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: ARABIDOPSIS THALIANA EXUDATES INDUCE GROWTH AND PROTEOMIC CHANGES IN GLUCONACETOBACTER DIAZOTROPHICUS
Review round: 1
Reviewer: 1

Output:
Basic reporting: Cruz dos Santos and col. submitted the manuscript “EttA and AccC are essential for the response of Gluconacetobacter diazotrophicus to Arabidopsis thaliana exudates”, which deals with the physiological and proteomic characterization of G. diazotrophicus response to root exudates from A. thaliana. G. diazotrophicus PAI5 is a model bacterium for the study of the interactions between non-symbiotic diazotrophic bacteria and plants, in particular, monocot plants. Several aspects of the interaction with sugarcane, the natural host of G. diazotrophicus, are known. However, the growth promotion exerted by G. diazotrophicus on the model plant A. thaliana allows a deeper characterization of the interaction mechanisms that take place between plants and beneficial bacteria. This is the main focus of the manuscript and the main strenght of the results presented by the authors.
Cruz dos Santos and col. first analyzed the bacterial growth in a culture medium were A. thaliana was growing, finding that the bacterial growth was enhanced, and that the production of plant metabolites increasing the bacterial growth was constitutive. After analyzing the bacterial proteome, the authors found a group of proteins up- and down-regulated by the plant exudates. Finally, the authors compared the response to plant exudates of bacterial mutants in selected genes, reporting that the proteins EttA and AccC are essential for the growth increase of G. diazotrophicus.
The manuscript is clearly written and the well structured according to PeerJ standards.
For this Basic Reporting section of the review, in my opinion, several aspects should be revised.
-Manuscript title put the focused specifically in the results from the last experiment, while the main findings are the overall proteomic results. The title could be reformulated in this sense.
The Introduction section is very general. The authors could present specific information of what is already known about determinants from G. diazotrophicus involved in the interactions with plants.
Figures are relevant, though Figure 2 could be included as supplementary material.
Experimental design: Results presented in the manuscript are interesting and deserve attention, considering the importance of G. diazotrophicus as a model bacterium.
Materials and Methods are well described. A few changes could improve the understanding of the methodology and the results.
-Authors should provide details about the preparation of the bacterial inoculums [Lines (L.) 115-117]. Were the cells washed before inoculation to prevent carryover of spent medium? Were the cells starved? These are key questions for understanding the proteomic response, considering the chemical differences between DYGS medium and Murashige Skoog medium.
-In L. 113-116, it is not clear how many seedlings were utilized in each Petri dish.
-In the subsection 2.4.1, it is not clear the protocol for the protein extraction. According to L. 128-129, the authors did not follow standard protocols for cell disruption, treating the cell pellets directly with an “extraction buffer” composed of trichloroacetic acid, acetone and DTT. This solution would hardly allow a good extraction of intracellular proteins, which could explain why only 450 proteins were indentified (L. 269).
-The utilization of STRING database and the parameters utilized (including the type of interaction analyzed) should be mentioned in the “2.4.4 Proteomic data analysis” subsection (L. 184).
Validity of the findings: As stated before (see other sections of this review), the aims of the work and the results obtained by the authors are interesting and valuable.
In the Results and in the Discussion sections, the authors should consider several points that can highly improve the manuscript:
- How do the culture conditions influence the results and, in consequence, the conclusions? According to what is described in Materials and Methods section, G. diazotrophicus strains were first precultured in DYGS medium (which is composed of peptone and yeast extract, among others), and then utilized to inoculate MS medium (mainly composed of mineral salts). Culture conditions are key aspects to be considered when comparing the growth of the wild type strain and the mutants affected in protein synthesis, the pentose phosphate pathway and fatty acid synthesis.
-It is not clear the criteria to select the mutants to be analyzed.
-Two Glucose-6-phosphate 1-dehydrogenase homologs were identified as significantly up-regulated by LC/MS, which correspond to the Accessions A9H0G0 and A9H326 (a third homolog not identified by LC/MS is also present in the genome of G. diazotrophicus). Which one was utilized in the experiments? How does the presence of the second homolog influence the results? In addition, in L. 381-383 these two homologs are mentioned as Zwf and GDI_3177.
- Only few proteins were considered in the Discussion section.
- Besides, it is not clear why, for instance, RpsA and RplW are considered in the Discussion and not others also related to protein synthesis.
- L. 343-347. MetE is involved in the Activated Methyl Cycle and is related to the production of AI-2 quorum sensing (QS) molecule in different microorganisms. However, similar to the rest of the Alphaproteobacteria, G. diazotrophicus lacks a LuxS homolog required for AI-2 synthesis. In consequence, the up-accumulation of MetE should not be related to QS activity.
- L370-372. The authors considered that after 24 of cocultivation of G. diazotrophicus with A. thaliana, the bacterial cells were under energy depletion, which is difficult to consider since the cells seems to be still in exponential phase, according to Figure 1. In any case, if according to the literature EttA is related to energy depletion, would it be expectable to find this protein down-accumulated in the presence of the plant, since root exudates provide extra carbon and energy sources?
- As mentioned in L291-292, ClpX plays a role in protein degradation. However, ClpX is the ATP-binding subunit of the ClpXP protease, and in consequence does not show degradation activity on acyl homoserine lactones (L292-295). Interestingly, it has been shown that a mutation in the ClpX coding sequence produces an increase in acyl homoserine lactone production by Burkholderia cenocepacia (see Microbiol. Res. 2016, 186-187:90-98). However, to date it is not clear the mechanism.
-The authors should include in the Discussion section about AccC, the presence of a second accC homolog in the genome of G. diazotrophicus.
Additional comments: No 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: ARABIDOPSIS THALIANA EXUDATES INDUCE GROWTH AND PROTEOMIC CHANGES IN GLUCONACETOBACTER DIAZOTROPHICUS
Review round: 1
Reviewer: 2

