insect pollination services, including bees, was estimated to worth about 153 billion and not 200 billion.

-	I understand that you the aim is to highlight the management practice by showing its importance in US. I would suggest that one line would be added (maybe in the first paragraph L47-57) to indicate why pollen and nectar are important for honey bee colonies. As example you could indicate that pollen is essential for providing the brood the protein needs for good development.

-	Since you are also assessing Varroa mites infestation here, I would suggest that you separate pathogens (Nosema, viruses and bacteria…) from parasite (Varroa destructor). This is much clearer for the naïve reader and accurate.

-	L82: Where did you find this value for theoretical number of bees (colonies) sharing the same resource patch?

-	L107: Here you are writing properly the name of the three viruses you tested but then in other section you directly write BQCV, IAPV and DWV. Remember that those acronyms are not general knowledge and please add abbreviation there.

Figures and table are good quality and generally well labelled. Some minor changes should be added.
Figure 1: Is it possible to add the geographical distance between your stationary and migratory colonies?
Figure 2: The blue label signification is indicated in the caption but since you made the effort to specify for the other treatments the legend in the figure, could you do the same for stationary colonies?

The paper generated data on health status of 48 Western honey bee colonies established in US before, during and after migration event. The raw data are all supplemented including weather measures. In your supplementary table 3, please indicate what is the value for each column. As an example I couldn’t understand what the value of BroodPattern meant. Follow the good example of your first supplementary table 1.
Experimental design: The experimental design is generally well organized but some details are difficult to understand. The number of colonies assessed here is well distributed among the treatments and reasonable for the research question. On a first sight, the three sampling points seems limited but given the period and the organism studied, it is more than reasonable (3 months, generation time honey bee 21 days). The methods used to detect pathogens and quantify parasite are following properly standards and seems reliable and replicable. Some minor changes, additions, modifications would be suggested as follows:

-	L130: I do not completely understand how the measure of frame of bees worked. Were you checking the number of frame with bees on it (with or without brood) only? Did you also estimate the brood availability and eventually of drone brood too, given that you studied Varroa prevalence and load? Varroa population an so load is highly associated with brood availability (Beaurepaire et al. 2017).

-	L134: Were all the queens A. m. carnica/A. m. ligustica genetic background check? How many colonies and apiaries are surrounding the stationary yard?

-	L182: I would indicate here directly rather than in your results (cf L249) the absence of IAPV in your data. This will avoid your reader to wonder if you forgot to write the IAPV test in the material methods after line 182.

-	Line 201: “We verified RT-PCR analysis through sequencing” – which sequencing? Could you specify the machine and length of sequences?

-	Line 231 – 239: in the result section is visible that GLMMs also tested for interaction effects (mentioned in line 270-277). Please make this clear in the Method part. In addition, isn’t it necessary to correct p-values for multiple testing?
Validity of the findings: The results are reasonable given the experiments and authors are really cautious in their conclusion. PeerJ standards requirements are met in this study.
Additional comments: It is a really interesting paper and you have done a really good experimental job in assessing the impact of migration in honey bee colonies.

I wish you could have also take some pictures of your bee colonies (if you haven’t done it already) so you could show the reason your colony size is so different before and after treatment. It is not always clear for me if it is only the foragers missing or maybe a stress in brood production. If you have it will always be valuable in the supplementary data to put some pictures to show the evolution overtime of FOB.

In general, you did a good job discussing the different factors but you did not mention that monoculture has been shown in several publications to increase host susceptibility towards pathogens and toxins. Can easily be a part of the sentence of Line 315 – 319.


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: HOME SICK: IMPACTS OF MIGRATORY BEEKEEPING ON HONEY BEE (APIS MELLIFERA) PESTS, PATHOGENS, AND COLONY SIZE
Review round: 2
Reviewer: 1

Output:
Basic reporting: The authors developed a design which makes it possible to explain the effects of our modern agricultural demand and pollination practices on the health of honey bees. Specifically, the effect of migration to and from almond monoculture pollination where bee colonies are facing tough conditions like poor diet, high pesticide loads and meeting thousands of bees from other locations, is studied. The strength of this study is the experimental design which is not only focusing on the bees exposed to the migration but also on the question if they will transmit disease to stationary colonies when shifted back home. This point is highly relevant in terms of disease spread among honey bee colonies over wide areas and has potential to answer many highly interesting questions in the future.
The introduction is clearly worded and well organized.

