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 CHARACTERIZATION AND EXPRESSION ANALYSIS OF SOYBEAN TRIHELIX GENE FAMILY
Review round: 1
Reviewer: 1

Output:
Basic reporting: The authors present a pretty comprehensive overview of the trihelix-family in soybean. The analysis includes standard analysis of the genomic localisation of trihelix genes, predicted protein domains and cis-regulatory elements in the promoter region of the 71 trihelix members. Furthermore, the expression of the trihelix factors after abiotic stress application is shown.  The depicted figures and overviews are easy to understand and very illustrating. In general, the manuscript is worth publishing in Peerj after implementing a couple of improvements.

The scientific writing style is sometimes quite awkward (362, 403,…) and partly not in a professional and unambiguous language, in parts grammatically wrong. Therefore, please revise the manuscript on that point to achieve the standard level.

Here a few examples:

in the abstract!!!: line 22 replace present by presented, line 24 suggested -->suggests, line 26 provided-->provide, line 27 in responding--> in response to
Writing mistake in line 261 (similar mistake in line 202) “These results indicating” is wrong….better: “These results indicate” or “These results are indicating”. However, the word “indicate” is too strong in this context…. better would be “suggest”. line 269, replace “were displaying” by “displayed” (results should be presented in simple past), line 271 replace “in” by “after” or “following”. Line 272 delete “There were”. Line 279, replace “The results suggested……genes was…” by “The results suggest……genes is”. (Conclusions should be presented in simple present). Rephrase line 307 “That was because” is an awkward expression. Line 357 replace “This fact indicated” by “This finding suggests” (be careful with the use of the word “indicate”). Line 366, delete “As we all know” (it is colloquial speaking), line 401 replace “This evidence strongly indicated” by “This finding suggests”. Line 244, replace “were exhibited” by “exhibited” (it should be AKTIVE, not PASSIVE)

In the manuscript are plenty of additional mistakes of that kind.

in the conclusion paragraph 419-429: delete “In addition, the expression pattern of the trihelix 425 genes in different tissues and the cis-elements of the promoters were also analyzed.”--> that is not a conclusion….

Please revise carefully the manuscript and consider rephrasing.
Experimental design: The paper is very theoretical and based on the in silico analysis of previously published raw data. Consequently, the evaluation of the trihelix factor function is very hypothetically and based mainly on already published trihelix members and the presented qPCR analysis (which gives only a rough hint about a putative biological function/ participation).

1. Please merge Table S5-S8, Data presentation would be more clear in this way.

2. Figure S4: The significance test is missing (Student’s test)
Validity of the findings: The validity of the finding is ok.

1. In Figure S3, cis-regulatory elements responsive to Jasmonate, Ethylene and Salicylic acid are depicted, which suggest an expression of corresponding genes after biotic stress perception. In Arabidopsis, trihelix transcription factors have been shown being involved in the plant immunity and being part of the MAP kinase signalling pathway (ASR3, GTL1).  I suggest to introduce and discuss this issue more in detail and to perform expression analysis of the 71 trihelix members or at least of a selection following flg22-application (and, or Jasmonate, ACC (precursor of Ethylene) and Salicylic acid treatment). This analysis would give the paper a more comprehensive overview of putative trihelix factor functions, besides the abiotic stress response.

2. In Arabidopsis, putative MAP kinase binding sites have been found in the trihelix members, thereby providing an important clue for the post-translational regulation by phosphorylation…. Can you identify MAP kinase binding sites in soybean trihelix factors? If yes…are they conserved and how is their distribution among the different groups?

3. Figure 8: Why these 12 trihelix genes have been chosen? any reason?

4. The indication in line 262 is incomplete. At least salicylic acid-responsive element and ethylene-responsive element refer to transcriptional control of the corresponding genes after biotic stress perception.
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 CHARACTERIZATION AND EXPRESSION ANALYSIS OF SOYBEAN TRIHELIX GENE FAMILY
Review round: 1
Reviewer: 2

Output:
Basic reporting: Comprehensive bioinformatic analysis of soybean trihelix transcriptional factors has been done in this paper. However, some of the details and analysis need to be made more accessible. The description of the results lacks the depth expected from a research article. They did phylogenetic analysis, protein motif and gene structure analysis, but there is very little biological function exploration. The legend of figures and supplemental figures was not well described. The English language should be improved for publication. Some examples include misspelling, no space between words, misuse of uppercase/lowercase.
Experimental design: Liu and colleagues used bioinformatic approaches to identify 71 trihelix transcriptional factors in soybean. They analyzed these gens and provide comprehensive genomic information including chromosomal distributions, gene duplication, motif compositions, gene structures and cis elements. They also did RNA-seq analysis of soybean plants with flood treatment and found a few of them are differentially expressed between conditions.
Validity of the findings: 71 trihelix transcription factors have been identified in soybean. RNA-seq data from submergence experiment indicates few trihelix genes likely involved in flooding stress.
Additional comments: 1, Line 64, analysis should be analyzed
2, Line 71-72, should make it clear that the data is from the publicly available RNA-seq database and in the result part the authors should cite the data source.
3, Line 133-134, what’s the q value?
4, Line 142 and 148, does root sample include all the root material or part or the root?
5, Line 165, what is the trihelix family size in other species
6, Line 164-190, Combine the two parts?
7, Trihelix factors also known as GT-factors because they bind to a typical GT domain. Could binding motif analysis be done?
8, Line 298-301, rephrase sentence
9, Line 306-309, rephrase sentence
10, please include reference for RNA-seq raw data in the text, figure legend and supplemental table.
11, Line 359-360, what are the two genes show different expression pattern? What the similar expression pattern that the 11 genes share?
12, Line 388-395, Literature was not well referenced. Xie et al (2009) reported that both GmGT-2A and GmGT-2B are involved in salt, freezing and drought stress, and GmGT-2B but not GmGT-2A affects ABA sensitivity.
For NaCl treatment, have the authors tried 150 mM?
13, Line 638-639, UTR is part of exon, not independent of exon.
14, Not all of the 15 genes are significantly differentially expressed genes.
15, What the difference between Figure 6 and Figure S6? It looks like that the same gene has different element.
 16, Combine Supplemental table S4, S5, S6, S7 and S8


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 CHARACTERIZATION AND EXPRESSION ANALYSIS OF SOYBEAN TRIHELIX GENE FAMILY
Review round: 2
Reviewer: 1

Output:
Basic reporting: English spelling and style have been significantly improved.

There is a mistake in reference to Völz et al, 2018.(GTL1). In the reference list, the citation is included, but cannot be found in the main text body...please update that and check carefully the references again for additional mistakes.
Experimental design: ok
Validity of the findings: ok
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 CHARACTERIZATION AND EXPRESSION ANALYSIS OF SOYBEAN TRIHELIX GENE FAMILY
Review round: 2
Reviewer: 2

Output:
Basic reporting: The English language still need to be improved for publication. Article structure is good but some of figures are not shown in a professional format. Some of the references are not cited properly. There are errors and mistakes in this manuscript that are not acceptable for a scientific article.
Experimental design: The authors used bioinformatic approaches to identify 71 trihelix transcriptional factors in soybean and provided genomic information of these genes which could be useful. They also did RNA-seq analysis of soybean plants with flood treatment and found a few of them are differentially expressed between conditions.
Validity of the findings: This article provide some useful information of soybean trihelix factors.
Additional comments: Please read your manuscript and check all details carefully before you submit it.
Minor comments
1, Could you reference the soybean RNA-seq data itself but not Phytozome?
2, Could you please label the domain name in Figure 4? Figure 4 legend is not updated. “Figure S1” should be Figure S2 since you added another supplemental figure.
3, Xie et al (2009) did state that GmGT-2A showed a higher expression level at 12h after the ABA treatment, but it is a side effect. Also, the 2A overexpression lines did not show any significant difference compared to wildtype with ABA treatment.  And Xie et al (2009) concluded that GmGT-2B affects ABA sensitivity.
4, Delete lines 357 to 362
5, Delete line 419
6, For Figure 9, could you use the same format for all the charts?


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 CHARACTERIZATION AND EXPRESSION ANALYSIS OF SOYBEAN TRIHELIX GENE FAMILY
Review round: 3
Reviewer: 1

Output:
Basic reporting: The manuscript has been improved. There are still some mistakes and errors.
Experimental design: No comment
Validity of the findings: No comment
Additional comments: 1, Line 152 and 158, please rephrase it. All root material from 5 plants was pooled and three…
Technically, we don’t pool samples unless we can’t get enough material from one sample/plant. Because pooling can reduce the effects of biological variation. Instead, we include more than three biological replicates.

2, Line 185, cDNA should CDS. cDNA includes UTR.

3, Line 193-194, 215-217 etc., please use present tense. There are plenty of mistakes of this kind.

4, Line 211, spices should be species

5, line 238, delete much

6, line 293-294, what (9) and (13) are?

7, Please rephrase line 316

8, line 320-339, the genes that response to different stress treatment are still confusing. “11 genes” in line 318 are the same as that in line 320? Are all the “12 genes” in line 327 from the 15 DEGs? How did the authors select the 12 genes?

9, line 357, should be 3 h, 12 h.

10, line 364, Osorio et al (2012) found 63 trihelix genes in soybean. Do the 71 genes include all those 63 genes? What the other 8 genes are? Could you please provide information (e.g. gene organization, gene expression pattern etc.) of the genes that were not found by Osorio et al (2012)?