Output:
Basic reporting: The paper entitled 'EttA and AccC are essential for the response of Gluconacetobacter diazotrophicus to Arabidopsis thaliana exudates' is very interesting. It explores the interaction between G. diazotrophicus and the model dicot Arabidopsis. The authors determine the bacterial translation response to the plant exudates. Their findings are very original.
Experimental design: My only concern is related to the complete demonstration of the participation of EttA and AccC in the bacterial response. I would ask the authors to either complement the mutants or resolve if the ettA and accC messengers do not include other transcripts or at least show in a map that those genes are not followed by other transcripts in operons.
Validity of the findings: The findings are almost validated, but it is necessary to be sure that only ettA and accC and not any other products are involved.
Additional comments: I have the additional following questions,

-were the seeds germinated in a liquid medium?

-why were the plantlets maintained under light all the time without darkness periods?

-Were the roots of the plantlets covered from the light?

Comments

I also would ask the authors to compare the published results of the G. diazotrophicus-sugarcane interaction with their results.

I would ask to discuss on transporters, like TonB which is also highly up-accumulated.

Line 216, "2.3" instead of "4.3"?

Line 223, "section 2.5" instead of "section 4.5"?


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: ARABIDOPSIS THALIANA EXUDATES INDUCE GROWTH AND PROTEOMIC CHANGES IN GLUCONACETOBACTER DIAZOTROPHICUS
Review round: 2
Reviewer: 1

Output:
Basic reporting: Tamires Cruz dos Santos, Gonçalo Apolinário de Souza Filho and col. submitted a revised version of the manuscript “Arabidopsis thaliana exudates induce growth and proteomic
changes in Gluconacetobacter diazotrophicus” (firstly entitled EttA and AccC are essential for the response of Gluconacetobacter diazotrophicus to Arabidopsis thaliana exudates).
I have carefully read the revised version and the corresponding letter with the point-by-point responses. I agree with all the changes the authors made on the manuscript, and I consider that it has been highly improved. I only have very minor comments that I include in the "General comments for the author" section.
Experimental design: No additional comments.
Validity of the findings: No additional comments.
Additional comments: Minor comments
- In Lines 104-105, the words “three times” are repeated.
- Use italics for A. thaliana in Lines 320, 322 and 413.
- Use lower case for “xylinus” in Line 334.
- Use upper case for Gram in Line 439.
- Check the references 18, 23, 24, 28, 37, 38, 47, 48, 49.