Line 72:  There are several publications which clearly show that almost all monocultures are less good for honey bees than diverse diets are. Although, the authors give such citations in the discussion (Line 352/353), it would be nice to also include them here, otherwise the “may” does not sound like there are already published data on that. And the next sentence points clearly to a publication about travel stress. I would prefer if all stress factors we have data about are being pointed out like this in the introduction.

Line 76/77 A short sentence what the other publications found, would be great.

Line 86: which are not but

Line 100 to America and Europe
Experimental design: As already stated above, the experimental design with the proper and elegantly planned controls is the strength of this work.
The method part is well described and points like supplemental feeding practices etc are clearly reported and also methods used to detect and quantify pathogens and parasites are reliable and reproducible.

The temporal scope of the study, although being long enough for the questions the authors want to address, is relatively short and a later time point would have been interesting to study long-term effects, e.g. it would be highly interesting to know if the overwinter success of the different treatments differ.

The focus lays on a small set of bee pathogens. While it is obvious that a decision for some has to be made, it would have been a more spectacular result if the authors would have tested for pathogens previously not present in the colonies and being brought back by the migratory colonies. This would have been the next dimension of the work cited in 406/407. But I understand that there is always a time and budget limit.

While the methods are very strong in terms of sample size and experimental design of planning the three treatment groups, I wish the authors would have measured brood size and pollen load in the frames at each sampling time point. This would have given a better indication about potential reasons for the population size decline and if the Varroa decrease in the migratory colonies has something to do with lower brood availability. Anyway, a careful discussion about this problem is provided by the authors which is the most important point when realizing gaps in the design.
Validity of the findings: The conclusion and discussion are supported by current research in the field and the authors do a good job discussing the limitations of their design and are careful with speculation and if they do, they state it clearly.
I for example really liked the discussion point that potentially a brought back more virulent Virus strain could explain infection pattern – this could be an amazing study in the future.
I also enjoyed that the authors are mentioning potential impact on wild bee populations.

The authors point out correctly that they did not measure pesticides in the bee tissue or colony but they give a statement about OSS. As there are likely also other pesticides which have been used and can have an influence on the bee e.g. homing ability of the forager, I would suggest to list all of them in the supplemental material for detailed information if they have access to this information (e.g. from the almond industry?). Otherwise, it sounds a bit biased to just pick out one for making a speculative point.

Line 441 large pollination events or a large event

Figure 2: please state what the circles around the centroids indicate
Additional comments: Do you have potentially taken pictures of the frames which might make a later rough estimation of bee bread and brood load possible? That would be very valuable and potentially worth to try to implement. Unfortunately, the Varroa load has no real meaning, now as the reason cannot be explained.
I know, it is always easy to point something like this out afterwards. Otherwise, I really liked the well-planned experiment in general and I think that you can tackle many highly interesting questions with this design in future studies. Good luck!


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 SEASONALITY ON CHEMICAL PROFILE AND ANTIFUNGAL ACTIVITY OF ESSENTIAL OIL ISOLATED FROM LEAVES PSIDIUM SALUTARE (KUNTH) O. BERG
Review round: 1
Reviewer: 1

Output:
Basic reporting: The manuscript describes the seasonal variations of Psidum salutare essential oils (yield and composition) and the antifungal activity of these.
The manuscript is well organized and well structured. It meets the expectations and standards of PeerJ. The subject discussed is very interesting because few studies have examined the seasonal effect of essential oils in this species. This is therefore an originality that is well emphasized in the introduction.
Experimental design: The experimental protocol is well done with approved and proven methods.
The only lack in this part is related to the lack of information on essential oil used for antifungal tests. What stage of development?
Validity of the findings: The results obtained are very interesting and can make it possible to have a modulation of the composition of the essential oils as a function of the stage of development and or seasons.
Additional comments: The manuscript suffers from several gaps
1- For a better follow-up of the different phenological stages, it is advisable to follow a standard scale of the species, if any, of a species of the same botanical family or by the use of the BBCH scale.
2- it is not specified the essential oil of which season is used for the antifungal tests. This is one of the weakest manuscript points. Since the title of the article is the seasonality of the composition of the essential oils of antifungal activity. However, if the seasonality of the composition is present, that of the antifungal activity is not.