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: SEED TYPE, HABITAT AND TIME OF DAY INFLUENCE POST-DISPERSAL SEED REMOVAL IN TEMPERATE ECOSYSTEMS
Review round: 1
Reviewer: 1

Output:
Basic reporting: This is a well-research manuscript which makes good use of the wide and varied literature regarding seed removal. The introduction and background give a good explanation of where the research currently stands, and why this study is important. However, there are sections where the language is unclear, or where more explanation would aid the reader. Possibly contrasting the findings of this paper with those reported in Fornara & Dalling, 2005, Journal of tropical Ecology, may be interesting, but are not essential. The data is supplied, and the figures are clear for the most part, although some of the symbols could be explained further and made larger to ease reading. Please find below more detailed information on suggested changes:

Line 38, 326 – Lambert & Champmann/Champman, 2005 – please check the spelling for this reference
Line 42 – “relative rate” instead of “relative importance”
Line 44 – “varies considerably” instead of “considerably varies”
Line 71 – missing the words “the presence of” in the section “the effect of ___ seed coat”
Line 73-74 – not clear how seed size plays a role when this paragraph has discussed the effect of the hull – perhaps clarify the relationship between dehulling and seed size more clearly.
Line 84 – section in parentheses can be its own sentence, but please provide justification e.g. difficulty of tracking seeds etc.
Lines 90-96 – please detail the mode of dispersal and basic morphology (especially any adaptations for modes of dispersal) for all species
Line 99 – why do you expect a lower rate of removal for intact sunflower seeds?
Line 102 – another good source to back up this claim is Fornara & Dalling, 2005, Journal of Tropical Ecology
Line 190 – “during the day” instead of “at day”
Line 194 – “removed at a higher rate” instead of “strongly removed”
Line 196 – “higher” instead of “stronger”
Line 198 – “removed at higher rate” instead of “stronger removed”
Lines 212-214 – this is a very good explanation. Perhaps refer to these points at line 99 to explain why you expect a lower rate of removal for hulled sunflower seeds.
 Line 220 – “primarily” instead of “primary”
Line 230 – “removed at the highest rate” instead of “removed best”
Line 231 – perhaps clarify how seed removal is an advantage, e.g. it increases the chances of dispersal away from the parent.
Line 242 – “inconsistent” instead of “inconstant”, and “may depend on” instead of “depends on”
Line 373 – author’s initials are punctuated, unlike rest of reference list – please check for consistency
Line 375 – font appears to change half-way through reference – please check for consistency

Figure 1 – It is not entirely clear what the ns and * below the bars mean. Please provide a more detailed explanation in the figure legend. I assume it indicates whether the differences in seed removal between habitats were significant, but I have no way of confirming this solely by looking at the graph and the figure legend.
Figure 2 – I am unable to distinguish between the lines on the graph. Perhaps use different types of dotted or dashed lines instead of shades of grey. Also, the legend is very hard to read and it is not obvious that the white-filled circles are C. majus. Perhaps increase the legend size.
Figure 3 – please indicate what the asterisks mean in the figure legend and increase their font size for ease of reading.
Experimental design: This manuscript reports the results of a well-thought-out experiment. The research question is clear and meaningful, and the rationale is well-thought-out. The methods are very well-described, although some further information and justification may be useful to the reader. Please find the details of suggested changes below:

Lines 118-120 – please provide the modes of dispersal for Tragopogon pratensis and Helianthus annuus
Line 142 – please consistently use either “meadow” or “grassland”, unless there is a difference between the two. If there is a difference, please explain what it is (although line 112 appears to indicate that they are the same)
Lines 142-143 – why was T. pratensis not used for this experiment?
Lines 145-146 – is there a possibility that moving the night-time seed plates away from the day-time plots may have influenced removal rates by, for example, moving the seed plates closer to/away from seed predators e.g. ant’s nests etc.?
Validity of the findings: The findings are interesting and well-reported, and any speculation is clearly labelled as such. The conclusions are relevant to the original research question and supported by the results. I only have two comments regarding the results, reported below. My only real issue is regarding Supplementary File 2. It is not clear why there is a set of NAs in the 8h-10h period in the Darmstadt data, as this is not mentioned in the manuscript. I understand that, while doing fieldwork, things outside our control occur which limit data collection, but a short explanation of why this occurred, or at least a statement that it occurred, will increase transparency. Otherwise, I believe the results are robust and fully support the conclusions drawn by the authors.

Further comments:
Lines 184-185 – how did removal rates change in time with habitat and seed type? A more detailed description of the results may be helpful.
Lines 254-256 – is there a way to access temperature data for the experimental period? It may provide another interesting layer of analysis.
Additional comments: This was an enjoyable study to read. I look forward to its publication.


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: SEED TYPE, HABITAT AND TIME OF DAY INFLUENCE POST-DISPERSAL SEED REMOVAL IN TEMPERATE ECOSYSTEMS
Review round: 1
Reviewer: 2

Output:
Basic reporting: Wehner et al.'s manuscript "Seed type, habitat and daytime influence post-dispersal seed removal in temperate ecosystems” attempts to disentangle the effects of seed traits, habitat type, and time of day on rates of seed removal for four species native to their study sites: Chelidonium majus, Lotus corniculatus, Tragopogon pratensis and Helianthus annuus. The authors attempt a tall order: to disentangle the effects of seed traits, habitat type, and time of day by presenting the results of two separate field studies. The first experiment compares seed removal across all 4 species in forests versus grasslands over a time period of 48 hours. The second experiment compares seed removal during the day versus night for the same suite of species in forests versus grasslands.

Overall, while the authors conclude that their study shows evidence that seed type and to a lesser extent habitat type influence seed removal, I think that their study design does not necessarily warrant such a general conclusion, and would benefit by adding in language to provide caveats to their overall conclusions. While I find their overall general conclusions problematic, I do think that they have completed two well-designed studies that fit well within this manuscript and within the scope of PeerJ.

Below, I list changes that I think should be made to improve the clarity of this manuscript and limit the scope to fit what is being tested with this study.

Please change “daytime” to “time of day” throughout the manuscript, including the title. The term daytime specifically refers to daylight hours, however, I think the authors really mean the time of day within a 24-hour period from the rest of the details provided.

Intro and background:
Consider starting the first paragraph of the intro at line 39 at post-dispersal seed removal. The focus of this manuscript is post-dispersal seed removal so it seems unnecessary to include what the authors mention before that for this manuscript.

Since the study focuses on seed traits, habitat type, and time of day I suggest restructuring the introduction around these factors. At the moment, the authors go into much detail about particular seed traits, but then include a single paragraph for both habitat type and time of day together. More detail is needed to build up to why the reader would predict a difference in seed removal rates as a result of these factors.

lines 84-86: The authors discuss how seed predators differ in preference for seed size. Seed dispersers also vary in in this preference. Consider adding in a sentence to describe this. Add additional references to back up your predictions  (at the moment only Brown et al. 1975 is used here).

line 92: Remove “seeds” after T. pratensis.

lines 94-96:  The authors mention that they compare seeds of C. majus and L. corniculatus to determine if there is an effect of elaiosomes. Given that so many other factors (chemistry, seed coat thickness, etc) potentially vary across these two species this is not a rigorous test of the effect of elaiosomes. Remove this  line. I think that it is okay to speculate about this within your discussion with a thorough discussion of the potentially confounding caveats, but this study is not set up to test this question.

line 98: The word  “greater” should be changed to larger.

Predictions about seed traits in general: I find this not to be a particularly good study design to be able to disentangle individual seed traits given the potential for confounding effects. Instead, the authors should make predictions about whether they expect difference overall among species and then describe why based on multiple traits at once.

Overall, the authors appear to conform to PeerJ standards and discipline norms when it comes to the basic structure of their manuscript. However, one change to the structure that may provide more clarity is in how the methods and results are structured around the two experiments. Use sub-titles to refer to each of the studies, instead of mentioning it in the text itself. Mirror this structure in the results.

I found that the figure captions need to be edited for clarity. For all figures, be clear about which experiment the data are from. Additionally, be clear about what you are showing with error bars (i.e. are they representing standard error, standard deviation, etc.?). What do the different parts of your boxplots indicate?

In figure 1, the authors refer to the colors as “dark grey” and “light grey”, however the color scheme appears to be white and grey.

Figure 1 shows seed removal as the proportion of seeds removed, while the next figures show the number of seeds removed. Be consistent in how you present this. Did you find the same patterns for proportion of seeds removed and total number of seeds removed in your statistical analyses?

It is unclear why time is presented on a logrhythmic scale on the x-axis of figure 2.

I find it difficult to distinguish among the points and lines in figure 2. This figure might benefit from use of color (making sure to use a color scheme that is color-blind friendly) to distinguish among seed types.

It would improve interpretation of patterns in the figures if there was some kind of indicator of the seed traits that were previously mentioned in the manuscript (seed size, dispersal syndrome, etc.). This could be done by reminding the reader of these in the figure captions or including it with pictures of the seeds or added words in the figure.

Dates in the raw data do not all appear to fall into June of 2018. Experiment 2 appears to have been completed in July of 2018. Confirm that the dates are correct in dataset from Experiment 2 and then make the necessary change to the methods section in the text.

It does appear that all of the data are present in the two supplements that are mentioned in  the methods. However, in the discussion the authors mention observations of ants and slugs removing seeds yet do not provide data or description in the methods of when they looked at this.
Experimental design: Wehner et al., present primary research that appears to be well within the scope of PeerJ. Additionally, from what is presented by Wehner et al., it appears that their work is performed to a high technical and ethical standard.

