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: ANTIBACTERIAL IMMUNE FUNCTIONS OF SUBADULTS AND ADULTS IN A SEMELPAROUS SPIDER
Review round: 1
Reviewer: 2

Output:
Basic reporting: 1.	BASIC REPORTING
a.	Clear, unambiguous, professional English language used throughout.
i.	The language throughout the manuscript requires improvement and would benefit from the following suggested revisions:
1.	Line 15: Phrase “were proposed” is used. Proposed by whom? By the authors, or someone else? If the latter, this needs a citation. If the former, the sentence should read “We propose” or “We proposed.” Unless this supposed to read “are proposed” as in, in general, this is what is commonly proposed in the literature. This would also require a citation.
2.	Line 17: Change “stage-level” to “stage-specific.”
3.	Line 17: Is the phrase “juveniles and subadults” redundant, since these are synonyms? If so, I would remove “juveniles” since subadults is used throughout.
4.	Lines 17-19: Revise wording. For example, phrases like “juveniles and subadults are yet unable to reproduce” could be stated more clearly by removing “yet,” while the word “certainly” could be removed from line 19.
5.	Lines 19-22: This sentence is saying several things and should be split into at least two sentences.
6.	Lines 61-63: Add the word “and” before “hence cannot trade off…”
7.	Line 81: Revise the language of the phrase “assay is based on that…” to read something like “assay is based on the fact that…”
8.	Line 92-93: The hemocoel encompasses the entire open body cavity, yet this sentence makes it seem as if it is restricted only to the abdomen. Is that really the case? Wouldn’t the hemocoel also extend into the cephalothorax in this spider? Or is that a separate body cavity?
9.	Line 103: Revise the phrase “with the used immune parameters” to something like “with the immune parameters we used” or “with the immune parameters we utilized.”
10.	Line 106: Use the word “Henceforth” rather than “Henceforward”?
11.	Line 106: Add “refer to” after the phrase “we use the term ‘subadult’ to” and add “to” after the phrase “one moult prior…”
12.	Line 108: Remove the word “have” after “We” at the start of the sentence. Similar corrections can be made throughout this section and elsewhere.
13.	Line 114: The phrase “ad libitum” should be italicized. Further, as it is used here the phrase describes a property of the water and food, when it should be a descriptor of the spiderlings’ feeding behavior (i.e., “the spiderlings fed on … ad libitum”). Please modify.
14.	Line 116: Remove the word “in” before the phrase “every two days…”
15.	Line 122: Remove the comma after “collected subadult…”
16.	Line 123: Remove the comma after “in 2017…”
17.	Line 129: The word “latesummer” should be “late summer.”
18.	Line 136: The phrase “killed in” should be “killed at” unless you specify the manner in which they were killed at -20˚C.
19.	Line 137: Remove “a” from the phrase “because a the heart…”
20.	Line 144: Remove the comma after “ice…”
21.	Line 145: Should say “at 4˚C” not “in 4˚C…”
22.	Line 146: “20-20 μl” a typo? If so, please correct.
23.	Lines 165-168: Revise this sentence, as the current wording is difficult to follow.
24.	Line 277: The phrase “being sad” should be “being said…”
25.	In general, the “Developmental stage differences in immune markers” and “Association between reproductive status, fecundity and immune markers” subsections of the materials and methods section could be more concisely.
26.	Line 284: Add “a” before the phrase “Chi-squared test…”
27.	Line 401: Revise “quasi showing a” phrase to “showing a quasi…”
28.	Line 473: Add “the fact” before the phrase “that adults…”
29.	Line 474: Remove the comma after “future reproduction” and add “and” in its place. Also, change “maximizing” to “maximal…”
b.	Intro & background to show context. Literature well referenced & relevant.
i.	Lines 72-78: The text introduces the study organism but does not provide its common name or other appropriate details about its life history (although some of this is mentioned in the abstract). So far, the reader knows that it is a semelparous species with adult and subadult stages, but a sentence or two here explaining the life history in greater detail would be helpful.
ii.	The introduction says much about immune trade-offs in adults due to investment in somatic maintenance and reproduction, but little is said of how trade-offs with reproduction and fecundity in females might differ with similar trade-offs observed in males. Consequently, the rationale for assessing fitness in both virgin and mated females as well as in males is not explicitly delineated in the text. Please explain the rationale for looking into both sexes (as well as both virgin and mated females) and how you might expect adult immunity to differ (if at all) between them.
c.	Figures are relevant, high quality, well labelled & described.
i.	Fig. 1: Panel (b) subadult and panel (d) adult box plots do not show the minus SD error bars. Level of significance for a single asterisk should also be mentioned in figure legend.
ii.	Fig. 2: Panel (a) and (c) box plots are missing what I assume to be minus SD error bars (the legend should specify what the error bars represent). Level of significance for a single asterisk should also be mentioned in figure legend. The panel (d) mated box plot appears to be missing and shows what appear to be individual activities (circles) unlike the other graphs. Please correct for consistency with the other panels.
iii.	Fig. 2 panel (a) shows a significant difference between virgin and mated spring females, but the box plots look nearly identical. However, the text states that “mated females showed significantly lower levels of bacterial growth inhibition (Fig. 2a-b, Table 2)” and Table 2 has a p value of P=0.030. Please double check that this is accurate and that the statistical test used is both appropriate and not yielding a false positive here.
Experimental design: 2.	EXPERIMENTAL DESIGN
a.	Research question well deﬁned, relevant & meaningful. It is stated how the research ﬁlls an identiﬁed knowledge gap.
i.	Line 106: Please explain the rationale for why penultimate subadults were chosen instead of earlier subadults.
b.	Rigorous investigation performed to a high technical & ethical standard.
i.	Can you further explain the rationale behind choosing two assays, a bacterial growth inhibition assay and bacterial cell wall lytic activity assay, that measure partially overlapping humoral immune parameters? As you stated earlier in the introduction with respect to the bacterial growth inhibition assay “These molecules [AMPs] mainly attack the integrity of the cell wall and cell membrane of the invading microbes.” However, as you note in the discussion (lines 458-460), lysozyme activity is also at play here. Likewise, the cell wall lytic activity assay may also be partially a result of AMP lytic activity, and not lysozymes alone, so you may need to qualify this somewhere in the text as well. In short, I believe your manuscript would be improved by providing your readers a stronger justification for using of both assays here and how they provide different information despite similar modes of action and partially overlapping activity. Distinguishing the two further should only require a few additional sentences in your introduction and/or discussion sections.
Validity of the findings: 3.	VALIDITY OF THE FINDINGS
a.	Data is robust, statistically sound, & controlled.
i.	Lines 119-121: This is a rather low sample size (n=38 overall) to subdivide into 4 groups. There were only 4 male adults from the 2016 collection, for example. Please explain how this sample size was sufficient given the variation within your groups.
ii.	At first glance, it appears somewhat concerning that of the 68 mated spring females, only 25 cocoons yielded fecundity data, whereas 28/28 of the mated late summer female cocoons yielded data. Was embryo viability and parasitism much higher in the spring compared to the late summer? If so, how would this potentially confounding factor be accounted for in your analyses, particularly those that compare data across seasons (for example, see the comparison made in lines 304-306)?
iii.	Lines 289-291: The sentence on the Bayesian analysis seems somewhat out of place here and its biological significance is not made clear to the readers.
iv.	Lines 296-299: Again, the biological significance of these statistical tests needs to be briefly stated here.
v.	Lines 314-319: Please explain the biological significance of these statistical measures.
b.	Conclusions are well stated, linked to original research question & limited to supporting results.
i.	Lines 340-344 state “On the other hand, mature individuals have to invest resources into gonad development and gamete production. Consequently, a higher proportion of the acquired energy and nutrients can be assigned to somatic tissues and to elements of somatic maintenance in subadults, while these limited resources will be partitioned into reproductive tissues and gamete production as well in adults.” Were the adults used in these experiments all virgins? If so, were the cell wall lytic activity results surprising, or would you expect lower immunity in adult females regardless of mating status? Also, when do females produce cocoons? Would you expect immune trade offs before, or only after process? And, in males, you saw similar declines in lytic activity as observed in females, but presumably males are investing fewer resources than females to gonad/gamete production. Can you explain why you saw similar trends in both sexes with the cell wall lytic activity assay? Apologies if I overlooked this.
ii.	Line 348: Could the lack of bacterial growth inhibition changes observed here be due to the bacterial strain used? This may explain why the lytic assay, which involved a different bacterium, did show ontogenetic differences.
iii.	Line 352: You may need a citation for the pollen beetle work here too and not just after the following sentence on line 356.
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: ANTIBACTERIAL IMMUNE FUNCTIONS OF SUBADULTS AND ADULTS IN A SEMELPAROUS SPIDER
Review round: 1
Reviewer: 3

Output:
Basic reporting: The manuscript meets the standards:
-The manuscript is written in clear and unambiguous, professional English.
- The manuscript present correct article structure, figures, tables.
- The manuscript is self-contained with relevant results to hypotheses.

I propose changes in the introduction to cover the field of knowledge in a not so general way, to express the main theories differently and rethink the basal hypotheses of the work.
Experimental design: The manuscript meets the standards:
-The research is within the 'Aims and Scope' of the journal.
- The research question is well defined, relevant & meaningful.
- The manuscript has been conducted rigorously and to a high technical standard, and in conformity with the prevailing ethical standards in the field.
- The methods described in the manuscript are sufficient detail & information to replicate.

It is not very well expressed how research will fill an identified knowledge gap, and there is little information about the taxonomic group evaluated as well as citations in related taxonomic groups.

The methodology of the manuscript is very thorough, the statistical analysis has been performed rigorously, and the experimental design is correct. I only suggest taking into account some considerations for the statistical model that evaluates fecundity in function of antibacterial immune functions.
Validity of the findings: The manuscript meets the standards:
-The data is robust, statistically sound, and controlled.