The other weak point concerns the climatic conditions. many parameters are missing, including temperatures. It is now well known that essential oils are produced and modulated according to climatic conditions, in particular in response to biotic and abiotic stresses. For these, there is not only water but especially temperatures. The authors have completely forgotten this parameter. Moreover, the variation of essential oils and the impact of climatic conditions on this variation is not well discussed


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 SEASONALITY ON CHEMICAL PROFILE AND ANTIFUNGAL ACTIVITY OF ESSENTIAL OIL ISOLATED FROM LEAVES PSIDIUM SALUTARE (KUNTH) O. BERG
Review round: 1
Reviewer: 2

Output:
Basic reporting: state novelty
Experimental design: fine
Validity of the findings: state reason for selecting the plant
Additional comments: All the experiments are well performed. The article can be accepted after stating its novelty in the abstract and conclusion


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 SEASONALITY ON CHEMICAL PROFILE AND ANTIFUNGAL ACTIVITY OF ESSENTIAL OIL ISOLATED FROM LEAVES PSIDIUM SALUTARE (KUNTH) O. BERG
Review round: 1
Reviewer: 3

Output:
Basic reporting: The manuscript is poorly written which makes the understanding of the data and interpretation difficult. I have highlighted sentences and example or where it must be re-phrased. The figure legends are very basic and incorrect if Figure 2 it seems (concentration units are not correct). The legend of the graph in Figure 1 are too small to read.
Experimental design: It is apparent that the authors have expertise in plants and essential oils. The concept of seasonality is relevant to plant extracts.

The antifungal assays however are more difficult to follow.

The method section needs extensive re-writing to adapt to standard procedures commonly used in the field of antifungals. It is not easy to reproduce the assays as they are described in the manuscript as essential details are missing.

Assays were performed three times according to the statistic section, which is good.
Validity of the findings: Conclusions should remain specific to the conditions used in the in vitro assays, as the choice of the conditions are atypical in the field. Assays that are typically used are recommended to the authors so their results can be extrapolated a bit further.
The drug test are statistically good and reliable.
The determination of the effect of the samples on Candida morphogenesis is less convincing. It is not clear how many times the assays were done. The concentration units appear to be wrong on the figure itself. Other hyphal inducers are typically used but they are not mentioned in the current manuscript.
The authors refer to fungicidal activity through out the text, yet the essential oil samples do not appear to be cidal even at the highest concentrations. Re-phrasing is required to make sure the appropriate wording is used. Recommendations were made to the authors in the General comments to authors section.
Additional comments: The manuscript aims to study the effect of seasonality on the composition and antifungal activity of plant material. The concept is relevant and attractive.

In abstract:
Line 29: “….effect on the Candida albicans virulence micromorphology”. Do the author mean the effect on Candida albicans morphogenesis (also known as the morphogenetic switch or dimorphic transition)? “Virulence micromorphology” should be replaced through the whole text by morphogenesis or morphogenetic switch.
Line 33: “through” should be replaced by “using the broth microdilution assay”
Line 34: “fungicidal” should be replaced by “inhibitory”, MIC instead of MFC, as it is not clear how the authors have demonstrated whether the essential oil kills the fungal cells or inhibits their growth. Killing and fungicidal activity would require CFUs analyses to demonstrate whether cells are alive and not growing, or dead.
Line 35: “…dimorphemic effect”..should be replaced by the “dimorphic switch/transition”
Line 38: “The antifungal activity revealed an action against..” should be replaced by “MIC assays revealed an antifungal activity against…”
Lines 39 to 41: The whole sentence starting from “in the antifungal modulating…” needs to be re-phrased. What is the antifungal modulating assays? Was the activity significant? Was this tested with statistics?
Lines 41 to 44: those sentences should be at the present tense: “The results show that…
Line 44: a sentence like this “The results show that P. salutare oil exhibits a significant antifungal activity against three Candida species and that it can act in synergy with fluconazole” would be clearer. What is a fluconazole potentiator?

Line 48 and Keywords: I would remove precipitation, which is included in seasonal variation; pathogenesis and micromorphology could be replaced by Candida species and morphogenesis respectively.