Below I give provide a list of things that I think need clarification or need to be added to the methods description:

line 110-111: It is unclear why this study was done in three locations from what is written here. Did the authors choose the three locations because each had grassland and forest habitat? For replication?

It would be good to add more general details about your study locations. How far away from each other are these sites? Are they predominantly grasslands or forests? What is known about their history of land-use? Add more detail about how they are currently managed.

lines 111-112: What time of year relative to the growing season are the meadows mowed?

line 113: Be more specific here about what a moderate amount of forestry entails. What forestry management techniques were used? Are they the same across sites?

lines 117-120: Be consistent with the information you present for each species so that the species are more easily compared. For example, you state that the seeds of speces a and b are small, but then make no similar mention to size for species c and d. Additionally, you mention that species a is dispersed by arthropods and species c is wind-dispersed, but then do not mention dispersal mode for the rest of the species.

line 121-123: Change the wording here. Instead of stating that the dry mass was “averaged with standard deviation”, state that you calculated the mean and standard deviations of each species seed dry mass.

line 128: How many wells are in the plastic plate? It is unclear whether each seed is placed in an individual well. Overall, I’m having a hard time picturing what these looked like making it difficult to envision how I would replicate this setup. Could you add some detail about the brand of plastic plate that you used?

line 130: How did you make sure seed plates were placed flat on the ground? Additionally, it is unclear how you chose the location where you placed the plates.  Were these locations random within a site?

line 154: It is unclear why the authors define removal rate as R = log(N removed/25+1). Authors need to provide clarification for why they use this calculation.

lines 159-165: Did the authors consider a non-linear fit for this analysis. When looking at figure 2, some of the species may be better fit by a different shape, but it doesn’t appear that the authors checked this.

line 161: Change “root-square” to square root. Add reason for transforming the data in this way.

line 163: It is unclear what the authors are specifically referring to when they say  “individual effects”.  Please clarify.

line 164 and 168. It is unclear how you are using one-way ANOVAs in these instances and why this was used. Please be more specific.

Note for statistical analysis in general: Authors need to be more specific about how they made sure their data fulfilled the assumptions of the statistical models that they used. They make no mention of this in their methods section. Additionally, while the authors mention interaction terms in their tables, they make no mention of including interactions in their methods.
Validity of the findings: line 173: It appears that the authors are using the term seed type instead of species. While it seems okay to speculate on the effect of variation among the different species as a potential result of the differences in their seeds, this study does not necessarily provide a rigorous test of this. There are lots of potential confounding variables that differ across the seeds of these four species. In order to provide rigorous evidence of the effects of seed type, the authors would need to add lots more species over a gradient of seed type variables. Change seed type to species.

Note for results in general: Authors need to describe the significant interaction terms present in table 2. Sometimes significant main effects cannot be safely interpreted when interaction terms are significant. Authors need to describe the significant interaction terms and provide a description of their significant main effects in the context of these significant interactions terms.

lines 209 and 215: The authors mention that they observed ants and slugs removing seeds, however nowhere in their methods or results do they mention that they observed who was removing seeds from their seed plates. For which study was this observed? Even if this was done opportunistically during the counting of seeds, it would benefit this manuscript to further describe this.

line 220: Change the term parachute to pappus. While these two terms are synonyms, pappus is more commonly used in the literature to describe this morphological feature on seeds that are wind dispersed.

lines 226-231: While seed size varies across your species, this trait is confounded by other traits such as morphology that lends seeds to being dispersed by animals (e.g. presence of an elaiosome) or wind (e.g. presence of pappus). Either remove this paragraph or add language that indicates this caveat.

line 232: Generally, it seems that you found not effect of habitat type, however the authors present this as having found little effect. The authors should change how this result is depicted here to be more in line with the evidence that they present which is that there was no effect of habitat type on seed removal for all but one species and that was only when the seed had been dehulled. Additionally, the discuss literature here about the effects of habitat type on seed removal, but it is unclear if they mean habitat type in general or if they are focusing on literature that measured seed removal in forests and grasslands. Add clarification.
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: SEED TYPE, HABITAT AND TIME OF DAY INFLUENCE POST-DISPERSAL SEED REMOVAL IN TEMPERATE ECOSYSTEMS
Review round: 2
Reviewer: 1

Output:
Basic reporting: The language is clear and the literature has been widely covered. The article is easy to read and well-written. The addition of diagrams of seed type to the figures is very helpful. Some attention should be given to the spelling of author names in-text and in the reference list.

Specific comments:
Line 26 – “removed at significantly higher rates” instead of “significantly removed at higher rates”
Line 74, 433 – check spelling of “Xiao, Zhand/Zhang & Wang, 2005)
Lines 85-86 – it is not entirely clear how competition between species drives the temporal differentiation, perhaps add another sentence to explain this more.
Line 111 – “generally considered as trait associated” doesn’t make sense
Line 114 – explain why you expect seed removal to be higher at night
Line 119 – “interested in comparing the two” instead of “interested to compare”
Line 168 – “12 hours during the day” not “12 hours at day”
Line 170 – “12 hours during the night” not “12 hours at night”
Line 232 – “myrmecochorous because of the presence of” instead of “through the presence  of”
Line 258 – add “for dispersal” to the end of “that depend on abiotic conditions”
Experimental design: The research question is clear and highlights the current gap in the knowledge relating to the predation and dispersal of seeds on the ground. The methods are coherent and clear, with the exception of the following:

Lines 168 – 170 – did day and night split evenly into 12 hours each? This is difficult to believe as the study locations and time would suggest that daylight hours would last longer than darkness (approximately 16 hours and 8 hours, respectively). Please instead report the correct periods of day/night -time and account for them in any statistical tests.
Validity of the findings: Study 1 is well-defined and the results are clear. However, I am hesitant to accept the findings from study 2, as the 12 hour day/12 hour night period does not appear to match the actual sunset/sunrise times in Germany during June and July. I suggest that the data is re-analysed using the actual daytime/night-time data, for currently the results do not correspond to the natural environment.
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: SEED TYPE, HABITAT AND TIME OF DAY INFLUENCE POST-DISPERSAL SEED REMOVAL IN TEMPERATE ECOSYSTEMS
Review round: 2
Reviewer: 2

Output:
Basic reporting: Lines 37-39 - Indicate that seed survival is also affected by seed size, dispersal mode, and annual seed production. As this sentence is currently written, it seems like the authors are drawing a distinction between the separate factors limiting seed dispersal and survival.

Line 18 - Consider removing “(micro-)” from before habitat. Given that the authors do not measure micro-habitat factors.

Line 53 - Change “consumption” to “predation” given that consumption itself doesn’t always mean seed death.

Lines 73-74 - Authors should elaborate further on how seed size affects seed dispersal (i.e. do smaller seeds move farther than larger seeds?). Authors could consider giving examples of when this has been found to be true.

Lines 75-82 -  Consider offering examples of each instance. Why do the authors believe that habitat should have an effect.

Lines  83-86 - Further along in the Introduction, the authors make the prediction that seed removal will be highest at night (versus during the day). The authors should add more details to this paragraph to build up to that prediction.

Lines 96-97 - Elaborate here on the range of seed dispersal syndromes and range in size. This could potentially be added within parentheses after each term.

Line 133 - Change “dispersion” to “dispersed” to match grammar used in a, c, and d.

Lines 200-206 - Authors state that seed removal did not differ between habitats in lines 200-201, but then go on to say that intact H. annuus were removed faster in grasslands than in forests. Change the first sentence to indicate this result, otherwise it seems as if there was never a difference detected across habitat types.

Lines 263-264 - This sentence is a bit awkward since the authors start off by saying habitat has no effect and then follow that up with saying but it they found higher removal in grasslands. The sentence could be changed to something like “Typically, habitat had no effect on seed removal, although removal of intact H. annuus was higher in grasslands.

Line 277 - Consider removing the word “different” from before seed types. It will improve the flow of the sentence.

Comments on figures:
Figure 2 - Consider  moving the legend such that it no longer overlaps data points. One option could be to move it to where the seed drawings and descriptions are now located.
Experimental design: Line 131 - Please report the source of your seeds (i.e. did the authors collect them, purchase them, etc?) to aid with providing sufficient detail for replication.

Line 146 - 161 - Here the authors should consider switching the order of these two paragraphs. As a reader, I was left  wondering what a subplot was until the following paragraph, but starting with the description of the sites and layout of the plots would let the reader know right away.

Lines 168-171 - It is unclear if the authors used the same methodology for their seed plates in Experiment 1 to set out the seeds in Experiment 2. Please clarify. Additionally, the authors shift from using the term plate to using the term panel (line 170). Be consistent in terminology to improve clarity. How did the authors choose the location for seed plates in Study 2?

Lines 174-196 - Make sure you include your models’ interaction terms in your descriptions of your statistical model. Additionally, make sure your results include mentioning what your models are showing for the interaction terms. If interaction terms are explaining a significant amount of the variation in your data, you additionally need to explain whether you can interpret the effects significant main effects when significant interaction terms are present.

Lines 183-184 - I think the blocking factor (e.g. the random factor) in the model should instead be plot and not subplot. The authors should fix this or add to this sentence to justify why it should be subplot instead. Additionally, this description should match up with the data provided in the supplemental.
Validity of the findings: No additional comments here.
Additional comments: Did the authors consider the possibility of a non-linear best-fit line for the analysis of seed removal over time (those shown in figure 2). Add whether this was done to your methods/results.