Also, although the manuscript gives good and stimulating reading, the discussion of the results obtained and the conclusions drawn by the authors must be re-examined in a new version of the manuscript.
Additional comments: Detailed remarks (numbers refer to lines in the manuscript):

37: ‘Immunocompetence is a critically important element of somatic maintenance’ Why? How? I think a broad definition of immunocompetence is missing.
48: Add: - Rachel Gilbert, Richard D. Karp, George W. Uetz, Effects of juvenile infection on adult immunity and secondary sexual characters in a wolf spider, Behavioral Ecology, Volume 27, Issue 3, May-June 2016, Pages 946-954 , [url]Https://doi.org/10.1093/beheco/arv241[url]
63-64: It's a rule? I believe that this approach is somewhat reductionist, and will depend on multiple factors (ecological, sexual, time of life of the species, mating system, sperm production, parental care, etc.). There is no citation on this topic in all this paragraph, it would be good to mention literature where this trade-off has been seen. The discussion on the differences in allocation of resources (somatic vs. reproductive) between species semelparous vs. Iteroparous is fine, but it can not be a central point or central hypothesis of the work since you are not comparing species with these reproductive characteristics. I think it is merely speculative to attribute the differences in immunological parameters observed between stages to this. I think it can be discussed and proposed speculatively, and perhaps discuss possible future comparative works among species of the genus that possess other reproductive tactics.
70: Will adults invest less in immunocompetence than juveniles? How would that be? Immunocompetence is the resource trade-off that individuals face between reproduction and other activities (as immunological traits), but in juvenils there would be no trade-off because there is no reproductive investment. Perhaps adults invest less in the production and maintenance of immunological parameters than in juveniles, but not if it is correct to talk about investment in differentiated immunocompetence between stages. Please review this concept throughout the entire work.
72: Why is Pardosa agrestis a good model for this study? What is known about this species that may be of interest for this study? I see that the Ph.D. thesis of Kiss, 2003 is cited but no other work.
75-76: ‘This semelparous spider species has, by definition, only one, short and well defined reproductive event during its lifetime’ What does that mean? Is it a monogamous species? Or is it that these species only have one reproductive season? Is anything known about the sperm production of this species or closely related species?.
83: Add a citation.
98-101: Sentence with repetitions, rewrite please.
111-112: Please provide the geographic coordinates for the collection area. The authors collected spiders in two nearby habitats, and this variable was not included in the models as a random effect, are the environments sufficiently similar? Could there be differences at the microhabitat level? Drapela et al., 2011 found that the environment can influence several reproductive characteristics of P. agrestis. I think it would be interesting to take this into account.
Drapela, T., Frank, T., Heer, X., Moser, D., & Zaller, J. G. (2011). Landscape structure affects activity density, body size and fecundity of Pardosa wolf spiders (Araneae: Lycosidae) in winter oilseed rape. European Journal of Entomology, 108(4).
122: The second collection was in the spring of 2017 and the third collection in the summer of 2017? What does this mean biologically for the species? Here I miss some basic information if it exists about when is the reproductive season, or about the lifetime duration of two sexes?
127: Out of topic but interesting to keep in mind: could you see what characteristics the parasitized cocoons had: were they smaller?  Were they less heavy? Did they have any special characteristics? Then see the females that produced these cocoons, if they had lower body condition for example? And finally, correlate the immunological parameters of the females with the occurrence of parasitism. Perhaps females with lower immunological values are more likely to be parasitized. It would also be interesting to see if there are differences between seasons or habitats in the occurrence of parasitism.
146: ‘20-20 μl from the supernatant’, clarify the quantity.
152: Add a citation with previous information about these periods of time have no consequences on the antimicrobial activity that will be measured.
243: Is there another work that has previously used this approach?.
256: The variable ‘season of collection’ was added as a variable confounding in this case? (as in the next analysis)? Or did you have any particular prediction or interest in the differences between seasons?.
257: The habitat variable (collection site) was not included in the models as a random effect, are the environments sufficiently similar? Could there be differences at the microhabitat level?.
264: I am not very familiar with Bayesian statistics although I see that there could be some problems with this model. The fecundity variable is represented by the number of offspring of each mother (only named at the end of the introduction) and is probably a non-parametric variable. Is there any Bayesian approach for data of this type? (e.g., Müller & Quintana, 2004; Hjort et al., 2010). Did you check the collinearity of the predictor variables (bacterial growth inhibition and cell wall lytic activity)? In the second model, it was found that the season had an effect on the cell wall lytic activity and the bacterial growth inhibition (304, 307-308). Now you are setting as predictor variables: 'season' and the antibacterial immune functions with which previously season had been related. There is a high risk of collinearity between the predictor variables, it is necessary to check this previously (perhaps controlling the variance Inflation factor). In the discussion, it is expressed that: ‘the number of observations in which we could regress fecundity on non-zero values of cell wall lytic activity was quite low’, which can also be clearly seen in figure 3b. Is there no way to deal with data that has many zeros? (something analogous to zero-inflated models) or make a model without the non-zero observations. Finally, according to Drapela et al., 2011 clutch size was positively correlated with female body size in P. agrestis. Do not you think it is necessary to control the number of offspring by the size of the female or add the condition as a predictor variable?.
Müller, P., & Quintana, F. A. (2004). Nonparametric Bayesian data analysis. Statistical science, 95-110.
Hjort, N.L., Holmes, C., Müller, P., & Walker, S.G. (Eds.). (2010). Bayesian nonparametrics (Vol. 28). Cambridge University Press.
276: ‘That being sad we dediced ..’ Rewrite.
335: See review of lines 63-64. The fact that juveniles invest more in somatic maintenance vs. adults could be because they should not allocate resources in reproduction as adults. However, attributing the differences found between age stages to which the species is semelparous is not possible with the experimental design proposed (you should compare adults vs. juveniles in species with the two reproductive tactics).
340: "On the other hand ..." Remove.
340-344:  Sentence with repetitions, rewrite please.
347: Male and female gametes have different costs, so how do you explain that no differences were found in the immunological parameters between sexes?
351: Too much information is given about the screened APTs in the work of Vogel et al., 2014. Compare in a more concise and meaningful way.
360: The experimental design is not to conclude on the costs of producing/storing lysozymes, please rewrite this phrase in a more speculative way. The example of Vogel et al., 2014 is not correct for this part of the discussion (they do not find an 'energetic conflict' but differences in the composition of lysozymes between stages).
374-381: The explanation of the differences found between the seasons is not clear to me. What does it mean that the females of these seasons belong to two separate generation cohorts? There is lacking basic information of the species.
Check: Samu, F., Németh, J., Tóth, F., Szita, É., Kiss, B., & Szinetár, C. (1998). Are two cohorts responsible for bimodal life history pattern in the wolf spider Pardosa agrestis in Hungary. In Proceedings 17th European Colloquium of Arachnology (pp. 215-221).
381: ‘segment of the spider's life history’ Rewrite
388-390  See review of line 264.
396: Add more recent literature.
397: See review of lines 63-64, 355. Please note that the experimental design does not support these ideas. Then I have a problem in the approach that the authors give to the ‘terminal investment’. In the work of Creighton et al., 2009 there is no mention of reproductive strategies of species semelparous vs. Iteroparous, but of the strategy of each individual for multiple matings and trade-offs with future matings (i.e., the shift in allocation towards current reproduction with increasing age). Iteroparous or semelparous species can make a terminal investment if they invest more resources and energy in their last mating compared to the previous ones, it is an intra-individual approach that has to do with the age of the individual and the senescence. Please review the concept and better explain its use for this case.
Check: Velando, A., H. Drummond, and R. Torres. 2006. Senescent birds redouble reproductive effort when ill: confirmation of the terminal investment hypothesis. Proceedings of the Royal Society B: Biological Sciences 273: 1443-1448
Bonneaud, C., Mazuc, J., Chastel, O., Westerdahl, H., & Sorci, G. (2004). Terminal investment induced by immune challenge and fitness traits associated with major histocompatibility complex in the house sparrow. Evolution, 58(12), 2823-2830.
Charlesworth, B., and J. A. Leon. 1976. The relation of reproductive effort with age. American Naturalist 110: 449-459.
Clutton-Brock, T. H. 1984. Reproductive effort and terminal investment in iteroparous animals. American Naturalist 123: 212-229.
412. Sentence with repetitions, rewrite please.
414: Add citations.
423: ‘Furthermore, in the species...’ change to ‘furthermore, in several species’.
Figure 2: Put n in capital letter to match with Figure 1. I am struck by the graph (d), why is the data structure like this for mated females in late summer? Do you have many similar data? Please explain.
Figure 3: Put n in capital letter to match with Figure 1.  See review of line 264.
Table 1: ‘For discrete predictors’, maybe better ‘for categorical predictors’.
Tables: I suggest unifying the tables (at least 1 and 2) into one.