In the introduction:
Line 58: remove “In this sense”
Lines 61-63: do the authors mean that the knowledge of the impact of seasonality on the essential oil composition can help identifying highly functionalized substances, which may have a greater commercial relevance?
Line 81: remove “especially”
Lines 80-82: Fungal infections caused….are a serious health problem in immunocompromised patients in particular and is aggravated by the increased resistance of clinical isolates to antifungals.
Lines 84 to 86: These derivatives of plant origin may represent alternative and less toxic treatments against …and the sentence that follows is incomplete, Lines 86 to 88.
Line 88: remove “In this sense”
Lines 88 to 91: “…..to describe the chemical profile of P. salutare leaf essential oil, and the influence of seasonal variation on its composition, antifungal activity and potency to inhibit the morphogenetic switch in Candida species.

In Material & Methods:
Lines 100-103: the sentence is missing a verb

In section Culture media and inocula: cells can be grown ON Sabouraud Dextrose agar, or IN Sabouraud dextrose liquid broth, but they cannot be grown IN Sabouraud Agar.

In Section 2.3.2: this section needs re-written as important clues are missing. The final cell density in the assay has to be indicated, the final concentration of DMSO in each wells must be indicated. Was DMSO used at 0.5% final, 1%, etc?. The way the authors chose to have their cells in eppendorfs or other recipients is not relevant here. Final concentrations, final dilutions, which temperature was used, for how long, etc. Is it IN sabouraud dextrose liquid medium? Or is it ON sabouraud agar?
Line 156: what is CDD?
In addition, typically one starts with measuring MIC50 and not MFC50 as it indicates growth inhibition as a first step. Whether a compound is cidal of static is determined by a different type of assay (refer to killing assays).

In section 2.3.3: This method is atypical. CFU counts by mean of killing assay over time is typically used for this kind of determination. Check killing assay for Candida species in published work.

In section 2.4:
What is CSD? What is the final cell density? What is the final fluconazole concentration? What is the natural product? Final concentration of DMSO? What temperature? For how long?
Use simpler writing to indicate important clues on how the assay was ran so someone else can reproduce it.

In section 2.5:
What is SEC? the first sentence is missing a verb. What is the final concentration of DMSO? What is the medium used? What is the hyphal inducer?

In Results:
The first sentence is incomplete. What about the month of August?
In Figure 1: the legends are a little too small to read. The essential oil samples were combined or used in combination (to adapt in legend).
Line 239: Do the authors mean the MFC of the essential oil samples themselves or in combination with fluconazole (which seems to be described from Line 254)? What do the author mean by Fluconazole modulation? Same line 254: what is a potential modulatory action? The authors presumably suspected and tested the activity of the essential oil samples in combination with the commonly used antifungal therapeutic agent fluconazole.
The essential oil samples are seemingly non-cidal to the Candida species tested. Is it then still appropriate to mention the “fungicidal” concentration? The inhibitory concentration seems more suited here, hence the suggestion to adapt the method to an MIC test rather than MFC.
In the experiment where the essential oil samples were combined to fluconazole: was the viability of the cells tested? Fluconazole inhibits growth of candida but does not kill. Was any of the combination cidal to the cells? The authors suggest a synergism between the plant-derived samples and the azole compound: do they render fluconazole cidal?
Could the authors refer to published data on how to preform drug combination assays? here is an example that may help: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3067183/
These assays have been fairly well established and standardized procedures are available. It may help in the interpretation of the data and in comparative analyses.

In Figure 2: are the indicated concentrations correct? Is it 8.192 ug/ml or 8.192 mg.ml? same for the bottom right figure.
The images are clear. However, it is difficult to interpret as it is not clear what the hyphal inducer was? Was it high temperature? Fluconazole typically blocks hyphal growth but does not block germ tube formation in liquid cultures at least. Typically, a good inducer is serum, but I understand that the authors kept the same medium as their drug assays for comparative reasons. However, shouldn’t the highest concentration inhibit growth itself? Could other hyphal inducing conditions bet tested to make final conclusions. Lines 263-266 are very general, yet they can only be made in that particular growth condition.


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 SEASONALITY ON CHEMICAL PROFILE AND ANTIFUNGAL ACTIVITY OF ESSENTIAL OIL ISOLATED FROM LEAVES PSIDIUM SALUTARE (KUNTH) O. BERG
Review round: 2
Reviewer: 1

Output:
Basic reporting: the manuscript is in the journal's scope and meets the standards of the journal
Experimental design: All methods and techniques were sound and fully respected
Validity of the findings: the results are original and are highlighted soundly.
Additional comments: All remarks made previously were taken into account


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 SEASONALITY ON CHEMICAL PROFILE AND ANTIFUNGAL ACTIVITY OF ESSENTIAL OIL ISOLATED FROM LEAVES PSIDIUM SALUTARE (KUNTH) O. BERG
Review round: 2
Reviewer: 2

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: The manuscript has been revised according to most comments made. Here are some minor comments, which are mainly typos:

In the abstract:
line.53: antifungal activity against three Candida species and that it can act in synergy with fluconazole would be clearer…please remove “would be cleare” which was part of my comment but should not be part of the sentence.