The authors should still consider adding language somewhere that indicates that variation in the traits that they measured may not necessarily be causal owing to the potential collinearity with other unmeasured traits. Additionally, authors should add to the discussion to explain the possibility of whether they think the patterns would  change if they added more species (i.e. would they expect to see the same general patterns if conducting this study at or closer to the scale of the whole plant community (i.e. what would be expected if they added more plant species?).

Authors should add transitions between paragraphs to improve flow of ideas from one paragraph to the next. Occasionally, there are places in the manuscript where grammar could be improved (e.g. lines 118-119 “Since we were interested to compare the two common habitats  forest and grasslands…” could be changed to “Since we were interested in comparing the two common habitats forest and grasslands…”).


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: PHYSIOLOGICAL PARAMETERS AND DIFFERENTIAL EXPRESSION ANALYSIS OF N-PHENYL-N′-[6-(2-CHLOROBENZOTHIAZOL)-YL] UREA-INDUCED CALLUS OF EUCALYPTUS UROPHYLLA × EUCALYPTUS GRANDIS
Review round: 1
Reviewer: 1

Output:
Basic reporting: .
Experimental design: .
Validity of the findings: .
Additional comments: Dear Authors,

Minor comments:

The line 47-48 something is wrong with the text, its much smaller than the rest.
Line 65 Please add space between – Embryogenic callus and (EC)
Line 120 the Authors have to add the name of the company for reverse transcriptase as for now I can see only the name for TRIZOL. In general the part of RNA sequencing have to be written in more details
Line 126 change from – Eucalyptus grandis to E. grandis, because you have already mentioned Eucalyptus before.
Lin3 137 Embryonic or embryogenic?
Line 187-199 No discussion here with the literature data at all!

Major comment

The part of the results is very short and in my opinion have to be combined with discussion. The discussion part is extremely week!

Materials and methods part have to be written in much more details.

Something wrong with the formatting! Please correct it, I can't really see some of the pictures.


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: PHYSIOLOGICAL PARAMETERS AND DIFFERENTIAL EXPRESSION ANALYSIS OF N-PHENYL-N′-[6-(2-CHLOROBENZOTHIAZOL)-YL] UREA-INDUCED CALLUS OF EUCALYPTUS UROPHYLLA × EUCALYPTUS GRANDIS
Review round: 1
Reviewer: 2

Output:
Basic reporting: See attached annotated copy for examples to illustrate these points:
English requires improvement in some parts. See attached annotated copy.
Insufficient references. Some relevant recent references were not included.
REferences style is inconsistent with journal requirements.
Figures and tables are adequate.
Results are explained but need expanding in some parts.
Experimental design: Improve presentation and indicate data anlaysis procedure
Validity of the findings: The finding is of scientific importance but the Discussion section is not well written. See the annotated copy.
Additional comments: I have made specific comments directly in the attached file.


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: PHYSIOLOGICAL PARAMETERS AND DIFFERENTIAL EXPRESSION ANALYSIS OF N-PHENYL-N′-[6-(2-CHLOROBENZOTHIAZOL)-YL] UREA-INDUCED CALLUS OF EUCALYPTUS UROPHYLLA × EUCALYPTUS GRANDIS
Review round: 2
Reviewer: 1

Output:
Basic reporting: .
Experimental design: .
Validity of the findings: .
Additional comments: Dear Authors,

Line 155-157 This is not enough to say they you have the pictures of the calli you have to describe it. This is the results. Please describe the color, the structure of the callus etc. This is quite basic for tissue culture works. For references see:
Betekhtin et al (20016) Spatial Distribution of Selected Chemical Cell Wall Components in the Embryogenic Callus of Brachypodium distachyon
Or
Luciani et al (2006) Effects of explants and growth regulators in garlic callus formation and plant regeneration

Best wishes


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: PHYSIOLOGICAL PARAMETERS AND DIFFERENTIAL EXPRESSION ANALYSIS OF N-PHENYL-N′-[6-(2-CHLOROBENZOTHIAZOL)-YL] UREA-INDUCED CALLUS OF EUCALYPTUS UROPHYLLA × EUCALYPTUS GRANDIS
Review round: 2
Reviewer: 2

Output:
Basic reporting: Satisfactory
Experimental design: Satisfactory
Validity of the findings: Satisfactory
Additional comments: None


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: TOP-DOWN AND BOTTOM-UP CONTROLS ON AN HERBIVORE ON A NATIVE AND INTRODUCED PLANT IN A TROPICAL AGRICULTURAL LANDSCAPE
Review round: 1
Reviewer: 1

Output:
Basic reporting: This is an interesting paper examining the performance of a native herbivore on native and exotic host plant species. The main finding is that on the exotic host, herbivore mortality is higher early in life. In contrast, on native host plants, herbivore mortality is higher on later instars, which was attributed to parasitism. Although the question is important for the study of insect-plant interactions, the manuscript needs substantial improvement.
Overall the paper doesn’t read well, therefore the authors should work on the flow. To facilitate the understanding, it would be important to keep reminding readers that S. myriacanthum is exotic and that S. acerifolium is native. This is particularly important for tables and figures, as well as, the discussion section.
Experimental design: It is stated that the role of habitat in defining the herbivore’s tritrophic niche was tested, however, I do not see how this was tested
Validity of the findings: The main conclusion stated in the paper is that the exotic plant is ameliorating the negative effects of pastures by interacting with a caterpillar. However, all the evidence provided showed that herbivores avoided open areas and performed poorly on plants from open habitats. Therefore, several conclusions are equivocal.

Something to take into consideration is that top-down control is largely influenced by plant traits. Predators and parasitoids would have a hard time finding their prey/host if the plant is sending mix messages. Therefore, the lack of parasitism on exotic hosts may be attributed to host quality, plant quality or plant signals. I am also curious about whether this Ithomiine butterfly sequesters plant defenses. Several Ithomiine species can sequester alkaloids from plants, so is it possible that on exotic hosts they were not parasitized because they were more toxic?
Additional comments: Introduction
Overall the introduction is very confusing, several topics are presented, but they didn’t seem match the actual results. It is stated that the role of habitat in defining the herbivore’s tritrophic niche was tested, however, I do not see how this was tested. If habitat is really being tested than I am concerned with replication because only one ecotone habitat and one pasture were surveyed. How big was the area sampled? How far apart were the plants?
L80: How or why was this species introduced? Wouldn’t this be a colonization event?
L51-52: why would forest loss benefit herbivores and not natural enemies? Is it because herbivores arrive first in the new environment? Please explain
L86-88: What are the hypotheses/predictions?
L80-93: Authors should clearly state their hypotheses and how they were they tested. I was unsure about how the authors tested the effects of habitats, especially because only one plant species occurs in both habitats. One alternative to clarify the hypotheses is to have them followed by the information in lines 94-102.
L105: why unlike most Ithomiine butterflies? These butterflies thrive in old field environments

Methods
Is it possible to correlate plant defenses and herbivore performance? It’s unclear which and how many plants were used for leaf trait analyses. If leaf traits were assessed on the same plants where caterpillars were found, then correlations could be made, and the discussion would be less speculative.
L121-123: Is there any data that would support this argument?
L126: Does this means that 600 plants were surveyed for S. myriacanthum? How many plants actually had eggs/caterpillars on them?
L130-131: Why repeated measures? From the description it sounded like 300 plants per sp were screened, but it didn’t mention that plants were revisited. If a repeated measure analyses was used were the plants marked and revisited? How do you know you are sampling the same individual? Were plants and caterpillars marked?
L133: italicized in situ
L136: Instar?
L139-140: was the habitat temperature and solar radiation recorded per habitat or plant?
L141-143: A GLM with logit distribution would be more adequate
L152: Why was survival analysis not used?
L158-160: Was the position of leaf discs standardized? How leaf was treated in the analyzes? It is stated that 10 leaves per plant was used, but from how many plants? Were the leaves pooled to create one value per plant? This is one reason why it is important to provide degrees of freedom for all statistics.

Results
S. myriacanthum was less colonized (fewer eggs) in open habitats compared to ecotone habitats. S. acerifolium: no comparison between open and ecotone habitats. Caterpillar mortality is attributed to parasitism, but the statistical comparison is not presented. When parasitized caterpillars are removed from the analysis, is mortality different?

L170-173: Please present your F-statistics and degrees of freedom. P-values are not informative.
L173-174: In both habitats?
L175-176: how many plants per habitat were inspected? If more plants are found in ecotone habitats, I assume more eggs will be found there as well, so how was this controlled in the analysis?
L181-183: Where is this result? No figure or table presented this information. Also, when only unparasitized larvae are taken into consideration, was the survival rate different?
L191-195: This result suggests that plant defenses might not be important
L195: What is the SE? Please provide this information for all results
L201: SLA means specific leaf area, so you can delete “leaf”

Discussion
Start your discussion by stating the purpose of the study and the questions and what was the key finding of the study.
L214: What is an important taxonomic trait?
L 220: it is unclear whether parasitism doesn’t occur because parasitoids avoid hosts feeding on S. myriacanthum or just because that plant species harbors lower host density.
L221: What do you mean by population controls?
L226-235: Murphy & Feeny’s paper (PNAS and Ecological monographs) should be included in the discussion of host shift and enemy space.
L236-238: But was this result controlled by the number adults? If adults are not found in pastures, then fewer eggs are expected in pastures as well. This result doesn’t seem surprising, unless there is a difference between the native host. The authors need to discuss alternative hypotheses for this result. Is the habitat effect similar for S. acerifolium?
L247-249: Confusing, please revise.
L250-252: It is unclear how this novel interaction is facilitated by the habitat. This seems an equivocal conclusion. The results showed that a reduced number of eggs and lower herbivore performance in open habitats. Therefore, I don’t understand how this can be beneficial for disturbed landscapes.
L262-264: What kind of edge species? Plants or herbivores? This study did not test the ability of species to colonize pastures.