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: ANTIBACTERIAL IMMUNE FUNCTIONS OF SUBADULTS AND ADULTS IN A SEMELPAROUS SPIDER
Review round: 2
Reviewer: 1

Output:
Basic reporting: i.	Lines 20-23: Sentence was split in two in an awkward manner. Please revise.
ii.	Line 67-68: Remove phrase “to see.”
iii.	Line 70: Remove comma before “because.”
iv.	Line 71: Add “a” before “critical impact.”
v.	Line 83-84: Consider placing the text “i.e. there are two separate adult peaks throughout the year” in parentheses.
vi.	The paragraph in lines 122-132 is a welcomed addition, but seems out of place in an introduction, as it now goes into far more detail on the specifics of the two immune assays than before. Either re-locate to the “Materials and methods” section or significantly shorten (or both).
vii.	Lines 125-126: Consider placing the text “i.e. are expected to be effective against microbes in general when released simultaneously” in parentheses.
viii.	Line 134: Delete “in the bacterial growth inhibition power and cell wall lytic activity, i.e.” If you’d like, you can add parenthetical references to each immune response mentioned in lines 135-136.
ix.	Line 215: Change “persistm" to “persist.”
x.	Line 448: Replace “Albeit” with “Although.”
xi.	Line 480: Add “a” before “stronger effect.”
xii.	Line 511: Add “the fact” before “that adults.”
xiii.	Line 515: Change phrase “no future reproductions” to “no future opportunities to reproduce” and consider removing the phrase “i.e. the first reproduction is also the last” as this phrase is redundant and the “i.e. …” pattern is overused in the revised portions of the text.
xiv.	Line 517: Change “duo” to “due.”
xv.	Line 531: Change “the same” to “similar.”
xvi.	Line 547: Change “, in physiological sense costly,” to “physiologically costly.”
xvii.	Line 554: Change “bacterial growth inhibition results from the presence of many types of peptides and proteins, which lysozyme is also a part of” to “bacterial growth inhibition also results from the presence of many other types of peptides and proteins.”
xviii.	Line 702: Change the informal “don’t” to “do not.”
xix.	Line 703: Add “and” before “therefore maximal.”
Experimental design: no comment
Validity of the findings: no comment
Additional comments: Most of the suggested edits were taken into account in the revised manuscript. When suggestions were not followed, the authors provided sufficient rationale for not doing so or for doing so in a modified manner. Clarifications on questions from the first review were satisfactory. The revised portions of the text generally read very nicely. I have provided a number of minor suggestions for revisions to improve the language in some of the revised portions of the document. If these additional changes to the text are made I recommend that the manuscript be accepted without further review.


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: ANTIBACTERIAL IMMUNE FUNCTIONS OF SUBADULTS AND ADULTS IN A SEMELPAROUS SPIDER
Review round: 2
Reviewer: 2

Output:
Basic reporting: The manuscript meets the standards:
-The manuscript is written in clear and unambiguous, professional English.
- The manuscript present correct article structure, figures, tables. The raw data is shared correctly.
- The manuscript is self-contained with relevant results to hypotheses.

I suggest covering in a better way and more completely some fields of knowledge in the intoduction (sexual dimorphism in immune response or mating relationship with immunity), to formulate more precise predictions regarding these topics.
Experimental design: The manuscript meets the standards:
-The research is within the 'Aims and Scope' of the journal.
- The research question is well defined, relevant & meaningful.
- The manuscript has been conducted rigorously and to a high technical standard, and in conformity with the prevailing ethical standards in the field.
- The methods described in the manuscript are sufficient detail & information to replicate.

The methodology of the manuscript is comprehensive, the statistical analysis has been performed rigorously, and the experimental design is correct.
Validity of the findings: The manuscript meets the standards:
-The data is robust, statistically sound, and controlled.

Conclusions are well stated, linked to original research question. However, several paragraphs should be re-ordered and re-examined in a new version of the manuscript. The addition of citations is requested in various parts of the discussion.
Additional comments: *The lines correspond to the final revised document (without tracked changes).

General comment:
It seems to me that the authors have correctly introduced, predicted and tested that individuals manage their resources in a stage-specific manner (subadults invest more in immune function and adults allocate their resources between reproduction and immune function). Probably this general phenomenon occurs in both semelparous and iteroparous species. It is possible, as the authors suggest that this trade-off between activities across ontogenetic stages is more pronounced in a semelparous species since only have one reproductive season. However, the authors are based on the fact that the species is semelparous to formulate its hypothesis and explain its results, which is incorrect since no specific design was made to test this (and it is excellent that the authors suggest it in the conclusions). Therefore, the authors should be even more careful with the approach of semelparity as an explanation of the patterns found. And in the excessive importance given to this strategy throughout the manuscript: TITLE, Abstract (21-25), Introduction (63-73), Discussion (394-397, 489-507), Conclusion (548-552).

Detailed remarks (numbers refer to lines in the manuscript):

Introduction:
67: "to be pronounced in semelparous species". Pronounced about what? The comparison is missing.
69: "no future reproductions". Better “no future reproductive seasons”.
73: "somatic maintenance" rewrite to avoid repetition.
62-76: This paragraph has some repetitions with the previous one, and has no citation.
78: "two separate adult peaks". Maybe better: two separate phenological peaks?
81-82: "sperm is then excreted and stored". I do not think that "excreted" is the best term, are you talking about sperm induction? Rewrite.
80-84: If there is no work to be cited on life history here put as personal observations of the authors.
88: "one would expect…" Better "we expect".
89: Unify concepts (e.g. 59: juvenile and subadults; 73: preadult; 89: premature).
90: "For proxies of immunocompetence…". I think the concept of immunocompetence was finally changed, since it can only be measured in adults. I suggest removing the term and writing better "For proxies of immune function" or something like that. If the term "immunocompetence" is maintained, it should be clarified that it will measure in adults (e.g. comparing virgins vs. mated, or males vs. females) and the concept must be previously defined.
110: "production of bacteria". Maybe "reproduction of bacteria?"
123-126: I understand that the axis of the manuscript is the comparison between adults and subadults. However, I notice that the introduction is extremely unbalanced in the presentation of the topics. Only in these lines the authors present some backround about the sexual dimorphism in immune response and there is no prediction about this topic. In some statistic models, sex was included as a predictor variable and in the discussion, there is a fairly extensive paragraph discussing the results. I suggest that the authors should introduce more extensively this topic. 126-130: Something similar to the previous comment. Only in these lines is the relationship between the immune response and fecundity. It seems to me that the theoretical framework on these points should be expanded, since the models include the reproductive state, there is a model with the fecundity as response variable, and another where the measurability of immune function is related to fecundity, and then in the discussion this topic is addressed in an extensive way.

Materials and methods:
197: Order the citations chronologically.
200: "persistm" correct typo.
352-353, 378-379: The interactions are not significant, but are they removed of the final models? The coefficients and estimates given are of the model without including the interactions?. If the interactions are excluded from the models, how are they excluded, with some particular method?
371-374: "the interaction terms were excluded from the final model". How is it excluded, with some particular method?
360-365: the values of Coefficients and PMCMC of the contrasts between virgin and mated females should coincide with the contrasts presented in table 2? Why do not they coincide?

Discussion:
410: "In M. aeneus it was…". As it is the first time that this species is named, put the scientific name without abbreviations.
414-416: Move the citation "(Gollas-Galvan et al., 2017)" at the end of the sentence.
416,446,451,464,518: the authors repeat the word "Notably", try to replace it sometimes with synonyms.
431: Sometimes a species is presented in brackets, sometimes not, sometimes with its vulgar name, sometimes not. Unify this in the examples.
438: "the cricket Gryllus texenis males", perhaps "adult males"?
429-454: I see the discussion somewhat messy. I suggest moving these paragraphs that talk about differences in immune function between subadults and adults to line 416 before you start talking about the relationship of immunity with fecundity.
462: Add more general citations
              e.g., Dewsbury, D. A. (1982). Ejaculate cost and male choice. The American Naturalist, 119
              (5), 601-610.
               Wedell, N., Gage, M.J., & Parker, G.A. (2002). Sperm competition, male prudence, and
               sperm-limited females. Trends in Ecology & Evolution, 17 (7), 313-320.
455-470: It's ok the discussion about the lack of differences between sexes, but I think you need to add citations of works with similar results
               e.g., Giglio et al., 2016 (citation again)
               Meylaers, K., Freitak, D., & Schoofs, L. (2007). Immunocompetence of Galleria mellonella:
               Sex- and stage-specific differences and the physiological cost of mounting an immune
               response during metamorphosis. Journal of Insect Physiology, 53 (2), 146-156.
             Yourth, C. P., Forbes, M. R., & Baker, R. L. (2002). Sex differences in melanotic encapsulation
              responses (immunocompetence) in the damselfly Lestes forcipatus Rambur. Canadian
              Journal of Zoology, 80 (9), 1578-1583.).
Something important and that was not mentioned is that the sexual comparisons were made with virgin individuals, who had not faced another reproductive cost than the production of gametes (both sexes). All costs associated with sexual attractiveness, mate search and encounter, courtship, post-copulatory mechanisms (if there are present) or oviposition associated costs are not present. I believe that if the sexes were compared but individuals who have already gone through a reproductive event could find differences in the trade-off that each sex displays between these reproductive activities and their immune function (immunocompetence).
488: Add citations in spiders
              e.g., Ahtiainen, J. J., Alatalo, R.V., Kortet, R., & Rantala, M. J. (2005). A trade-off between
             sexual signaling and immune function in a natural population of the drumming wolf spider
             Hygrolycosa rubrofasciata. Journal of Evolutionary Biology, 18 (4), 985-991.
             Calbacho-Rosa, L., Moreno-Garcia, M.A., Lanz-Mendoza, H., Peretti, A.V., & Córdoba-Aguilar,
             A. (2012). Reproductive activities impair immunocompetence in Physocyclus dugesi (Araneae:
             Pholcidae). The Journal of Arachnology, 40 (1), 18-23.
416-423 and  471-488: I suggest that the paragraphs be unified. Also, this paragraph should be simplified and rewritten more clearly and without repetition. Present the main results at the beginning of the paragraph and compare it with other taxa with similar results.
496-507: I never heard about the "quasi-terminal investment" and I would like to have some citation. The theory of "terminal investment" is about the strategy of each individual for multiple matings and trade-offs with future matings (i.e., the shift in allocation towards current reproduction with increasing age). Iteroparous or semelparous species can make a terminal investment if they invest more resources and energy in their last compared to the previous ones, it is an intra-individual approach that has to do with the age of the individual and the senescence. You could talk about "terminal investment" if you saw for example that the trade-off (somatic vs. reproductive) is different in the last mating regarding the first or towards the end of the reproductive season with respect to the beginning of the same. I think that here the concept is incorrectly handled.
508-513: This paragraph would place after 454.
541: Order the citations chronologically.