In intro:
line.100: is the first study to describe .to describe the chemical….please correct the typo here

In methods:
Line 111: It presents a Table 1 and receipt of an average of 1.043 mm (mm) of rainfall per year (FUNCEME, 2016), where they concentrate between January and May with a dry period that lasts between….it presents a Table 1 does not sound correct: adjust that whole sentence.

Line 122: Fresh leaves of the species Psidum salutare were collected from three individuals…..do you mean that three people collected the leafes? It does not seem like it is an information that is needed in method. Please adjust this sentence

Line 170: The antifungal fluconazole (from Sigma - F8929 ≥ 98% (HPLC), powder) it was diluted in sterile water (16.384 μg/mL), and oil were previously diluted in dimethyl sulfoxide (DMSO - Dynamic) and sterile distilled water was added in order to obtain the desired concentration for the tests (16384 μg/mL) both oil and fluconazole were posteriorly microdiluted in Sabouraud Dextrose Broth (SDB) medium in a serial concentration manner ranging from 8192 to 8 μg/mL in 96-well plates…..this sentence is a little too long and there are several typos in it.
Line 177: the fact that DMSO concentration was not constant through out the experiments may affect the results as DMSO alone at high concentration can affect cell viability. Did the authors test the effect of 5% DMSO on Candida cells in the same conditions as the ones used for the MIC assay for example?

Table 3 Title: “Modulation of the essential oil of salutare on Candida (ug/ml) e IC50 (ug.ml)”. What is the meaning of “modulation” here? Do you mean the effect? Add P. salutare, and not salutare. And is it IC50 and not e IC50?


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: HETEROLOGOUS EXPRESSION OF A GLYOXALASE I GENE FROM SUGARCANE CONFERS TOLERANCE TO SEVERAL ENVIRONMENTAL STRESSES IN BACTERIA
Review round: 1
Reviewer: 1

Output:
Basic reporting: Literature reporting was not sufficient.
Experimental design: fine.
Validity of the findings: It could be further validated and improved.
Additional comments: The manuscript titled ‘A novel glyoxalase I gene from sugarcane plays an important role in response to environmental stresses’ by Wu et al provided detailed characterization of one sugarcane glyoxalase I member. The manuscript needs further modification to publish.
1.	Firstly, the review was very back dated. The authors did not add any reference after 2013. While after 2013, there are a lot of other reports on glyoxalase pathway from rice, Arabidopsis, soybean and medicago plant. So try to incorporate the recent reports on glyoxalase pathway.
2.	The result and discussion are very short. The authors could elaborate their findings and could compare with available information from other organisms.
3.	The figures are not organized and seem to be scattered. Please concise the figures and try to reduce figure number.
4.	Moreover, the authors have purified the recombinant protein in hand, but they not perform enzyme kinetics for the enzyme. They should perform enzyme kinetics of ScGlo1 and compare with previously reported members.


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: HETEROLOGOUS EXPRESSION OF A GLYOXALASE I GENE FROM SUGARCANE CONFERS TOLERANCE TO SEVERAL ENVIRONMENTAL STRESSES IN BACTERIA
Review round: 1
Reviewer: 2

Output:
Basic reporting: The manuscript reports characterization of a glyoxalase I enzymes from Sugarcane with potential role in stress response.
At some places, text is difficult to understand. For instance, lines 154, 162-163, 186, 196-198 and 234. Authors should correct grammatical errors and language.