Table 1: Provide SE and Chi-square value
Table 2: What is overall? Survival from hatching to pupa? Only 9% and 6% of the caterpillars survived?
Fig: 2- It is stated that 300 plants were observed. Were all the plants in the same habitat?
Fig. 3 - Post hoc test comparisons needed. Why was toughness data for open habitat not available?


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: TOP-DOWN AND BOTTOM-UP CONTROLS ON AN HERBIVORE ON A NATIVE AND INTRODUCED PLANT IN A TROPICAL AGRICULTURAL LANDSCAPE
Review round: 1
Reviewer: 2

Output:
Basic reporting: Many sentences were not in professional English and many were structured poorly. For these reasons, this paper was difficult to follow. I attempted to rewrite some sentences and provide example sentences to make the paper read more clearly and with less ambiguity. I have also provided comments asking for additional information.

The authors have sufficient references and background relating to this study. However, the authors could improve the paper by adding a couple sentences defining a host plant and what is meant by the term “novel” (see line 61). I also suggest rewriting the sentence on lines 71-72 to be clearer, see my comment.

The article structure was good. However, I have provided edits and feedback for the tables and figures.

Overall, the paper is meaningful but needs to be written more clearly and in professional English.
Experimental design: I believe this paper has merit for the journal, but I recommend a statistician to review the analyses and results. The research questions should be restated to be clearer (I attempted to rewrite the objectives in the abstract and introduction to give the authors a starting point). The materials and methods section is well organized and detailed. However, the authors should state what statistical software program was used and what alpha (significance level) was set to (i.e. 0.05)—see comments within this section.
Validity of the findings: Findings are rationale and beneficial to literature pertaining to trophic relationships as well as plant-insect interactions. The authors do a good job relating their finding to the study objectives in the discussion and conclusion section. Again, I suggest having a statistician review the analyses and results prior to accepting the paper into the journal.
Additional comments: Suggestions to improve Figures and Tables:

Figure 1: Scientific names imbedded in the photos need to be italicized and ‘photo credit’ needs to be stated in the figure as well. See figure and table referencing in the journal guidelines.

Figure 2: Good figure! Replace the word “stadium” with “stage” and define L1, L2, L3, etc. in the caption (similar to my comments for Tables 1-3). Also, italic the species names within the figure. Overall, the table is clear and can be easily interpreted.

Figure 3: Replace “#” with “Number of” for the appropriate y-axes. Missing a period after the “S” in the species scientific name in the last sentence.

Tables 1: Add a note below the table or in the table description that clearly explains what L1 to L2, L2 to L3, … represents. I also suggest stating L1 to L2 as “Instar 1-2”. Tables should be stand alone and some readers who are not entomologist may not understand the larvae development. Also, add the “rates” to the left of the p-values and put the p-values in parentheses. Please see my comments associated with the table.

Tables 2 and 3: I strongly suggest making the same revisions discussed about Table 1 to Tables 2 and 3.


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

Input:
Title: A SEVEN-YEAR SURVEILLANCE OF EPIDEMIOLOGY OF MALARIA REVEALS TRAVEL AND GENDER ARE THE KEY DRIVERS OF DISPERSION OF DRUG RESISTANT GENOTYPES IN KENYA
Review round: 1
Reviewer: 1

Output:
Basic reporting: The article as it stands, is interesting but needs further improvement to achieve sufficient scientific relevance for publication. The paper describes the frequency of chloroquine resistance markers present in P. falciparum from distinct areas of Kenya throughout 7 years. This gives a broad view of the parasite selection happening in this region.
The resistant markers studied here, although authors emphasize its relevance for chloroquine, introduction should be changed for the importance of this markers in regards to the first line treatment used in Kenya throughout these 7 years. This work shows a clear selection of this markers and its relevance should be discussed towards the present treatment used in the region.
The PfCRT K76T SNP has been considered the major parasite risk factor for partner drug lumefantrine (Sisowath et al. 2009; Venkatesan et al. 2014) that the authors inform to be used in Kenya as one of the first line treatment (artemether-lumefantrine). Regarding PfMDR1 N86Y and Y184F, their impact on lumefantrine has been studied through gene editing strategies (Veiga et al. 2016). Its impact should be discussed considering its selection in Kenya through the years.
Moreover, PfCRT and PfMDR1 are both transporter proteins located in the food vacuole of the parasite. They are thought to regulate the flux of solutes within this membrane. Their functional relatedness is evidenced by the linkage disequilibrium observed between certain combination of alleles (Happi et al. 2006; Holmgren et al. 2006; Sisowath et al. 2009). With this in mind, the manuscript misses a haplotype analysis, through time, of the herein studied alleles.

What’s the second-line treatment, could this influence the selection of the alleles here in studied?

Suggestion: Copy number variation (CNV) of pfmdr1 has been linked to lumefantrine increased tolerance (Sidhu et al. 2006; Veiga et al. 2011). Although its prevalence in Africa is low, it will be interesting to study it. I would recommend at least to mention this type of polymorphism and its contribution to first-line treatment used in Kenya, in the discussion.

The discussion section should contain mentions to the figures and tables described in the manuscript.

Table 3 is not mentioned in any place of the manuscript.
Experimental design: Genotyping of the herein described molecular markers of resistance are not well described. Line 149: A fragment containing the PfCRT K76T SNP was amplified by nested PCR but there is no description of the genotyping discrimination. What was the method used to discriminate between amplified fragments with wildtype or mutant allele? RFLP, sanger sequencing…? What was the control used to discriminate between mutant and wildtype? Line 178: same problem for the markers present in PfMDR1. Authors describe the use of realtime PCR but Amplitaq Gold polymerase is not suitable for realtime PCR. How was the allele discrimination done? Realtime SNP genotyping (if this was the method, PCR conditions described are wrong) or RFLPs or sanger sequencing? What was the control used to discriminate between mutant and wildtype? The reference used to describe the methodology (Akala et al. 2011) is not correct since Akala et al 2011, refers to other papers for the allelic discrimination methodology used in their work.
Validity of the findings: "No comments"
Additional comments: Minor comments:

Line 64: “associated with single nucleotide polymorphisms in Pfcrt 76 gene”. Pfcrt 76 is not the name of the gene. Sentence should be rephrased. Suggestion: “associated with PfCRT K76T single nucleotide polymorphism.”
Line 67: “monitor possible confounding factors concurrently”. Describe some of the relevant confounding factors.

Line 105: “attending outpatient clinics from 2007 to 2014”. Results show data from 2008 to 2014.

Line 106: change “P f” to “P. falciparum”

Line 109: “2ml of blood was collected”… “Additionally, FTA filter paper was used to collect three blood spots”… . in this manuscript blood was only used for molecular analysis and therefore used only the blood collected into the FTA filter paper. For what they have used the 2mL collected blood?

Line 116: “100 ml each”. I believe author meant 100 µl.
Line 132: DNA extraction was done with QIAamp DNA Mini Kit. Therefore, I find it unnecessary to detail the extraction protocol (line 138-line 148) unless its differs from manufacture.

Line 148: “-200C until required.” The symbol used to describe the degrees Celsius is not correct. Please amend that throughout the paper.

Line 149: “Pfcrt 76 genotyping: Pfcrt genotyping was amplified by…”. Again the terminology is not correct. Suggestion: PfCRT K76T SNP analysis: A fragment of pfcrt gene spanning K76T SNP was amplified by…

Line 152: the reference paper for the genotyping condicions described (Lakshmanan et al. 2005), uses reverse transcription PCR followed by nested and sanger sequencing. This methodology might not be the one used in this manuscript once they extracted DNA and not RNA from clinical samples.

Line 169: “para film.”. amend to parafilm.

Line 174: “Negative and positive control was used”. Please describe the type of controls used.

Line 178:  “Pfmdr1 86 and Pfmdr1 184 genotyping. Sequences of Pfmdr1 86 and Pfmdr1 184 genes were…” same comment as the one made for line 149. “of Pfmdr1 86 and Pfmdr1 184 genes” With this sentence it gives the impression the authors don’t know what is a gene and what is an allele!

Line 201: “clinical factors to chloroquine resistance.” Chloroquine resistance was not accessed in this manuscript. I believe the author meant to inform about the genotyping in the region, therefore, I suggest to complete with “clinical factors to chloroquine resistance molecular markers.”
Line 365: Parasite density discussed in this paragraph has been shown to fluctuate even in the very first hours of sample collection (Carlsson et al. 2011) alerting for caution when discussing its burden. Considering this manuscript with 2 figures (figure 2 and 3) on this subject, I suggest to add on the discussion this concern.

Line 415: SP needs detailed description.

Figure 5: the spatial distribution considers samples from the 7 years? If so, and considering the fluctuation of alleles prevalence shown in figure 4, to discriminate spatial distribution only for the recent year since average of all year does not give relevant information.

End of reviewer comments.