Figures and tables: in the captions add the level of significance considered.
Tables: unify number of decimals (table 1: 2-3 decimals, table 2: 3 decimals, table 3: 2-3 decimals). I recommend 3 decimals.
Table 1: in the title of the caption add "effect of developmental stage AND SEX". In "for discrete predictors" change to "for categorical predictors".
Table 3: unify "" or '' throughout the manuscript. Better "p-value" instead of P (to match with t-value). To unify with previous tables (1) put in the last row: Season (S - LS) for example and add a reference in table caption (S: spring females, LS: late summer females).

References: scientific names must be in italics.


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

Input:
Title: GENOME-WIDE IDENTIFICATION OF ZMHMAS AND ASSOCIATION OF NATURAL VARIATION IN ZMHMA2 AND ZMHMA3 WITH LEAF CADMIUM ACCUMULATION IN MAIZE
Review round: 1
Reviewer: 1

Output:
Basic reporting: The objective of the study was not clear whether to address food safety or Cd toxicity. Do they intended to develop marker-assisted selection from food safety perspective or  identification of genes responsible for Cd toxicity?

Materials and Methods are not self explanatory. Results and discussions are poorly written.  There are also very confusing words in both MM and result sections
For example :
line 123  what does it mean 79 growth stages ?  There is no 79 growth stages in maize or other plants
line 124  Table S2 is not the correct document
219 : non limiting condition?
line 278: What does mean 20 sites ?
Experimental design: The study lacks basic concept of experimental design including replications, field experiment design and no clear evidence why they chose the soil Cd levels.
Examples :
1) Plant materials and phenotyping  lines 74-95

Both field and greenhouse experiments do not have any biological replications. There is no clear experimental design for both field and greenhouse experiments, and do not include how they analyzed the field and greenhouse data.
No clear evidence why they chose the mentioned soil Cd concentration

2)  Gene expression analysis  lines 120-138

The experiment lacks biological replications. Furthermore, there is not clear experimental design, how they chose these time points and Cd concentration and analyzed the data. The experimental design was also poorly described or set up. It is not clear how many factors and levels of each factor were used.

3) DNA sequencing lines 139-148
 Read depth or sequence coverage is not provided

4) lines 149-161 Gene based GWAS

In order to apply, MLM model with accounting the Q and K parameters, it is not clear how they estimated  Q and K parameters.
Do they estimate the two parameters from the 279 SNP or  from previous studies?
Do the number of SNPs sufficient enough for Gene based GWAS?
Validity of the findings: My major concern is that the data included in the article is not helpful to address the main aim of the article i.e. identify potential markers and develop marker-assisted selection. The candidate genes reported here are superficial, not explained more than 16% of the phenotypic variance. Furthermore, the experiments lack most the basic experimental design and analysis.
Additional comments: No additional comments


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

Input:
Title: GENOME-WIDE IDENTIFICATION OF ZMHMAS AND ASSOCIATION OF NATURAL VARIATION IN ZMHMA2 AND ZMHMA3 WITH LEAF CADMIUM ACCUMULATION IN MAIZE
Review round: 1
Reviewer: 2

Output:
Basic reporting: Good
Experimental design: Good
Validity of the findings: Good
Additional comments: Line 28, In abstract, what is “seeding”?
Line 30, Please define “SNPs” in the abstract
Line 35, What does mean by “low Cd accumulation” in the abstract. Do you mean below permissible limit?
Line 397-398, please reframe the sentence
Line 402, please see above comment at line 35
I could not find phenotypic data like fresh mass, length of root and shoot, etc. of maize seedlings


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

Input:
Title: GENOME-WIDE IDENTIFICATION OF ZMHMAS AND ASSOCIATION OF NATURAL VARIATION IN ZMHMA2 AND ZMHMA3 WITH LEAF CADMIUM ACCUMULATION IN MAIZE
Review round: 2
Reviewer: 1

Output:
Basic reporting: Good
Experimental design: Good
Validity of the findings: Good
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: MANURE APPLICATION INCREASED DENITRIFYING GENE ABUNDANCE IN A DRIP-IRRIGATED COTTON FIELD
Review round: 1
Reviewer: 1

Output:
Basic reporting: Overall, the article is well written and clear. However, there is a little spelling and format mistakes.

L30, “(r=0.70-084, P<0.01)” should have space between letter, character, and value, “(r = 0.70 - 084, P < 0.01)”, please check the whole article.
L40, “Denitrifying enzyme activitie” should be “Denitrifying enzyme activities”.
L47, “inorganic” better to be “inorganic sources”.
L99, “NW China” better to be “northwestern China”.
L134, “The used manure” should be “The manure used for this study”.
L175, “Santa Clara, CA” should be “Santa Clara, CA, USA”, please consistently list the machine name, company name, company location (city, state, country), or part of these information, but should be consistent in whole article.
L247, “Manure application increased soil total N content” better to be “Manure application (both Manure and U+M treatments) increased soil total N content”.
L262, L287, please consistently use comma before “and”, for example, “being 88.7, 27.6, 15.8, and 17.0 for Control, Urea, Manure and U+M, respectively.”  should be “being 88.7, 27.6, 15.8, and 17.0 for Control, Urea, Manure, and U+M, respectively.” Please check the whole article.
L365, “the nitrate reducer (narG), and nirK and nosZ-type denitrifiers” should be “the nitrate reducer (narG), and nirK- and nosZ-type denitrifiers”.
L387-388, “The absence of urea treatment on microbial community was not unexpected as the soil was low in organic matter of only 6.6 g kg-1”, not clear, please reorganize this sentence.
L417, “nirK- and -nosZ type denitrifiers” should be “nirK- and nosZ-type denitrifiers”
L428 & L432, consistent in format, “Lewis Publishers, Boca Raton, FL” or “Lewis Publ. CRC Press, Boca Raton, Florida”. Please check all references, make sure in uniform format.
Table 3, “Chao” should be “Chao1”
Table 5, “*, **, *** indicate significance at P < 0.05%, < 0.01% and < 0.001%, respectively.” should be “*, **, *** indicate significance at P < 0.05, < 0.01 and < 0.001, respectively.”
Fig. 3, better to increase the spacing between columns

The author provided sufficient information and all the information the author provided are clear and sticks to the main idea. For example, the authors provided sufficient introduction and background, in order to state the research question and objective. However, it is better to provide bacteria or fungi species when cited prior studies, which can provide some information about the richness and diversity of soil microbial community so that match the findings (Fig. 2 and Table 3&4). In addition, all prior literature were appropriately referenced.

L238, “(Oksanen et al., 2011)”, I didn’t find it in reference section, the one I found is “Oksanen, 2011”, it should be “(Oksanen, 2011)” or add the correct one.

The article is well organized, especially the introduction and experimental design sections, which is eligible and well organized. And the hypothesis and objectives are well matched to each other, which is clear for us to read. All the tables and figures are relevant the content of the article, and appropriately described and labeled. And all raw data are appropriate and available in accordance.
Experimental design: The subject that is appropriate for publication in PeerJ. The author clearly state the research question and list the objective, which is meaningful. There is an ever increasing body of knowledge published in scientific literature, which showed application of organic manure increased N2O emission under greenhouse and filed condition. However, litter information about how application of organic manure and inorganic fertilizer effect on the gene abundances and activity of nitrifier and denitrifier communities under drip irrigated conditions. This article indicated that manure application increased the abundances of denitrifing functional genes and denitrification potential, which could contribute to the increased N2O emissions.

The materials and methods used are appropriate and no repeat information. While, some detail information were missing.

 L119, the author gave the mean annual air temperature, it’s better to provide the air temperature during the growing seasons.
L129, should provide more plot information, buffer separating plots within block and buffer separating the blocks.
L130, please provide the available N content for urea and manure. For example, urea (46% N).
L136, the N, P, K content of manure based on dry basis or wet basis, what is the moisture of the manure, and what is the pH of the manure?
L140, cotton seed from what company, and what is the planted density of cotton seeds?
L235 & L237, the Vegan package for PCoA and CCA, are they in the same version? If it is, please move the Version and reference from L237 to L235.
Validity of the findings: Overall, the findings are valid and meaningful. All results and conclusion are well stated and based on the data. All underlying data are provided and analyzed by appropriate statistical methods. The conclusion are matched the research question (hypothesis and objectives) and indicated the limitation of this study and future questions needed to investigate.

L251-252, “soil C:N ratio were 37-100% greater in Manure and U+M than in Urea and Control treatments”, while no significant difference between Manure and Control treatments, better to change this sentence, separate Manure and U+M into two sentences, or just keep U+M, for example “soil C:N ratio were 48-100% greater in Manure than in Urea and Control treatments”.
L270-274. PCoA1 explained 61% of the total variation and the microbial communities under different fertilizer treatments were separated into two groups along axis PCoA1, so it is better to provide what features contribute to PCoA1.
L300-301, “NEA, however, was not correlated with the abundance of any functional gene” is not consistent to Table 5, which showed that NEA was positively correlated to nirK.
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: MANURE APPLICATION INCREASED DENITRIFYING GENE ABUNDANCE IN A DRIP-IRRIGATED COTTON FIELD
Review round: 1
Reviewer: 2

Output:
Basic reporting: No comment
Experimental design: The objective stated in the abstract should be improved.  As it stand it is not clearly indicated that this study also evaluated the diversity of bacterial and archaeal communities in this system.