In abstract as well as in introduction (line 94) and discussion (line 309), authors state that SA, MeJA, PEG and ABA treatments were given to mimic simulated pathogen stress conditions. This seems to be incorrect as PEG treatment is given to induce osmotic stress. And these hormones are not only involved in biotic stress but are important for abiotic stress response as well. Authors should be careful with their reporting and are advised to revise the text.
Importantly, authors refer to glyoxalase I from Sugar cane as ScGLo1 but the same name has been used for glyoxalases from S. cerevisiae also. Pl revise the nomenclature to avoid confusions.
Title should be modified to clearly indicate that response to stress has been tested in bacterial systems and not plants. In fact, title can be revised to “Heterologous expression of a Glyoxalase I gene from Sugarcane confers tolerance to salinity and heavy metal stresses in bacteria”.
Line 41: References should be cited in chronological order or cited just after the relevant context. Philips and Thornalley 1993 can be cited after the phrase “MG is a cytotoxic by-product produced mainly from triose-phosphate step’.
Line 45-48: The statement is incorrect. Glyoxalase system does not produces more free GSH, it uses and then recycles one molecule per cycle.
Lines 49-54: Authors have cited glyoxalases characterized from various species but have not mentioned recently characterized literature such as by Kaur et al. 2016, Plant J (rice), Mustafiz et al. 2014 Plant J (rice) Jain et al. 2016, Plos one (Arabidopsis).
Experimental design: Methods
Line 133: What does (+1) represents? Pl elaborate
Line 155: Why have ethanol and Tween 20 been used with 100 uM MeJA and 5 mM SA treatment?
Line 165: Why references have been cited after Table 1? Is it that sequences of primers have been retrieved from these references? If so, then include as footnotes in Table 1 otherwise remove these references.
Line 166: G-QF/G-QR represent primers of which genes?
Line 172: Correct to 2-ddct.
Line 209-210. Cite reference of transformation protocol.

Results
Line 230: Change the title to Sequence analysis of ScGLO1 (Note: revise the name of ScGlo1)
Figure 4: Why have authors shown growth curve in response to only NaCl and ZnSO4 and not CuCl2 and CdCl2. All should be shown. Y axis should be changed to “Absorbance (600 nm)” as it is not a direct measure of cell growth.
Figure 6: Authors have shown GLYI expression in response to different elicitors in two panels. PEG treated samples should be shown along with NaCl, CuCl2 and CdCl2 and not with SA, MeJA or ABA. The lower panel can be said to represent expression in response to hormonal treatments and upper panel representing abiotic stress treatments. Again, here now why have authors not shown expression upon ZnSO4 treatment. Same treatments should have been used in all experiments.
Line 271-276: Again, why have activities been demonstrated only in response to CdCl2 and NaCl and not ZnSO4 and CuCl2?
Also, what has been taken as 100% here, i.e. in what respect have the activities been calculated. What is the reference here?
Importantly, how can authors say that the activity they are measuring is specific to ScGlo1. Plants contain multiple glyoxalases. And thus, sugarcane is also possessing multiple glyoxalases. While measuring activity, we cannot distinguish between activities of specific glyoxalases. Authors should, thus, revise the section and state that they measured ‘specific Glyoxalase I activity’ in plant extracts.
Figure 8: Revise the caption to “Determination of Subcellular localization of ScGloI”. Details can follow after the caption.
Validity of the findings: Line 292: How can the presence of two glyoxalase domains confirm that it is an active glyoxalase I enzyme. The word “genuine” is incorrect and should be avoided. To determine whether it is a functional glyoxalase, authors should have checked the activity of purified glyoxalase I enzyme which will confirm that the gene which they transformed in E. coli is functional in nature and that tolerance is due to over-production of the corresponding protein. This will also determine whether this ScGlo1 is Ni or Zn dependent enzyme.
Lines 308-316: Mere upregulation of transcript levels does not mean that the gene has role in relieving stress. Authors should rephrase these statements.
Since, authors have characterized a two-domain glyoxalase I enzyme, they should cite reference of its recently characterized rice homolog OsGLYI-11.2 (Mustafiz et al. 2014, Plant J) indicating similarity between the two.
Line 329: Rephrase the sentence. Can’t conclusively state from the results that ScGlo1 plays an important role in sugarcane's response to salt stress as no transgenics have been raised and validated under salt stress here.
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: HETEROLOGOUS EXPRESSION OF A GLYOXALASE I GENE FROM SUGARCANE CONFERS TOLERANCE TO SEVERAL ENVIRONMENTAL STRESSES IN BACTERIA
Review round: 2
Reviewer: 1

Output:
Basic reporting: Text still has many language mistakes. Authors should get the manuscript checked by a native English speaker.
References have been now cited but not discussed properly. Moreover, if more than one reference has been cited after a statement, these have not been arranged in the chronological order.
Experimental design: Discussed in the next section
Validity of the findings: Regarding response to previous queries:
Query: Figure 8: Revise the caption to “Determination of Subcellular localization of ScGloI”. Details can follow after the caption
Authors haven’t yet revised the figure caption.
Query: While measuring activity, we cannot distinguish between activities of specific glyoxalases. Authors should, thus, revise the section and state that they measured ‘specific Glyoxalase I activity’ in plant extracts.