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: ARABIDOPSIS THALIANA EXUDATES INDUCE GROWTH AND PROTEOMIC CHANGES IN GLUCONACETOBACTER DIAZOTROPHICUS
Review round: 2
Reviewer: 2

Output:
Basic reporting: The authors have achieved the main concerns. My opinion is that the manuscript has the quality to be accepted for publication.
Experimental design: No comment
Validity of the findings: No comment
Additional comments: I consider that the authors have included most of the demands.


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: ENERGY BALANCE OF FOOD IN A DETRITO-BRYOPHAGOUS GROUNDHOPPER (ORTHOPTERA: TETRIGIDAE)
Review round: 1
Reviewer: 1

Output:
Basic reporting: 1. First check on figures: ok
2. First check on tables:

Table 3 – I have not understood Table 3: apparently the authors opted to organize a three-way table (actually 3 x 2 = six way, i.e., three types of food, multiplied by two sexes), in a 4 by 2 table (dis-considering the two variables “Weights of defecated feces” and “Energy food budgets”, which led the table to be pretty confusing and difficult to interpret. I would suggest a more straightforward organization, with the type of food organized as three distinct columns, sex as two lines, in two horizontal groups of lines, one pair (males and females) for “Weights of defecated feces” and another pair (males and females) for “Energy food budgets”. If I misunderstood the table, I am terribly sorry, but this denotes that it has a rather awkward presentation.

Table 4 – Here again, the author opted for a the same awkward organization used in Table 3. Please, I would ask the authors to consider remodeling of these tables’ organization.

Table 5 – Here the table’s organization is much better!

Tables were excessive and confusing. I recommend that the authors substitute most table by figures, showing more clearly what treatment levels (Food items) were different from each other. It was not possible to understand clearly the results of the a multiple average tests (Tukey multiple comparisons). I would suggest that, in state of this approach, the authors  could use a more straightforward contrast analysis (see, for example, Crawley (2005): chap. 11), testing explicit hypotheses. They would probably also be a posteriori, but the presentation and interpretation of the results would be easier to follow, and thus, to interpret.

The authors presented two figures, which presented quite interesting and relevant results, but awkwardly they cited them only in the discussion section. I would recommend them to include the citation and presentation of these figures in the results section.

I would suggest that, after transforming all or most of the tables in figures, the authors should reword the manuscript, so as to make it more clear. Furthermore, I would suggest them to ask for a thorough native English speaker review, so as to facilitate comprehension by the reader.

I considered the experiment and the results extremely relevant and interesting, but the wording and presentation of the manuscript were very confusing and difficult to follow. At the same time, the text was repetitive. Worst of all, the results were not clearly presented, neither in the text, nor, much worse, in the tables.

I would encourage the authors to invest in this manuscript, through a more straightforward  statistical analysis and presentation of the results, and through a rewording of the text, so as to make it more direct and clear.
I would like to emphasize that the manuscript is scientifically valid and suitable, but it requires further improvement in the presentation and wording, so as to be more clear to the readers.
Raw data can be opened, but are not in English, but Czech. This is not a critical problem, as far as the interested reader can use automatic translator sites, which work reasonably well, especially considering there are few and rather simple words, whose meaning can be checked and eventual translation errors can be pinpointed.

English language  is not clear, difficult to understand and rather repetitive. I would recommend that the authors ask for a native English revision.