References used in the reviewer comments:
Akala HM, Eyase FL, Cheruiyot AC, Omondi AA, Ogutu BR, Waters NC, Johnson JD, Polhemus ME, Schnabel DC, and Walsh DS. 2011. Antimalarial drug sensitivity profile of western Kenya Plasmodium falciparum field isolates determined by a SYBR Green I in vitro assay and molecular analysis. Am J Trop Med Hyg 85:34-41. 10.4269/ajtmh.2011.10-0674
Carlsson AM, Ngasala BE, Dahlstrom S, Membi C, Veiga IM, Rombo L, Abdulla S, Premji Z, Gil JP, Bjorkman A, and Martensson A. 2011. Plasmodium falciparum population dynamics during the early phase of anti-malarial drug treatment in Tanzanian children with acute uncomplicated malaria. Malar J 10:380. 10.1186/1475-2875-10-380
Happi CT, Gbotosho GO, Folarin OA, Sowunmi A, Bolaji OM, Fateye BA, Kyle DE, Milhous W, Wirth DF, and Oduola AM. 2006. Linkage disequilibrium between two distinct loci in chromosomes 5 and 7 of Plasmodium falciparum and in vivo chloroquine resistance in Southwest Nigeria. Parasitol Res 100:141-148. 10.1007/s00436-006-0246-4
Holmgren G, Gil JP, Ferreira PM, Veiga MI, Obonyo CO, and Bjorkman A. 2006. Amodiaquine resistant Plasmodium falciparum malaria in vivo is associated with selection of pfcrt 76T and pfmdr1 86Y. Infect Genet Evol 6:309-314. 10.1016/j.meegid.2005.09.001
Lakshmanan V, Bray PG, Verdier-Pinard D, Johnson DJ, Horrocks P, Muhle RA, Alakpa GE, Hughes RH, Ward SA, Krogstad DJ, Sidhu AB, and Fidock DA. 2005. A critical role for PfCRT K76T in Plasmodium falciparum verapamil-reversible chloroquine resistance. EMBO J 24:2294-2305. 10.1038/sj.emboj.7600681
Sidhu AB, Uhlemann AC, Valderramos SG, Valderramos JC, Krishna S, and Fidock DA. 2006. Decreasing pfmdr1 copy number in plasmodium falciparum malaria heightens susceptibility to mefloquine, lumefantrine, halofantrine, quinine, and artemisinin. J Infect Dis 194:528-535. 10.1086/507115
Sisowath C, Petersen I, Veiga MI, Martensson A, Premji Z, Bjorkman A, Fidock DA, and Gil JP. 2009. In vivo selection of Plasmodium falciparum parasites carrying the chloroquine-susceptible pfcrt K76 allele after treatment with artemether-lumefantrine in Africa. J Infect Dis 199:750-757. 10.1086/596738
Veiga MI, Dhingra SK, Henrich PP, Straimer J, Gnadig N, Uhlemann AC, Martin RE, Lehane AM, and Fidock DA. 2016. Globally prevalent PfMDR1 mutations modulate Plasmodium falciparum susceptibility to artemisinin-based combination therapies. Nat Commun 7:11553. 10.1038/ncomms11553
Veiga MI, Ferreira PE, Jornhagen L, Malmberg M, Kone A, Schmidt BA, Petzold M, Bjorkman A, Nosten F, and Gil JP. 2011. Novel polymorphisms in Plasmodium falciparum ABC transporter genes are associated with major ACT antimalarial drug resistance. PLoS One 6:e20212. 10.1371/journal.pone.0020212
Venkatesan M, Gadalla NB, Stepniewska K, Dahal P, Nsanzabana C, Moriera C, Price RN, Martensson A, Rosenthal PJ, Dorsey G, Sutherland CJ, Guerin P, Davis TM, Menard D, Adam I, Ademowo G, Arze C, Baliraine FN, Berens-Riha N, Bjorkman A, Borrmann S, Checchi F, Desai M, Dhorda M, Djimde AA, El-Sayed BB, Eshetu T, Eyase F, Falade C, Faucher JF, Froberg G, Grivoyannis A, Hamour S, Houze S, Johnson J, Kamugisha E, Kariuki S, Kiechel JR, Kironde F, Kofoed PE, LeBras J, Malmberg M, Mwai L, Ngasala B, Nosten F, Nsobya SL, Nzila A, Oguike M, Otienoburu SD, Ogutu B, Ouedraogo JB, Piola P, Rombo L, Schramm B, Some AF, Thwing J, Ursing J, Wong RP, Zeynudin A, Zongo I, Plowe CV, Sibley CH, Group AMMS, and Wwarn AL. 2014. Polymorphisms in Plasmodium falciparum chloroquine resistance transporter and multidrug resistance 1 genes: parasite risk factors that affect treatment outcomes for P. falciparum malaria after artemether-lumefantrine and artesunate-amodiaquine. Am J Trop Med Hyg 91:833-843. 10.4269/ajtmh.14-0031


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

Input:
Title: A SEVEN-YEAR SURVEILLANCE OF EPIDEMIOLOGY OF MALARIA REVEALS TRAVEL AND GENDER ARE THE KEY DRIVERS OF DISPERSION OF DRUG RESISTANT GENOTYPES IN KENYA
Review round: 1
Reviewer: 2

Output:
Basic reporting: The manuscript addresses a very relevant question on the role of demographic factors for the spread of drug resistance and used comprehensive analyses to identify these factors. The manuscript is written very clearly and raw data is made available.
Experimental design: The study aims to establish the association of demographic factors with the spread of drug (at least chloroquine) resistance in Kenya. This manuscript presents a comprehensive analysis of factors that are associated with CQ resistance. The authors reported response to the question "Ever travelled in the last 2 months", however, the risk of travel is highly dependent on the destination  (eg. malaria prevalence at the destination) and duration (eg. for how long they traveled? how likely are the travelers to get infected at the destination?).

Did the authors collect more information to characterize travel data? Can this be incorporated as a stratified analysis?
Validity of the findings: Minor comment:

1. Line 73: “These factors could consequently be associated with chloroquine resistance.” Do the authors refer to association with the dispersal of resistance? Please clarify

Major comments:

2. Line 219 – Is this log2 transformation? If the parasite density is reported as log10 (as stated in Figure 2 and 3) – Please clarify or correct accordingly. If the authors reported log10 values, the parasite density measured as “the number of parasites diagnosed by microscopy per microliter of whole blood” is extremely high.

In average human blood contains about ~ 5 million RBC per microliter of whole blood (notwithstanding anemia/malnutrition etc), therefore a parasite density of 5,000,000 (in log10=6.7) is almost 100% parasitemia. Although Pf can cause high parasitemia infections (rarely up to 20%) and certainly anything close to 100% (or more, in this case, log10 of 14.2) should be treated with caution. Based on the description provided, I suggest the authors clarify how the data was log-transformed
Additional comments: No additional comments


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

Input:
Title: A SEVEN-YEAR SURVEILLANCE OF EPIDEMIOLOGY OF MALARIA REVEALS TRAVEL AND GENDER ARE THE KEY DRIVERS OF DISPERSION OF DRUG RESISTANT GENOTYPES IN KENYA
Review round: 2
Reviewer: 1

Output:
Basic reporting: No Comment.
Experimental design: No comment.
Validity of the findings: No comment
Additional comments: Line 420: Type error "zones pf Kenya." pf - of
Line 425: "treatment (Carlsson et al., 2011), It is therefore" change the comma for a dot.
Line 448 - 450: The sentence "The implication of Pfmdr1 Y184F mutation has been reported to be minimal on antimalarial drug susceptibilities (Veiga et al., 2016)." should be taken with caution since this relates to specific genetic background and could lead the reader to misinterpret it. I would suggest to delete it since does not complement the discussion taken in this paragraph.


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: DIFFERENCES OF SOIL ENZYME ACTIVITIES AND ITS INFLUENCING FACTORS UNDER DIFFERENT FLOODING CONDITIONS IN ILI VALLEY, XINJIANG
Review round: 1
Reviewer: 1

Output:
Basic reporting: THE paper is a primary data collection,in which authors discuss about water roles under different types of conditions. More revisions are needed to focus on the given target by good logic and recent references.
Experimental design: ok
Validity of the findings: ok
Additional comments: The paper needs major revision in terms of writing,more recent citations and concrete conclusions and recommendations.


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: DIFFERENCES OF SOIL ENZYME ACTIVITIES AND ITS INFLUENCING FACTORS UNDER DIFFERENT FLOODING CONDITIONS IN ILI VALLEY, XINJIANG
Review round: 1
Reviewer: 2

Output:
Basic reporting: This paper is interesting and brings important subject. However, some points should be addressed. The English must be corrected. Some parts of paper are not clear and with no sense. Please check the paper carefully and send it to an English native service.
Experimental design: No comment
Validity of the findings: Wetland is a topic of interest globally, this paper deals with an interesting research area.
The objective is fulfilled, since a very meticulous analysis of the soil microbial biomass and enzyme activity. The work was made with precise and reliable techniques.
It is a work of great importance that generates very valuable information especially for future analyzes that give an account of the state of health of the soils wetlands regions.
Additional comments: This paper is interesting and brings important subject. However, some points should be addressed.

Abstract
I suggest rewriting the results presentation, it's too long and hard to understand.
I suggest a conclusion in the Abstract.
Line 18 - Water is an important component of wetland.
This sentence isn´t necessary.
Line 48 and 49 - Keywords: Yili valley; wetland; flooding conditions; differences in enzyme activity; microbial biomass carbon.
Remove differences in

Introduction

Line 77 - The name of the valley is incorrected.

Line 81- soil is with lowercase letter

Line 106 - Sampling point setting and field sample collection
I suggest change this section to “Study site and sample collection”.

Line 111-113 - The flooding period of one year is about 2-3 months, while PAW belongs to long-term flooded habitat, and the flooding.
Which months? Year? Precipitation data.