Authors haven’t understood my query properly. What I meant was that the GLYI activity being tested here is contributed by all glyoxalase I enzymes of sugarcane (as sugarcane like other plants is having multiple glyoxalase I enzymes), how can authors say that they have specifically measured SoGLYI activity. This GLYI activity is contributed by other glyoxalase I enzymes as well which are present in sugarcane. So authors should state the activity as Specific GLYI activity (not referring to any particular enzyme i.e. SoGLYI in this case).

New queries:
Figure 5A: Describes expression in different tissues and not different stresses. Pl correct in figure legend.
Figure 5A, B, C: Authors have calculated relative expression in respect to which sample as control. What is the reference here? Pl specify.
Figure 5D: change x axis from treat time to treatment time. Secondly, “0” of x and y axis should coincide. Also, authors have not shown empty vector control’s activity in response to different heavy metal treatments which must be shown as it would serve as a control for comparison. Authors should include details about whether stressors were added right from the time zero i.e. start of the experiment or later.
Figure 6: Authors should have used DAPI dye as a nuclear marker as authors state that SoGLYI is getting localized to both nucleus and cytosol.
Lines 257-263: Since information about accession IDs of glyoxalases has been mentioned in figure 1 legend, authors need not repeat this information here. Can be omitted.
Line 275:18.3 kDa is not merely His tag but instead Trx-His-S-tag. Authors should correct it.
Line 299: Increased expression under stresses doesn’t mean that SoGloI confers stress tolerance. Authors should revise or omit this sentence.
Line 304: Authors have mentioned heavy metal treatments as stresses everywhere in the text, as also written in this particular sentence. Authors should refer these as treatments and not stresses everywhere in the text. Though these treatments confer stress on plants but there are no terms as CuCl2 stress etc.
Discussion: many repetitions are there in discussion section like in Lines 324-333 and 334-340. These have already been mentioned in the introduction section. Pl revise. Also carefully, check the entire section for other possible repetitions.

Line 365: ABA mainly mimics abiotic stress, how can then authors say that it stimulates specifically pathogenic stresses. Pl revise.
Line 367: Again, ABA is a treatment and not stress. Pl revise
Line 387-389: What do authors mean from “hosts’ tolerance to Zn and Zn accumulation”. How is Zn and Zn accumulation separate? Pl rephrase.
Line 407-408: Statement is incorrect. Glyoxalase system doesn’t recycle GSH with MG to form hemithioacetal. Instead, GSH spontaneously forms a hemithioacetal adduct with MG, which is the actual substrate for glyoxalase I enzyme. GSH is used in the first reaction catalyzed by glyoxalase I and recycled back in the second step catalyzed by glyoxalase II.
Figure 2: Change the caption to “Heterologous expression of recombinant SoGloI in E. coli cells”. Following the caption, details can be mentioned.
Figure 4: Change caption to “Growth assessment of SoGloI overexpressing E. coli cells to salt and heavy metal treatments. Following which, details should be mentioned about the time stressors were added and their concentrations.
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: HIGH-LEVEL EXPRESSION AND MOLECULAR CHARACTERIZATION OF A RECOMBINANT PROLIDASE FROM ESCHERICHIA COLI NOVABLUE
Review round: 1
Reviewer: 1

Output:
Basic reporting: The authors used clear language to describe their design rationales. Figures are well labeled and described, the main text is well referenced.
Experimental design: Methods are described in detail.
Validity of the findings: Findings are valid with robust data presentation.
Additional comments: The article describes a protocol to purify prodilase from E.coli. After purification, the authors employed various biochemical and biophysical techniques to interrogate the properties of the purified prodilase, suggesting promising progress toward the rational adjustment of reaction conditions for prodilase -catalyzed reactions.

The authors used clear language to describe their design rationales. Figures are well labeled and described, the main text is well referenced. Methods are described in detail and the findings are valid with robust data presentation.