There was a single date in one year used to assess the effect of fertiliser sources on the abundance of nitrifier and denitrifier and bacterial-archaeal communities.   Given that nitrification and denitrification are episodic and that environmental conditions affect both abundance, microbial diversity and denitrification and nitrification activity, the use of a single date to understand this system is insufficient.

The relationships between the abundance of nitrifier and denitrifier and bacterial-archaeal communities and DEA and NEA were explored but not with actual N2O emissions from this system.  DEA and NEA are measurements of the potential denitrification and nitrification but it is not the measurements of the actual N2O emissions of this system.  N2O emissions were measured using static chambers in Kuang et al 2018.  It is unfortunate that the authors did not try to use this data and explore correlation with the abundance of denitrifier and nitrifiers with actual.

There is insufficient details and rationale on the crop production system including if this is short term or long term effect of fertilizer application that is under study, the crop production system and practice (planting date).
Validity of the findings: The discussion is too speculative with inferring that the results from this study explains in situ N2O emissions previously published under Kuang et al. 2018.  DEA and NEA are measurements of potential denitrification and nitrification so it should not be interpreted as the actual measurement of these processes.
Additional comments: Specific comments
Line 25: Microbial is vague.  Is this bacterial or fungal communities?
Line 29-34: The results in the abstract need to be presented more systematically.  The changes in the abundance of denitrifiers, nitrifiers and nitrate reducers in this system should be first described. The possible relationships between the abundance of denitrifiers, nitrifiers and nitrate reducers with DEA, NEA and soil properties should then be described.
Line 29-31:  This sentence does not make sense:  Real-time quantitative PCR’ does not reveal an increased in DEA (please remove ‘Real-time quantitative PCR’), nor is the correlation with increased abundance (remove ‘increased’).
Line 31: denitrifiers
Line 47: inorganic fertilizer.  Furthermore, please be consistent in the term used in the study, either used inorganic, mineral or synthetic fertiliser. I suggest to select one and change throughout the manuscript.
Line 71: It is correct that both denitrifiers and nitrate reducers can reduce nitrate to nitrite but here you are specifically stating the steps for denitrification so I suggest removing the nitrate reducers from this sentence.
Line 74-77: this information is correct but irrelevant to the study since fungal denitrifiers are not studied so I suggest removing.
Line 101: water and nutrient use efficiency by the crop
Line 126: when did manure application starts in this system?  Basically, are we seeing the short-term or long-term effect of application of manure and inorganic fertilizer?
Line 134: remove ‘used’
Line 147: Why was a single date sampled? What is the rationale for choosing this particular date?  It would have been logical to use a date at which N2O emissions were measured in the Kuang et al 2018 paper.
Line 152: what was the rational to freeze the soils for DEA and NEA measurements?  These measurements are measuring enzyme activity and it is less than ideal to freeze the samples since it would affect their activity.
Line 168: change defrost for thawed.  Explain how the samples were thawed i.e. what temperature and for how long.
Line 190: use quantitative PCR not real-time PCR
Line 195-196: There is not enough information as to what genes were cloned as standard.  Is this from specific denitrifier and nitrifier species or an environmental sequence?  Difference in the performance of the standards can be observed, given that it is important to be specific as to what was used.
Line 199-201: The range of PCR efficiency, the slope and the intercept should be stated for each PCR reactions.
Line 203: Was the number of sequences normalized across all samples?  If not, there would be no way to know if differences among treatments are due to the treatments or the unequal amount of sequences across the samples.
Furthermore, the singletons and low abundance OTUs should be removed to avoid spurious OTUs as suggested by Bokulich et al. 2013.
Line 240-242: This phrase makes no sense (abundance of categories between treatment?), please rephrase.
Line 242-243: corrected p-values of what?  Welch’s t-test?
Please use phyla for plural or phylum for singular.
Table 4: The Proteobacteria is among the three most abundant bacterial phyla thus it should be presented.
Line 313: change ‘communication’ to ‘abundance and diversity’

Line 316-318:  That DEA is correlated with the abundance of denitrifiers on a single date in this study is not evidence that denitrification was the major process involved at the field level in Kuang et al. 2018. The statement should be removed.
Line 332-334: this statement is irrelevant: this is about findings reported under Kuang et al. 2018 and it should be removed.
Line 342: compared to what? The control?
Line 344: what does denitrification means here, N2O emissions or denitrification rate or DEA?  Please make sure that specify this and that you are not comparing with different measurement.  DEA is a potential, not the actual measurement.
Line 383: why would they nirS play a more minor role for DEA compared to other denitrifier groups?
Line 367-368: This is incorrect: DEA is not more associated with nitrate, nitrite and N2O.   What was measured was the abundance of denitrifiers in soils, while the DEA measured the enzymatic activity under optimal conditions i.e. with carbon and nitrate addition.  The conclusion here is that the abundance of denitrifiers has increased with manure application to soils and that also increased the pool of denitrification enzymes in soils so DEA increased too.
Line 381: change Agreement with to similarly to
Line 382: please be specific, it is the bacterial and archaeal community that has been studied.  Also what are you referring to alpha or beta-diversity, or relative abundance of phyla?
Line 384: remove reproduction
Line 388: ‘suggesting that C other than N’ – what does this means?
Line 390: this statement contradict the statement made a the start of this paragraph.
Line 393: this is the relative abundance, it is important to mention this.
Line 408: potential NEA and DEA


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: MANURE APPLICATION INCREASED DENITRIFYING GENE ABUNDANCE IN A DRIP-IRRIGATED COTTON FIELD
Review round: 1
Reviewer: 3

Output:
Basic reporting: The article has adequate literature references and the authors clearly state the research problem and objective in the introduction. The results are well presented using appropriate figures and tables for the types of data.

The manuscript should undergo editing for proper English.

The versions of bioinformatics software, including PRINSEQ, UCHIME, and mother are not specified. The software also needs to be cited.

The conclusion states that pyrosequencing was the sequencing technology used, however, the materials and methods clearly state MiSeq sequencing.
Experimental design: Three replicates for each treatment is the absolute minimum, this was perhaps done due to the costs of sequencing. Fortunately they were composites of 4 soil samples but in the future consider including more replicates.

More details regarding the experimental methodology would be beneficial. More specifically:
How was the auger cleaned between samples? How much soil was used for DNA extraction?

Details on the analysis of sequencing data is also lacking. Please elaborate on the parameters used for sequence quality assessment control with PRINSEQ. If defaults were used, specify.

What is the rationale for performing 5 PCR cycles with the 45 ° C annealing temperature prior to 20 cycles with the typical 55 °C temp.?

Line 188: In the future, consider storing DNA TE (or Tris with low EDTA for sequencing applications) as it is a buffered solution and EDTA protects against nucleases.

narG and nirK have very low qPCR efficiencies; is this the case for other studies also? This should probably be addressed.
Validity of the findings: The qPCR results are concerning as no internal amplification control was used to detect PCR inhibition. As such, it is unknown if the difference between organic and inorganic N treatments is due to greater inhibitors in the organic sample or due to an actual difference in gene abundance. Inhibition should be addressed.

In line 385-386, the authors state “It is also likely that manure application had caused high loads of exogenous microbes and thus resulted in the community disturbance”. There is no question of this; manure is loaded with microbial activity. This statement should be clarified to reflect this.

Richness and diversity were not impacted by any of the treatments. Given what we know about soil microbial community structure and organic versus inorganic N addition, this is surprising and contrary to previous studies. This should be explored in the discussion as it is presently not mentioned.

As there was no detected difference between the number of OTUs across any treatment, is it possible the 97% species identity cutoff was too low for the V3-V4 region of the 16S rRNA gene? Please justify the cutoff and consider rerunning with a greater cutoff. Conversely, it is possible that inadequate parameters were used in PRINSEQ resulting in error prone reads being included in the analysis and obscuring differences between treatments. Please include the parameters used for PRINSEQ.
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: MANURE APPLICATION INCREASED DENITRIFYING GENE ABUNDANCE IN A DRIP-IRRIGATED COTTON FIELD
Review round: 2
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: Thank you for responding to my comments. I am recommending acceptance of the manuscript to the technical editor.


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: EFFECTS ON DEVELOPMENTAL OUTCOMES AFTER CESAREAN BIRTH VERSUS VAGINAL BIRTH IN CHINESE CHILDREN AGED 1–59 MONTHS: A CROSS-SECTIONAL COMMUNITY-BASED SURVEY
Review round: 1
Reviewer: 1

Output:
Basic reporting: There are a few sentences that need rephrasing for clarity.

Introduction. I suggest that you improve the description of the objective at lines 112-115.

Consider the objective of the abstract.

Although your results are compelling, the interpretation should be improved in the following ways:

Table 1. Among 1755 children, 102(22.8%) of them were delivered by cesarean section.

According to Table 1, they are 401 (22.8%).

The results must be presented in text or table, but not both. Your results are repetitive.

Lines 182-186 and 188-190 are already included in Table 1, I suggest deleting those paragraphs.

I suggest to the authors should include all results relevant to the hypothesis (significant p-values). For example, “A total of 1755 children aged 1-59 months were included in our study, 401 (22.8%) of them were delivered by cesarean section. There were significant differences in gestational age weeks (p<0.001) and the caregiver’s education (p<0.001) between cesarean and vaginal delivered children (Table 1)”.

Figure 1. Lines 194-197 are already included in Figure 1, I suggest deleting those paragraphs. Consider "The prevalence of suspected developmental delay in cesarean and vaginal delivered groups was similar (23.4 % vs 21.3%; p=0.37) (Figure 1). There was no significant difference in the five separate ASQ domains between the two groups (Figure 1)."