Line 116 – How soils samples were collected?

Line 126 – Research method
I suggest change this section to “Laboratory analysis or Analysis of soil properties”

Line 127 - Soil basic physical and chemical properties
Remove the word basic.

Line 128 – I suggest: "Soil organic carbon (SOC) was analyzed or determined by REFERENCE method."

Line 138 – Remove the word soil.

Line 139- I suggest: "Microbial biomass C was analyzed or determined REFERENCE method."

Line 144 - Remove the word soil

Line 145 - I suggest: " Urease, catalase, sucrase activities were analyzed according to the methodology used previously by REFERENCES”.

Line 161- Remove the word basic.

Results

I felt that the authors could have explored the results more and discussed their findings more thoroughly. Particularly, I believe that the authors should highlight how their results compare to other wetland studies.
Examples: Shao et al. 2015 (Seasonal Dynamics of Soil Labile Organic Carbon and Enzyme Activities in Relation to Vegetation Types in Hangzhou Bay Tidal Flat Wetland - doi.org/10.1371/journal.pone.0142677); Ma et al. 2018 (Bacterial and Fungal Community Composition and Functional Activity Associated with Lake Wetland Water Level Gradients - doi: 10.1038/s41598-018-19153-z); Gu et al. 2019 (Soil Enzyme Activity in Soils Subjected to Flooding and the Effect on Nitrogen and Phosphorus Uptake by Oilseed Rape - https://doi.org/10.3389/fpls.2019.00368)
Line 202 – Remove “(MBC) under different flooding conditions”.

Line 221 - Remove “in wetlands under different flooding conditions”.

Line 332 – I suggest a review about this reference because this paper (Powlson et al. 1987) it is not specific about this subject. The reference can be used in the discussion in another way.

Conclusions
Conclusions should be improved.

References
References format needs to be revised.

Line 445 – This reference needs to be corrected

Table 5 – correct the legend

Figure 3 - correct the legend


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: DIFFERENCES OF SOIL ENZYME ACTIVITIES AND ITS INFLUENCING FACTORS UNDER DIFFERENT FLOODING CONDITIONS IN ILI VALLEY, XINJIANG
Review round: 1
Reviewer: 3

Output:
Basic reporting: The manuscript needs to be revised thoroughly, in which a language advisor should be involved.
Experimental design: No comments
Validity of the findings: No comments
Additional comments: 1)	The first part of material and methods, which gave depiction on Yily valley should be integrate into Introduction;
2)	Sampling site has been showed in a map, however, this is no coordinate. These data should be provide.
3)	As stated in the introduction, there is a risk of soil degradation threatening Yily Valley eco. However, in the discussion part, the results have not been discussed together with any aspect overlapping with the risk;
4)	There are some advises in attachment.


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: DIFFERENCES OF SOIL ENZYME ACTIVITIES AND ITS INFLUENCING FACTORS UNDER DIFFERENT FLOODING CONDITIONS IN ILI VALLEY, XINJIANG
Review round: 2
Reviewer: 1

Output:
Basic reporting: The authors made corrections as suggested, which made the article more complete and easy to read.
Experimental design: All correction suggestions have been accepted, the paper is now complete.
Validity of the findings: The authors made corrections as suggested, which made the paper complete.
Additional comments: I would like to say that for my part no compliment is needed in its correction.
Congratulations to the authors for their dedication.


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: PROGNOSTIC ANALYSIS OF HISTOPATHOLOGICAL IMAGES USING PRE-TRAINED CONVOLUTIONAL NEURAL NETWORKS: APPLICATION TO HEPATOCELLULAR CARCINOMA
Review round: 1
Reviewer: 1

Output:
Basic reporting: While I believe the underlying concepts of transfer learning was appropriately applied, I have several concerns about the study.

-Please clarify the building and training process of concatenated networks. In my experience, random forest has been generally used to integrate feature information. Could the authors compare the proposed aggregation method and random forest? "The possibility of the combination of OCT and fundus images for improving the diagnostic accuracy of deep learning for age-related macular degeneration: a preliminary experiment" described the random forest feature aggregation.

-Three subplots in Figure 3 look the same to each other. The differnece should be emphasized.

-The configuration of Table 2 looks abnormal. Please review this table to be clear.
Experimental design: -Recent deep learning studies have revealed CAMs or attention maps. If possible, demonstrate the feature map examples for HCC sampels. Is there new features for HCC? More specific demonstration for deep learning application to histopathology is needed becuase the figures and discussion about deep learnign are too general.

-Is there other clinical information such as age, gender, diabetes, and hypertension? The combination of clinical information and deep learning features will
improve the prediction accuracy in survival analysis.

-The comparison between the traditional training model (using ADAM optimizer) and pretrained model should be discussed.
Validity of the findings: -PARADIGM combined with deep learning should be explained in more detail. Is it the first research to use both image-based deep learning and pathway analysis in HCC samples? The combination of deep learning and PARADIGM was the most interesting part in this manuscript, however the novelty was not highlighted. Please strengthen the explaination about Figure 6, because it failed to demonstrate the meaning of this study. Which pathways were activated and were associated with deep learning features (in  attention maps)?

-Please disccuss about recent AI researches in the histopathology fields, such as "Similar image search for histopathology: SMILY" from Google.

-Study limitations were not disccussed. No external validation, relatively small number of dataset, and no multiclass grading should be addressed.
Additional comments: Although this study contain a novel approach for HCC samples, the manuscript looks old-fashioned. I recommend a more comprehensive exploration about interpretability in histopathological images and pathyway analysis.


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: PROGNOSTIC ANALYSIS OF HISTOPATHOLOGICAL IMAGES USING PRE-TRAINED CONVOLUTIONAL NEURAL NETWORKS: APPLICATION TO HEPATOCELLULAR CARCINOMA
Review round: 1
Reviewer: 2

Output:
Basic reporting: 1.	Although this paper is generally well written, further proofing is required. For instance, text in lines 52-53 and in 165 is nonsensical.

2.	Figure 1 could be much clearer and better presented. Moreover, for other figures some axis labels are missing. The text to the far right in Figure 6 appears to be misaligned.

3.	Table 1 and 2 require re-formatting. Table 2 appears to be incomplete.

4.	I would suggest segregating the Background section into two sections entitled Introduction/Background and Related Works.
Experimental design: 1.	VGG-16, Inception V3 and ResNet-50 are quite large CNNs, and as I’m sure you are aware, they are quite computationally expensive. An ablation study is necessary to determine if simpler networks can be used to achieve similar performance. It’s difficult to deduce if the models used are well-fitting. Does an increase in regularization (by means of drop-out or by other regularization techniques) decrease or increase performance?

2.	It is not entirely clear how the (89640 x 35870) and (5601 x 22495) images were down-sampled and randomly cropped to (256 x 256) and (512 x 512). During the down-sampling process, was the aspect ratio of the input images preserved? Inputs to VGG-16, Inception V3 and ResNet-50 commonly have a size of (3 x 224 x 224). At what image-preprocessing stages were images down-sampled and/or cropped, and what image sizes were fed into each network?

3.	The image augmentation steps need to be more clearly detailed. How exactly was strain-color optimization performed? What exact modifications were made to images to augment their color? To what extent does image augmentation affect performance? I would suggest comparing performance with and without further image-augmentation.

4.	The authors state that ‘We used these models to extract features in an unsupervised manner to avoid the challenges of training an entire CNN model scratch’. I believe that transfer learning could easily be used to improve the performance of the networks used- if this is to be left to future works, I suggest that this is stated transparently. While the usage of transfer learning is briefly discussed in the Discussion section, the authors do not make mention of Generative Adversarial Networks (GANs) or other systems that can be used to generate synthetic images.

5.	How was the C parameter of the linear Support Vector Machine (SVM) determined?

6.	ResNeXt has been demonstrated to obtain better results on both the ImageNet-5K set and the COCO detection set than its ResNet counterpart. Is there any reason as to why ResNet was used instead of ResNeXt?
Validity of the findings: No comment. Based solely on the approaches and methodology used, the findings are well stated, robust, statistically sound, controlled, and adequately linked to the research aims.
Additional comments: This paper uses pretrained Convolutional Neural Networks (CNNs) to extract features from Hepatocellular Carcinoma (HCC) histopathological images, and makes attempts to correlate them with relevant biological pathways. I would like to commend the authors on their efforts. The paper is interesting and well-written; however, I believe my questions and concerns should be addressed prior to publication.


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: PROGNOSTIC ANALYSIS OF HISTOPATHOLOGICAL IMAGES USING PRE-TRAINED CONVOLUTIONAL NEURAL NETWORKS: APPLICATION TO HEPATOCELLULAR CARCINOMA
Review round: 2
Reviewer: 1

Output:
Basic reporting: I appreciate the authors for the effort to revise manuscript.
Experimental design: no addtional comments
Validity of the findings: fig4 & 5
groups were described as "0" and "1".
they should be corrected.
Additional comments: The title is too broad.
I think "HCC" should be commented.