This is a nice biophysical study on a protein of interest, and I support its publication if the following minor issues could be adjusted:

1)	The abstract needs to be significantly changed. Generally speaking the authors should start with a problem and state rationales why it is important. The current version of abstract simply stated what the authors did, without stating the significance of their work.
2)	In the caption for Fig. 2A, the authors should give more information regarding “purified”. Was it after Ni-NTA or after gel filtration? Same goes to 2C.
3)	More information should be given to the figure captions in Figs. 3 through 8. In general readers should be able to understand an article just by studying the figures and reading their captions.
4)	In lines 304-305, the authors state that “It is clear that the transition point at a heating rate of 0.5 C/min occurred at lower temperature than those of 1 and 2 C/min”. It is however not clear why the authors made this argument; the data appears to be too noisy to draw this conclusion. Please comment.
5)	In line 309, “down to 20C”, I suppose the temperature was dropped from 80C? The authors should make the starting temperature clearer.
6)	A calculationpeople typically perform from GdnHCl unfolding experimental data is the delta_G of folding of a protein. If the authors could make this calculation it will give readers a direct visualization on the stability of this prodilase compared to other proteins.
7)	Axis labels are too small to see in Fig 7. Please modify this figure to make it more readable.


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: HIGH-LEVEL EXPRESSION AND MOLECULAR CHARACTERIZATION OF A RECOMBINANT PROLIDASE FROM ESCHERICHIA COLI NOVABLUE
Review round: 1
Reviewer: 2

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: This manuscript by Wang et al. demonstrated the overexpression and molecular characterization of a recombinant prolidase from E. coli NovaBlue (EcPepQ). The authors also determined the biophysical and biochemical properties of EcPepQ, which is interesting and informative. However, I recommend the authors to address the following issues to improve the quality of manuscript.

1. The authors claimed that the His-tagged enzyme could be purified nearly 3.0-fold with a yield of 68.4% by single-step affinity purification (lines 255-256). Please describe how the authors calculated the values ‘3.0-fold’ and ‘68.4%’.

2. For the clarity of the figure 2B, it would be better to mark the molecular weights of four reference proteins instead of the numbers (e.g., ‘670 kDa’ instead of ‘1’, ‘158 kDa’ instead of ‘2’, etc.).

3. In figure 3, effects of temperature and pH on activity and stability of EcPepQ were presented. However, the related sentences were a little confused as follows; ‘the maximum activity for EcPepQ was at 60 C (line 265)’, ‘The recombinant enzyme was fully active at temperatures below 40 C (lines 268-269).’ Please explain the difference between relative activity (fig. 3a, b) and residual activity (fig. 3c, d). In addition, for the clarity of the manuscript, I recommend that those paragraphs should be polished up.

4. In figure 7, please indicate the unit of x axis.

5. The legends of figures 3-8 are too simple to fully understand the figures. I recommend that the authors should describe the data in more detail.

6. In figure 8, the authors measured CD and fluorescence spectra of EcPepQ in the presence of various co-solvents with concentrations resulting in >90% decreases in the prolidase activity. Interestingly, although all co-solvents decrease the prolidase activity of EcPepQ with similar levels, several solvents, including acetonitrile, methanol, and ethanol, significantly affected the secondary structure of EcPepQ, but other co-solvents (e.g., glycerol, ethylene glycol, THF, isopropanol) little affected the EcPepQ structure. Please discuss the different effects of those co-solvents on the structure of EcPepQ.

7. Typos
(line 100) (Weaver et al., 2004) should be changed to (Weaver et al., 2014).
(line 190) ‘250 nm’ should be changed to ‘260 nm’.
(lines 356, 366) ‘far-UV spectra’ should be changed to ‘far-UV CD spectra’.

(legend of figure 2) ‘B lue Dextran’ should be changed to ‘Blue Dextran’.
(legend of figure 3) ‘onactivity’ should be changed to ‘on activity’.
(legend of figure 4) ‘ofdivalent’ should be changed to ‘of divalent’.
(legend of figure 4) ‘thecatalytic’ should be changed to ‘the catalytic’.
(legend of figure 6) ‘thecorresponding’ should be changed to ‘the corresponding’.
(legend of figure 7) ‘miscibleorganic’ should be changed to ‘miscible organic’.
(legend of figure 8) ‘tryptophanfluorescence’ should be changed to ‘tryptophan fluorescence’.