The p-values that were included in the Abstract are not included in the results section. I suggest to authors include those p-values in Figure 1 "ASQ domains of communication (7.7% vs. 7.8%, p=0.949), fine motor (7.0% vs. 6.1%, p=0.538), gross motor (8.5% vs. 6.4%, p=0.154), problem solving (7.2% vs. 6.7%, p=0.722) and personal social (8.0% vs. 7.9%, p=0.960)."

In Tables 2 and 3, I suggest deleting following sentences: “Among these children, 21.3% of them had abnormal ASQ scores” and “Among these children, 23.4% of them had abnormal ASQ scores.
Experimental design: The main limitation of this study is the design. A cross-sectional study is not the most appropriate to measure the development of both cesarean and vaginal-delivered children. The design should be a cohort study (Asztalos et al. Twin Birth Study: 2-year neurodevelopmental follow-up of the randomized trial of planned cesarean or planned vaginal delivery for twin pregnancy. Am J Obstet Gynecol. 2016;214(3):371.e1-371.e19). However, the authors describe this in the study limitation and conclusion section. Despite this limitation, the methods “cross-sectional study” were described with sufficient detail and information to replicate.
Validity of the findings: The conclusions should be appropriately stated and should be limited to those supported by the results. However, in this study, the conclusions are not well stated. These are not limited to supporting results.

If the prevalence of suspected developmental delay in cesarean and vaginal delivered groups was similar (23.4 % vs 21.3%; p=0.37), Why do they conclude that cesarean birth had no effects on children’s growth and development?

I suggest that you improve the description of conclusion, for example, “Our study showed the prevalence of suspected developmental delay in cesarean and vaginal delivered were similar in poor rural areas in China. Therefore, our findings suggest that cesarean delivery does not increase or decrease the risk of suspected developmental in children delay as compared with vaginal delivery”
Additional comments: I suggest you include the study design in the descriptive title, for example, "Effects on developmental outcomes after cesarean birth versus vaginal birth in Chinese children aged 1-59 months: A cross-sectional community-based survey"

Consider in the manuscript the Methods and Discussion sections.


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: EFFECTS ON DEVELOPMENTAL OUTCOMES AFTER CESAREAN BIRTH VERSUS VAGINAL BIRTH IN CHINESE CHILDREN AGED 1–59 MONTHS: A CROSS-SECTIONAL COMMUNITY-BASED SURVEY
Review round: 1
Reviewer: 2

Output:
Basic reporting: Is an interesting and well writing paper, with a huge number of patients.
In a cross sectional design, with the limitations discussed by the authors, the aim of the study is to compare the prevalence of developmental delay risk in cesarean and vaginal born children, and to constructo a multivariate model for both groups.

If there is no differences in the prevalence of developmental delay (DD) risk between cesarean and vaginal born children, no interaction is expected between DD risk and way of delivery, for this reason I think that authors can construct a global multivariate model, instead of different model for each group.

In figure 1: please add a foot note explaining better the figure and analysis. ASQ has 5 domain, nos 6. In the figure there is one column called "stunting", is not clear what does it means, neither this concept is explained in methodology section.

The paper lack of references about ASQ validation in China. Please add them.
Experimental design: Is not an experimental review
Validity of the findings: I agree with the validity of the finding
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: EFFECTS ON DEVELOPMENTAL OUTCOMES AFTER CESAREAN BIRTH VERSUS VAGINAL BIRTH IN CHINESE CHILDREN AGED 1–59 MONTHS: A CROSS-SECTIONAL COMMUNITY-BASED SURVEY
Review round: 2
Reviewer: 1

Output:
Basic reporting: I have no commens.
Experimental design: I have no comments.
Validity of the findings: I have no comments.
Additional comments: The authors have corrected the comments. I suggest accept the manuscript.


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

Input:
Title: EFFECTS ON DEVELOPMENTAL OUTCOMES AFTER CESAREAN BIRTH VERSUS VAGINAL BIRTH IN CHINESE CHILDREN AGED 1–59 MONTHS: A CROSS-SECTIONAL COMMUNITY-BASED SURVEY
Review round: 2
Reviewer: 2

Output:
Basic reporting: I agree with the changes made by the authors.

I recommend to erase the sentence in the discussion: "Our study did not find evidence to support cesarean section would improve child’s growth and neurodevelopment." (second sentence of discussion, lane 212), because in the same sentence it is stated: "Our results are consistent with other studies regarding full-term, preterm, breech presentationchildren or twins, and these studies also found no significant difference in developmental and behavioral outcomes between cesarean and vaginal-delivered groups"

Please review the footnote in table 2, there are asterisks, a and b, and it is not clear the difference between b and asterisks. >=3 child birth order is not significant in the adjusted analysis.

In other hand, the recommendation to construct a multivariable analysis is including only the significant variables in the crude analysis, not all of them, please review with a statistician.
Experimental design: Adequate
Validity of the findings: Adequate
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: LENDATUS, A NEW GENUS OF XANTHOPYGINA (COLEOPTERA: STAPHYLINIDAE: STAPHYLININAE) WITH DESCRIPTION OF THREE NEW SPECIES
Review round: 1
Reviewer: 1

Output:
Basic reporting: The manuscript succeeds in all these areas of basic reporting.
Experimental design: The manuscript succeeds in all these areas of experimental design.
Validity of the findings: The manuscript succeeds in all areas of these standards.
Additional comments: Overall the manuscript is clear, concise and well-illustrated. I have some minor linguistic corrections in the attached file and some important (but simple) suggestions for improvement:

-in the abstract, it is said that the genus can be identified using a character on the male genitalia. I suggest swapping this for the very distinctive external character found in both sexes (pronotal punctation); I would also emphasize this elsewhere in the paper to clarify the message that it is actually easy to recognize the genus
-for L. platys: there is a single record on the west slopes on the Andes and this is a single female. I think this should be commented (whether there is any doubt about this record)


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: LENDATUS, A NEW GENUS OF XANTHOPYGINA (COLEOPTERA: STAPHYLINIDAE: STAPHYLININAE) WITH DESCRIPTION OF THREE NEW SPECIES
Review round: 1
Reviewer: 2

Output:
Basic reporting: English is good, sufficient references for the topic, good structure and relevant figures, tables and data accessibility.

My only structural comment would be the diagnosis sections, which to me fell more like comparison sections. I like having a diagnosis section containing the unique combination of characters within the genus and a separate comparison section focusing on the differences between the species and potential similar species (both within genus and in other genera). The diagnosis can also help define new taxa, as this combination can be compared to a potential new taxa.
Experimental design: For a taxonomic revision the article follows current traditions and is up to data in methodology.
Validity of the findings: Taxonomic revision are of high importance, especially in Neotropical settings, where much is still unrevised and unknown. This will bring much needed and useful information on new taxa from a poorly known region. It will be of importance for many other fields, amongst others, ecological assessments and biodiversity studies.
Additional comments: Only very minor comments:

* Would add a key to Isanopus group of genera
* Abdominal segments are usually termed in Roman numerals, e.g. segment V not 5.
* I have added a few comments, missing italics, etc. to attached pdf.


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

Input:
Title: LENDATUS, A NEW GENUS OF XANTHOPYGINA (COLEOPTERA: STAPHYLINIDAE: STAPHYLININAE) WITH DESCRIPTION OF THREE NEW SPECIES
Review round: 1
Reviewer: 3

Output:
Basic reporting: 1. The author of the article has English as a native language.
2. The literature in the manuscript is referenced appropriately, the "Introduction" section is sufficient enough.
3. The whole article has a proper acceptable structure format. All the pictures are relevant to the content.
4. The obtained results of the article are relevant and confirm the previous hypotheses of the author.
Experimental design: The questions aroused in the article are meaningful and relevant for the modern systematics of the discussed group of beetles. The manuscript is well illustrated by original images. No ethical issues within the article. The methods used by Author are well described in the "Materials & Methods" section.
Validity of the findings: The manuscript is dedicated to description of a new genus and three species of the mega diverse beetle family Staphylinidae. The Author bring clear evidences for delimitation of the new genus and species. The article meets the PeerJ criteria and should be accepted as is.
Additional comments: Dear Stylianos,

It was a pleasure for me to make a review of your manuscript. I have only a minor comment. Could you please add markings in the pictures which indicate specific parts mentioned in the manuscript? It may help some readers to navigate the discussed structures.

All the best!