Author comments are impressively better written that the manuscript. I would be happy to find such wording along the manuscript itself.
Experimental design: I missed an explicit presentation of tested hypotheses, and an explicit connection between the research questions and the methodology. It was difficult to understand which were the so-called feeding groups (there were three diets, as far as I understood – detritus and two moss species): the authors mentioned 240 individual groundhoppers, being 20 males and 20 females in each feeding group, which sums to 120 individuals, not 240. I certainly missed something here. This seemingly minor doubt shows the difficulties I had to understand the experimental design. By the way, the authors seem to have confounded experimental design, related to replications, factors and factor levels (treatments, sex) with the description of the laboratory methodology itself.
Validity of the findings: The findings are valid and relevant. Very little is known on the diet and energy balance of feeding in orthopterans (although there were the publications cited by the authors, including themselves). Such information is of utmost importance to understand the ecological processes involved in this insects’ survival, besides providing information that can be extrapolated, certainly with caution, to other related insects, for example other orthopterans that also inhabit the same microhabitats.
Additional comments: I would recommend the acceptance of this manuscript, but not in the present form. The manuscript necessitates thorough rewording, reduction, reorganization and probably substitution of most tables by figures, inclusion of the figures in the results section – and consequently, inclusion of the reasoning, hypotheses and methodologies for the comparisons, in the appropriate sections, as well as a thorough English revision, so as to facilitate comprehension. Nevertheless I would instate the authors to invest this effort to ameliorate the manuscript, and thus, provide an important contribution to science on orthopterans and insect ecology.


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: ENERGY BALANCE OF FOOD IN A DETRITO-BRYOPHAGOUS GROUNDHOPPER (ORTHOPTERA: TETRIGIDAE)
Review round: 1
Reviewer: 2