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: PROGNOSTIC ANALYSIS OF HISTOPATHOLOGICAL IMAGES USING PRE-TRAINED CONVOLUTIONAL NEURAL NETWORKS: APPLICATION TO HEPATOCELLULAR CARCINOMA
Review round: 2
Reviewer: 2

Output:
Basic reporting: The authors' have addressed all my comments.
Experimental design: I would like to acknowledge the authors' efforts towards addressing my comments, however, I still have some concerns:

1.	An ablation study is still required- do simpler and/or smaller pretrained CNNs perform similarly to VGG-16, Inception V3 and ResNet-50?
2.	Pretrained ResNeXt models for Keras are easily accessible: https://github.com/qubvel/classification_models. I suggest that the authors use either ResNeXt-50 or ResNeXt-101 instead of ResNet-50, which should improve performance.
3.	Why was ResNet-50 used instead of ResNet-18, 24, 101 or 152? Does it exhibit better performance?
Validity of the findings: The findings are well stated, robust, statistically sound, controlled, and adequately linked to the research aims.
Additional comments: I would like to acknowledge the author’s efforts towards addressing my comments. Although the manuscript has significantly improved, I still have some concerns.


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

Input:
Title: PROGNOSTIC ANALYSIS OF HISTOPATHOLOGICAL IMAGES USING PRE-TRAINED CONVOLUTIONAL NEURAL NETWORKS: APPLICATION TO HEPATOCELLULAR CARCINOMA
Review round: 3
Reviewer: 1

Output:
Basic reporting: I appreciate the authors for revision and codes. Based on this research, many researchers will be inspired for histopathological image analysis.
Experimental design: MobileNet is a good option for the light-weight model.
No further comment.
Validity of the findings: No further comment.
Additional comments: No further 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: IN VITRO ANTICANCER ACTIVITY OF METHANOLIC EXTRACT OF GRANULOCYSTOPSIS SP., A MICROALGAE FROM AN OLIGOTROPHIC OASIS IN THE CHIHUAHUAN DESERT
Review round: 1
Reviewer: 1

Output:
Basic reporting: Structure of the manuscript is acceptable.
Experimental design: There are general screening experiments toplam understand apoptotic effect of the extract.
Validity of the findings: Findings are acceptable for the manuscript.
Additional comments: The manuscript is acceptable but it needs more detail abort signal transduction of the extract. Discussion should be extended.


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: IN VITRO ANTICANCER ACTIVITY OF METHANOLIC EXTRACT OF GRANULOCYSTOPSIS SP., A MICROALGAE FROM AN OLIGOTROPHIC OASIS IN THE CHIHUAHUAN DESERT
Review round: 1
Reviewer: 2

Output:
Basic reporting: Clear and unambiguous, professional English used throughout
Literature references, sufficient field background/context provided
Professional article structure, figures, tables.
Experimental design: aim of work is very clear but it can be complete by identification of bioactive compounds that found in the methanolic extract
Validity of the findings: the novelty of this study can be complete by connect the cytotoxicity by compounds that cause that, but the work is very important in isolation, purification and identified Granulocystopsis sp. and  by morphological (where is the reference of morphological identification?) and genetic. then extraction bioactive metabolites, then study the effect of this extract on cancer cell lines.
Additional comments: Title: In vitro anticancer activity of methanolic extract from Granulocystopsis sp., a microalgae from an oligotrophic oasis in the Chihuahuan desert
Very expressive
Abstract: Lines 26 – 29: Delete "Many types of biologically active components have been identified in microalgae and used in medicinal applications, but the success of biotechnological potential rises when biological diversity allows the discovery of novel chemical diversity such as the one that can be found in extremophiles or endemic organisms of untapped environments". This is abstract not introduction begin with the objective or the aim of work followed by summarized methodology and results and end by brief conclusion. The abstract should be rewrite.
Key words: very long, can you summarized it"cytotoxic activity=anticancer activity" also if you write microalgae you should not write Granulocystopsis
Introduction:  Prepared well, but it is very long
Materials and methods: writing very well but it should summaried
Also, where is the reference and manuals of morphological identification?
Results: the methanolic extract contains mainly ohenolic compounds. why the authors did not identified the compounds in this extracts using HPLC-MS or at least identified the phenolic compounds by HPLC.
Conclusion: very long, summaried it main points that observed the impact of your study and the novelty of your data. The conclusion should be rewrite.


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: IN VITRO ANTICANCER ACTIVITY OF METHANOLIC EXTRACT OF GRANULOCYSTOPSIS SP., A MICROALGAE FROM AN OLIGOTROPHIC OASIS IN THE CHIHUAHUAN DESERT
Review round: 1
Reviewer: 3

Output:
Basic reporting: The title should be changed to: In vitro anticancer activity of methanolic extract of
Granulocystopsis sp., a microalgae from an oligotrophic oasis in the Chihuahuan desert.
Generally, it is an important idea and can improve anticancer drugs by using natural resources from algae. but it needs to improve the languages. A lot of verbs in present and it should be in past tense.
Phrases are too long and should be more clear and shorter as in the abstract (Example: First one phrase).
Using the form we is not appropriate and it is better to use the passive tense.
Line 31: delete "we decide" and add "was conducted" in line 32 after the word"Mexico.
lines 32-26: too long phrase
line 40: mention to the methanolic extract
line 40: of instead of from.
41: except that in
41: MTT should be written in whole abbreviation
84-88: rewrite and correct grammatically
91: "methanolic extract instead of" instead of "methanolic extract from"
126: we is not appropriate
225: write the reference of the manufacture instruction
Experimental design: Is well designed but should be in brief not like in thesis.
Reference of morphological identification should be added
Validity of the findings: good but the titles of all finding should not be written as a normal phrases. Titles should not contains verbs.
Additional comments: Check it grammatically well and carefully


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: TUMOUR-ASSOCIATED MACROPHAGES MEDIATE THE INVASION AND METASTASIS OF BLADDER CANCER CELLS THROUGH CXCL8
Review round: 1
Reviewer: 1

Output:
Basic reporting: The English language should be improved to ensure that an international audience can clearly understand your text. Some examples where the language could be improved include sentence in introduction and discussion part. The reference, table, and figures are appropriate expect figure 2a.
Experimental design: no comment
Validity of the findings: no comment
Additional comments: This manuscript submitted by Prof. Tian aimed to investigate the effect of TAM on invasion and metastasis in bladder cancer and potential mechanism. After searching the Pubmed, it is the first report on TAM mediates invasion and metastasis through CXCL8. Although it is of some significance for treatment of bladder cancer, but there are some comments:
1.	They should analysis relationship between expression of CXCL8 and clinic factors.
2.	It is better to show the survival data.


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: TUMOUR-ASSOCIATED MACROPHAGES MEDIATE THE INVASION AND METASTASIS OF BLADDER CANCER CELLS THROUGH CXCL8
Review round: 1
Reviewer: 2

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: This study is novel, the author investigate and clarify the tumour-promoting role of CXCL8 secreted by TAMs in the tumour microenvironment of urothelial carcinoma of the bladder. And find tumour-associated macrophages mediates invasion and metastasis of bladder cancer cells through CXCL8.
Questions
1.Fig1, could the author show us the table, we want to see the expression of these marker in the patients
2.Fig3, the background of this pictures are not the same, and please provide us the westren blot data
3.Fig4 and fig5 please provide us the western blot data


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: TUMOUR-ASSOCIATED MACROPHAGES MEDIATE THE INVASION AND METASTASIS OF BLADDER CANCER CELLS THROUGH CXCL8
Review round: 1
Reviewer: 3

Output:
Basic reporting: Wu and colleagues in their manuscript titled “Tumour-associated macrophages mediates invasion and metastasis of bladder cancer cells through CXCL8” studied the role of CXCL8 derived from TAMs in the para-epithelial carcinoma network of the human urinary tract in the bladder tumour microenvironment and also analyzed the critical role and biological effects of TAMs-derived CXCL8 on bladder cancer cell production.
Experimental design: The experimental design is good. How did the authors achieve 100x and 400x magnification? The clarity of the figures is not good.
Validity of the findings: The findings are good and in line with the known roles of CXCL8 in other cancers. Please discuss these results based on the paper “ShRNA-mediated knock-down of CXCL8 inhibits tumor growth in colorectal liver metastasis” where the authors showed  that CXCL8 secreted by tumor cells at the invasion front were able to promote migration through angiogenesis by upregulating VEGFA and invasion via the AKT/GSK3β/β-catenin/MMP7 pathway by upregulating BCL-2 confirming the key role of CXCL8 during tumor progression
Additional comments: The abstract needs to be rewritten briefly. It looks like material and methods section.


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

Input:
Title: TUMOUR-ASSOCIATED MACROPHAGES MEDIATE THE INVASION AND METASTASIS OF BLADDER CANCER CELLS THROUGH CXCL8
Review round: 2
Reviewer: 1

Output:
Basic reporting: No comments
Experimental design: No comments
Validity of the findings: No comments
Additional comments: The authors have changed the manuscript according to the comments, I think it is acceptalbe in its current version.


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: TUMOUR-ASSOCIATED MACROPHAGES MEDIATE THE INVASION AND METASTASIS OF BLADDER CANCER CELLS THROUGH CXCL8
Review round: 2
Reviewer: 2

Output:
Basic reporting: N/A
Experimental design: N/A
Validity of the findings: N/A
Additional comments: The authors have answered all my questions.


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: TUMOUR-ASSOCIATED MACROPHAGES MEDIATE THE INVASION AND METASTASIS OF BLADDER CANCER CELLS THROUGH CXCL8
Review round: 2
Reviewer: 3

Output:
Basic reporting: Th authors have answered all my queries and the manuscript has improved and no further comments from me.
Experimental design: Th authors have answered all my queries and the manuscript has improved and no further comments from me.
Validity of the findings: Th authors have answered all my queries and the manuscript has improved and no further comments from me.
Additional comments: Th authors have answered all my queries and the manuscript has improved and no further comments from me.