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: IDENTIFICATION OF A FIVE-MIRNA SIGNATURE PREDICTING SURVIVAL IN CUTANEOUS MELANOMA CANCER PATIENTS
Review round: 1
Reviewer: 1

Output:
Basic reporting: The English language needs some revisions performed by an English native speaker. (e.g. Page 6 line 96, substitute the word “programme” with “program”, the same for Figure Legend 3; Page 6 line 86 miRNAs is not uppercase; Page 5 line 70 substitute the sentence “Melanoma is produced by the malignant transformation...” as follows “Melanoma is the result of the malignant transformation...”).
In the Introduction Section some information are lacking and some references are not appropriate for the contents of the manuscript (See General Comments).
Figures and tables are of good quality and clearly describe the results achieved by the authors. Raw data is correctly provided.
Experimental design: The manuscript proposed by Tao L and co-workers fits well with the aim of the journal. The scientific question proposed by the authors is clearly expressed and is aimed at demonstrating the usefulness of the evaluation of the expression levels of specific miRNAs as prognostic biomarkers for cutaneous melanoma. The scientific idea is very current and attractive because of the lack of effective prognostic biomarkers for cutaneous melanoma. Despite the idea is quite innovative and original, the investigation performed is not fully satisfactory because the authors analyze only one GSE Datasets excluding other datasets potentially interesting (see General Comments). In addition, some information about the differential and statistical analyses performed are lacking.  Therefore, the description of methods could be improved.
Validity of the findings: The results obtained by the authors will pave the way to new experimental and observational study performed on in vitro, in vivo and clinical models in order to validate the prognostic significance of the five selected miRNAs. The data obtained would have been more significant if the authors had taken into account more GSE datasets of miRNAs expression. Overall, the presentation of the data is clear and of interest for the researchers involved in this field of study. The statistical analyses performed seem appropriate but it can be improved. Finally, the conclusion appears clear and summarizes well the main findings of the study.
Additional comments: Below are reported some minor/major revisions that the authors have to address:

1) In the introduction section, the authors should describe the importance of bioinformatics prediction analyses for the identification of putative diagnostic and prognostic biomarkers and describe other bioinformatics study performed on GEO DataSets and TCGA regarding the involvement of miRNA in cancer development. The authors should also explain why their study is different compared to those performed by other authors. For this purpose see the methods used in other similar studies:
- 10.18632/oncotarget.18222
- 10.1038/JID.2015.355
- 10.3892/ol.2015.4031
- 10.3390/cancers11050610

2) “Although the treatment of melanoma is constantly developing, the results are still unsatisfactory, and life-threatening if metastasis occurs, prompting clinicians to pursue new predictive markers and targeted therapeutic genes (Dyduch et al. 2017; Shain & Bastian 2016)” The references indicated for in the above sentences do not deal with this topic in detail. It is suggested to provide more appropriate references, for this purpose see:
- 10.3389/fphar.2018.01300
- 10.3390/healthcare2010001
- 10.3892/ijo.2018.4287

3) In the subheading “Acquisition of microarray data source and DEMs” of the “Materials & Methods” section, authors have to specify why they do not use the GEO2R tools provided by GEO DataSets for the differential analysis. Furthermore, the authors should specify why they selected the GSE35579 dataset. Have they applied inclusion or exclusion criteria for datasets selection? Clarify why the authors do not analyze other melanoma miRNA expression datasets, such as GSE62370 (tissues), GSE34460 (tissues), GSE18509 (tissues), GSE100508 (exosomes), GSE88727 (lymph nodes), GSE61741 (RNA from blood) and GSE20994 (RNA from blood). At least GSE62370, GSE34460 and GSE18509 should be included because built with expression data derived from melanoma tissue samples as the GSE35579 dataset.

4) How the GEO DataSets and TCGA miRNA expression data were compared and normalized?

5) In the first paragraph of the subheading “DEM identification and construction of a five-miRNA signature”, the authors talk about 186 differentially expressed genes of which 80 up-regulated and 105 down-regulated. Did the authors mean 186 differentially expressed miRNAs? Please check this paragraph and correct the errors, if any.

6) It is not clear how the authors select the five miRNAs. Are these miRNAs the most up-regulated or down-regulated? Did the authors only refer to the survival data for the choice of miRNAs?

7) Lines 243-246 page 10, sentences repeated.


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: IDENTIFICATION OF A FIVE-MIRNA SIGNATURE PREDICTING SURVIVAL IN CUTANEOUS MELANOMA CANCER PATIENTS
Review round: 1
Reviewer: 2

Output:
Basic reporting: The authors proposed to identify signature miRNAs to improve prognostic markers that can be useful therapeutic targets during the future therapy development. Their subject was clear without unambiguousity, and overall studies were performed well to answer their scientific questions. English language used throughout their manuscript was proper level to be published. Introduction provided enough background to understand what the authors aimed to in this project. Literatures were well referenced by using most relevant and up-to-date works. Figures were well-organized with relevance to their findings, and the figure legends were well written.
Experimental design: The authors used GEO database to identify DEMs from melanoma patients, and crossed them to TCGA database to correlate their contribution to prognosis. Enough statistical consideration was made to rule out any unreasonable observations.
Validity of the findings: The authors identified five DEMs that were most up or down-regulated in the database, and showed the clinical relationship by comparing the expression levels to survival rates in melanoma patients. Based on their findings, it seems those five DEMs can serve as prognostic markers in clinical application. Although they did not perform wet laboratory studies to validate the functional role of the five DEMs, the authors justified well their findings by critically discuss their findings to a great volume of references in the discussion. Therefore, their findings can be described as a groundwork for future studies where the five DEMs should be further studied or considered as signature miRNAs in melanoma development and progression.
Additional comments: The authors has redefined the signature miRNAs in melanoma patients by re-analyzing two databases GEO and TCGA with enough statistical consideration. Overall research was performed well with clear description about their methodologies and findings with enough figures. Although they did not perform wet laboratory studies to validate the functional role of the five DEMs, the authors justified well their findings by critically discuss their findings to a great volume of references in the discussion. Therefore, their findings can be described as a groundwork for future studies where the five DEMs should be further studied or considered as signature miRNAs in melanoma development and progression. The manuscript will need only minor work on the formatting. For examples, many places of period or comma has space gaps after the last letter in the sentence.


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: IDENTIFICATION OF A FIVE-MIRNA SIGNATURE PREDICTING SURVIVAL IN CUTANEOUS MELANOMA CANCER PATIENTS
Review round: 2
Reviewer: 1

Output:
Basic reporting: The revised version of the manuscript meet the standards of the journal, also the English language was improved.
Experimental design: The authors provided more details about the experimental design and workflow. They well addressed all the reviewers' comments.
Validity of the findings: The achieved results are satisfactory and of interest for the readers.
Additional comments: The authors well addressed all the reviewers' 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: THE ROLE OF AMBIENT TEMPERATURE AND BODY MASS ON BODY TEMPERATURE, STANDARD METABOLIC RATE AND EVAPORATIVE WATER LOSS IN SOUTHERN AFRICAN ANURANS OF DIFFERENT HABITAT SPECIALISATION
Review round: 1
Reviewer: 1

Output:
Basic reporting: The authors need to explore the consequences of the interaction between temperature, vapor pressure, and evaporative water loss (see specific comments below). Also, resistance to water loss is not discussed properly. Exploring these ideas might help hypothesis development, which was not very well developed.

Figure 2 and 3. The bubble plots are confusing and unnecessary. I would explore a simple regression. Use statistics to talk about why body size is important. The reader gets nothing from the bubbles.
Experimental design: Major criticism: The authors state that flow rates varied from 100 to 600 mL/min. This is troubling because flow rates can have a big influence on the rate of EWL. The authors might prove that this wasn’t the case by regressing flow rate against EWL in the supplemental material. Otherwise, flow rate must be included as a covariate. Can you explain why you varied flow rate so greatly? (Wind speed is calculated by multiplying flow rates by the cross sectional surface area of your chamber.)

line 196: By scrubbing water vapor from the system, you did not control anything about vapor pressure. In fact, you confounded your results with the vapor pressure deficit (VPD). The VPD is the factor that drives rates of evaporative water loss, and VPDs increase exponentially with temperature. Therefore, the results from EWL are primarily driven by the VPD, a direct consequence of warming temperatures. What we know, however, is that when controlling for different VPDs, amphibians respond independently to temperature and VPD (Riddell and Sears 2015). So controlling for these effects is critical in experimental physiology. If you do not control for it, then you cannot determine whether a physiological response of water loss is due to temperature or VPDs. It’s not a death sentence, by any means. Most scientists don’t control for it, but you cannot state that you controlled humidity because you cannot determine whether the change in water loss rate is due to temperature or VPD.
Validity of the findings: The conclusion that “ecology matters” is not adequately supported, and based on the narrative in the discussion, the authors understand why they cannot make this argument. They selected three highly divergent species; any physiological differences could have been equally due to evolutionary history as ecological pressure. Perhaps the authors can discuss the ecological and evolutionary implications in the discussion, but they certainly cannot conclude anything about the how ecology matters in this case. These conclusions must be removed from the paper, particularly without an appropriate analysis. That being said, I’m not entirely sure what the message of the manuscript will be. The authors should think more carefully about the message.

I am also concerned about their analysis comparing two species. I understand why they did not include the third species in their analysis, but including mass as a covariate in your analysis could have statistically controlled for these differences. If the authors want to compare slopes and intercepts of the relationships, they will also need to standardize covariates and center over zero. As an alternative, they could examine the residuals of physiological traits regressed against mass. Finally, their two species analysis conflicts with the prevailing literature that has determined two species analyses are inherently flawed (see Adolph and Garland 1994). So removing the third species was not necessary, and in doing so, the authors ignore important literature regarding multiple species comparisons. I would either analyze each species separately or include species as a random factor in your mixed model analysis. You can then look at the standard error of intercepts (and whether they overlap zero) to see if including species as a random effect mattered in your analysis.

In general, I have an issue with the dozens of t-tests in your results. Why not use a ANCOVA or similar hypothesis test to evaluate these differences? Then you can calculate effect sizes to look at the relative influence of all of your factors and covariates. Without some sort of effect size or measure of fit, I have no idea how important your covariates are. As you can tell, I am not a big fan of solely relying on p-values to interpret data.
Additional comments: Line-by-line

Abstract:

23: What does “available” ambient temperatures mean? Ecologically-relevant?

36: I would remove the last line about sexual dimorphism being important because you did not mention that it mattered in the results portion of your abstract. Or tell us why sex mattered in your experiments.

41: See Riddell et al 2017 for explicit evidence that water loss physiology structures the fundamental niche of amphibians.

72: but see Burggren and Vitals 2005 and Dohm et al. 2001.

76: Waxy secretions are actually quite rare. What’s more likely are differences in skin resistance to water loss due to changes in blood delivery to the skin due to perfusion or lipid composition of the skin.

78: It also feels like you just contradicted yourself. Check the logic here.

194: Variable flow rates are a potential methodological issue. See major criticism.

240: Was species a random effect? See major criticism.

254: Change ‘did differ’ to differed - more clear.

256: I think body mass didn’t influence either of these because of flow rate issues, especially water loss. Please include flow rates as a covariate.

357-359: Your expectations are missing a critical components about the role of temperature and water loss (see comment for line 196).

364: Skin resistance is not an ability. It’s simply a property of the animal’s skin, specifically the resistance of their skin to water loss. It’s analogous to resistances and conductances from circuit theory in physics.

365: The authors should remove this conclusion because they didn’t calculate resistances. Therefore they can’t make any conclusions on the relative importance of body mass on water loss physiology - at least not completely. If you calculated resistances you could get an entirely different pattern than what you are reporting. Your ability to discuss the physiology of these animals is therefore limited.

369: Remember that resistance does not necessarily indicate the rate of water loss. The rate of water loss is determined but the combination of resistance to water loss and the evaporative demand of the air. Resistance units are time per distance (i.e., seconds per centimeter is commonly used). A rate of water loss would be grams of water lost per unit of time. I would make that clear to the reader; otherwise, your language is confusing.

393: I find it hard to believe that food isn’t limiting in aquatic systems. Provide citation.

395: citation format issue.

416: or evolution. Your conclusion is arbitrary.


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

Input:
Title: THE ROLE OF AMBIENT TEMPERATURE AND BODY MASS ON BODY TEMPERATURE, STANDARD METABOLIC RATE AND EVAPORATIVE WATER LOSS IN SOUTHERN AFRICAN ANURANS OF DIFFERENT HABITAT SPECIALISATION
Review round: 1
Reviewer: 2

Output:
Basic reporting: Mokhatla et al. – The role of ambient temperature and body mass on body temperature, standard metabolic rate and evaporative water loss in southern African anurans of different ecologies.

In general, I found the dataset very interesting but a bit confusing in the manner that authors showed them in the manuscript at its present format. I had to struggle to follow some of the text of the paper. For example, it is not clearly stated in a logical sense and sequence in the text the direction of what authors expected as a whole with what they reached in terms of novel findings. It is very noticeable when reading the main goal of the paper (Lines 26-27) and then reading the presentation of all the results (in several directions sometimes without connection, Lines 30-33) in the Abstract, i.e. many of the results do not answer the main hypothesis(es) of the paper in a logical sense (many factors precluding what is central and what is peripheral). In this way, the idea behind the paper became explorative rather than hypothetico-deductive, as can be observed in the Discussion section (many parts hard to show a logical sequence). Making things clear, concise and concatenated in the text may improve the comprehension of the text for a broader audience of readers as well may highlight even more the importance of the main results found by the authors. I suggest to keep the manuscript straightforward and as simple as possible, with temperature as central and other factors with potential to affect temperature changes on physiological traits (for example, with your experimental design you cannot say if it was body mass or gender the most important factor affecting physiological traits under temperature changes). My impression when reading was that authors were trying to introduce many factors to justify the study and highlight it as unique. I would be cautious about saying there are few studies (Line 22) on topics that you are exploring; I left some references below. In addition, I am afraid at its present form the study suffers from some methodological problems, which prevent the interpretation some findings. I made specific comments and suggestions below. Hope these are clear.

Abstract and Introduction with consequences in the manuscript as a whole

In their own words, authors aimed to determine the role of ambient temperature (Ta) and on body temperature (Tb), whole-animal standard metabolic rate (SMR) and whole-animal EWL (Evaporative Water Loss) in three amphibian species with differential ecologies from Africa (see Lines 26-28; I comment below on two misconceptions presented here). They expected that differences in species' response would be associated with differences in ecological specialization of each species, and body mass and sexual dimorphism would also be important factors influencing such responses. Authors specified they expected that the aquatic species would show higher SMR and EWL in comparison with the semi-aquatic species (intermediary SMR and EWL) and with the terrestrial species (reduced SMR and EWL) (Lines 94-108). What authors expected to happen in relation to the Tb of the amphibians was not clearly presented as hypothesis (at least in the initial sections of the paper – Abstract, Introduction – in which all ideas are, or should be clearly, presented). Moreover, although they described certain effects of temperature changes on Tb, SMR and EWL in some parts of the Introduction, they did not clearly state the direction of what was expected both in terms of specific or general results as a whole in regard to temperature changes; see the Abstract (Line 27: “…determine the role of Ta…”) and also the final of the Introduction (Line 94: “…determine how ambient temperature affects…”, in the Lines 98-99: “…tested how the variation in Ta affects…”, and in the Lines 99-100: “…determine whether these species differ in their responses to Ta fluctuations…”).

Likewise, it is hard to the reader to disentangle what is essential to pay attention in regard to the main hypothesis/es of the manuscript, if it was/were not clearly stated. As far as I understood, expected variations in Tb, SMR and EWL due to changes in an experimental abiotic factor (Ta) and/or in other factors (body mass, sexual dimorphism, habitat use). But see that only habitat use is mentioned at the end of the Introduction regarding to the expected results, which should answer the hypothesis/es. In this context, would body mass and gender be only factors of control for tests of temperature effects on physiological traits or would they being also tested as factors as well temperature? In addition, variation in the “whole-animal” and “mass-specific” SMR and EWL unexpectedly appear for the first time in the text at the final of the Introduction, also contributing to make the reader confused about where authors are and where they are going. At this point, I was again, with an impression that authors were trying to promote their study. It is not a problem at all, but it should be clear why whole-animal or specific-mass differed and what are the implications for that.

Thus, the introduction needs more focus on what are authors really planning to answer. I suggest the use of the points mentioned above to improve the manuscript goals and hypothesis/es in a clear and conciseness manner to provide more justification for your study. This will also help you expand upon the knowledge gap being filled. Specifically, the authors’ interpretation (and justification for carrying out this study) that “few studies have tested…taking species ecology” is not, at my point of view, suitable. Several studies may contradict this statement since many of them have tested SMR (Gatten et al. 1992 for a review), Tb (Brattstrom, 1963, Ecology), EWL (Tracy et al, 2007, Ecology; Lillywhite 2006, JEB for a review) under a set of experimental conditions, body masses, species ecology. Therefore, try to build logical hypothesis and predictions to match your results (with temperature variation as central). You may also reinforce the importance of including sexual dimorphism (this is rare in the literature) as factor influencing temperature changes on your physiological traits (at least for amphibians).

Minor (but not least important):

Lines 26-28: Change “determine” to “investigate” or “evaluate” or “assess” or “estimate” or a similar term. “Determine” is not suitable or proper in these circumstances since no one can determine animal responses other than them. In addition, different “habitat use” should be used instead of differential “ecologies”, a term that is vague in the context that was presented.
Experimental design: Methods:

Why the methods for obtaining Tb’s did not appear in the Methods section? I confess I still can not clearly understand how the authors obtained or derived the body temperatures (Tb’s). The Tb’s are show for the first time as results in the Results section, including the way that authors obtained/derived them. Moreover, if I have interpreted correctly, there is no sense what authors tried to do since the relationship between air, skin and core temperature can change a lot depending on the conditions of measurement and the range of temperatures over which you look. For example, the air or substrate at a given location does not predict the body temperature of the amphibian at that location, because it doesn’t include particular conduction with the substrate, summed radiation from across the entire substrate and the rest of the environment plus convection and so forth. Thus, I think you cannot oversimplify that relationship and then deriving Tb from Ta since air temperature interacts with the body of an amphibian to set its body temperature in a non linear or simplistic way. Also, depending on the interplay with hydration level (my comment below, Line 217), Tb may be very different from expected from a linear regression with Ta. My suggestion is to the authors delete these Tb from the paper results (see that Tb is not well contextualized in the hypotheses; I made comments above). Maybe try to explore the rates of Tb exchange (cooling and warming) and the levels of hydration (initial and final body mass measurements).

Lines 139-145: Anuran species were fed with different diet. Please explain why you used this treatment since different acclimation conditions may affect physiological traits in a differential way. Also, explain why you are confident that individuals did not get food differentially since there were more than one per plastic boxes at least for 2 species (there is no information on the third, Xenopus laevis). Different captivity conditions ("treatments") may imply in differences when analyzing physiological responses.

Lines 169-171: Please provide an explanation on why chambers with different sizes would not affect the final results when comparing rates of SMR among species.

Lines 156-223: Please separate in two sections: SMR and EWL measurements. It is very confusing at the present form.

Lines 213-216: Why did you assume that stable vapour pressure and temperature trace suggest equilibrium of Tb with test temperature? It is not logical. Thus, provide a rational explanation and, if possible, data to confirm that.

Line 217: Why did you used 2 hours for EWL measurements for all individuals independent of their body size? Do you have evidence that this time period did not caused dehydration, and then you include another factor other than temperature influencing the rates of EWL?

Minor (but not least important):

Line 138: Returning animals kept in captivity for 2-4 months to nature should be avoided. There is very little knowledge about what could be being introduced in terms of new microorganisms, pathogens, etc.
Validity of the findings: Results, Discussion, Conclusions

Since I have several concerns on the way the manuscript was organized and also that data was obtained, impact and novelty can not be entirely assessed at its present form.

Lines 300-308: Thermoregulation and Tb relationships should not be discussed without referring their interplay with hydro-regulation. Even if you mention it in some lines below at EWL sub-section, the influence of body mass on Tb and metabolism can not be decoupled from hydration state. If so, please provide rational explanation.

Lines 356-387: Why discuss cutaneous resistance and not rates of EWL if you only measured rates of EWL? Please provide rational explanation.

Line 418: “…Ta influence on..” in which way or direction? Describe it better.

Figures: I found Figs 2 and 3 very ingenious. But I can not obtain simple information as main descriptive tendencies. Would it be possible to include such information as central tendencies (mean or median) and variances (SD or SE)?
Additional comments: All my comments and suggestions are covered above.