Output:
Basic reporting: The authors have studied the energy balance of Tetrix subulata in the lab. The results are very interesting and worth publishing. However, there are numerous linguistic issues with the manuscript. I have made some suggestions for changes below, but it would be great if could be checked by a native-speaker. There are only a few sentences, which do not need corrections. I found the discussion rather weak. In most cases, only the own studies are cited, while the content of similar studies on insects is not well integrated in the text (just a list is provided).
Experimental design: The experimental design of the study was good and the research question was interesting. The methods are described with sufficient detail.
Validity of the findings: The findings are quite novel and provide new insight into the feeding ecology of groundhoppers. The underlying data are robust and statistically sound. The conclusions are well stated and linked to the original research questions (although there are a couple of redundancies in the dicussion). The authors also present some alternative hypotheses regarding the value of bryophyte feeding in this species.
Additional comments: Specific comments:
L 23: Either it should be “budgets of detrito-bryovorous grasshoppers” or “budget of the detrito-bryovorous grasshopper”.
L 23: Not clear what is meant here with “comparable with the small herbivorous grasshoppers” (is this related to other grasshopper species?)
L 26: Add “The” before “groundhopper”.
L 28: Replace “passes” by “pass”.
L 30: Replace “are unimportant source” by “are not an important source”.
L 30-31: Not clear what is meant with “whereby their significance in the diet is different”.
L 35: Replace “to make sense of” by “understand”.
L 36: Can you replace “the relevant inputs” with something more precise?
L 42-43: Replace “and they combine various choose in quality or quantity” by “, which may vary substantially in quality or quantity”.
L 44-45: Better start this sentence with “However,” and add a reference at the end of it.
L 46: Replace “show” by “have a”.
L 49: Replace “conditioned by” by “as a result of”.
L 49: Add “The” before “proportions”.
L 50: Replace “is” by “are”.
L 50: Replace “occupied” by “occupying”.
L 52: Replace “of moss tissues” by “moss tissue”.
L 53: Replace “of residual matters” by “residual matter”.
L 55: Replace “moss tissues” by “moss tissue”.
L 67: Replace “near rivers on the moist habitats (wet meadows)” by “in moist habitats, such as wet meadows, river banks, ephemeral ponds etc.”
L 73: Replace “of meadow” by “of a meadow”.
L 76: Replace “by” by “using the”.
L 80: Replace “had been” by “were”.
L 82: Replace “had been” by “were”.
L 82: Add “a” before “textile membrane”.
L 83: Replace “contained the soil to 7 cm depth” by “contained a 7 cm layer of soil”
L 85: Replace “and grass” by “as well as the grass”.
L 85: Add “the” before “substrate”.
L 86: Remove “covered” before “by grass”.
L 87: Add “The” before “substrate” and before “insectaria”.
L 92: I don’t know what an “inert fabric” is.
L 97: Remove “moss”.
L 102: Remove “moss”.
L 103: Remove “moss”.
L 119: Add “a” before “30 days”.
L 120: Replace “freeze” by “frozen”.
L 124: Add “The” before “calorific value”.
L 124: Add “each” before the first “sample”.
L 124: Replace “sample of ash-free” by “ash-free sample”.
L 127: Replace “collections” by “collection”.
L 139: Add “each” before “specimen”.
L 144: Add “each” before “specimen”.
L 148: Add “the” before “groundhopper”.
L 150: Add “each” before “specimen”.
L 152: Add “the” before “conversion factor”.
L 152: Replace “transfer” by “converting”.
L 152: Add “the” before “type of food”.
L 156: Add “the” before “weight”.
L 157: Add “each” before “feeding group”.
L 158: This sentence needs to be completely re-written.
L 162: Change to “We used Tukey’s HSD test to compare differences…”.
L 168: This sentence is somewhat unnecessary. Better just explain the differences as in the next sentence and refer to the table at the end of this sentence.
L 171: Replace “not differed” by “did not differ significantly”.
L 171: Replace “consumed” by “consuming”.
L 172: Delete the sentence “Mean weights…” and just add the reference to table 2 in the parentheses of the next sentence.
L 173: Replace “significantly differed” by “differed significantly”.
L 174: Replace “have” by “had”.
L 175: Replace “consumed” by “consuming” (or just “fed on different diets” instead of “consumed different type of food”).
L 176: Add “The” before “Tukey”.
L 177: Add “the” before “weight”.
L 177: Delete “defaecated” (it should be clear that “faeces” is defecated).
L 177: Replace “between served food” by “between different food types”
L 178: Replace “but no between two served moss species” by “but not between moss species”.
L 178-180: This sentence is not clear. Do the numbers refer to the different sexes or the different food types?
L 181-182: Replace this sentence by “The calorific value of the different food types (Table 1) differed significantly (df = 2….).
L 182: Add “The” before “Tukey”.
L 182: Add “the” before “calorific”.
L 183: Add “the” before “moss” (2 times).
L 184: Add “the” before “served food”
L 184: Delete “shows Table 1 and its matter” and add “Table 1” to the parentheses at the end of the sentence.
L 185: Change “significantly differed” to “differed significantly”.
L 186: Add “The” before “Tukey HSD test”.
L 187-188: Delete this sentence.
L 188: Add “The” before “Energy”.
L 189: Delete “show Tables 3, 5 and this parameter” (add the reference to the tables in parentheses at the end of the sentence).
L 189: Change “significantly differed” to “differed significantly”.
L 191: Add “The” before “Tukey HSD test”.
L 195: Replace “Base on” by “Based upon”.
L 195: Replace “are different” by “differ”
L 196: Add “the” before “detrito-bryovorous”.
L 198: Change “the moss tissues” to “moss tissue”.
L 198: Change “Mosses are unimportant” to “Mosses appear not to be important”.
L 199: What is meant with “their portion in the food seems different”. Could mosses have any other benefit for digestion (e.g. comparable to dietary fibres?).
L 204: Replace “consumed” by “consume”.
L 205: Delete “the” at the end of the line.
L 208-209: Delete the sentence “We provided…” (the reader should know this by now).
L 209-210: Rewrite this sentence as follows: “Our analyses show that detritus has a higher percentage of silicon, while moss is richer in other elements (particularly carbon, phosphor, Table 1).”
L 211: Add “The” before “Results”.
L 213: Delete “the” before “both food components”.
L 215-218: Rather than just listing this high number of references, it would be good to compare your results with those from these studies.
L 220-221: Not clear what this sentence refers to. I assume it refers to the references given before, but this may not be clear to the reader.
L 222-223: Delete this sentence (the reader should know this meanwhile).
L 223-224: Rather than stating that you “can compare” the results, just do it!
L 227: Replace “feeding” by “feed upon”.
L 227: Replace “to growth” by “for growth”.
L 228: Add “The” before “Theory”.
L 231: What is meant with “better balanced food components per time”?
L 232: Add “The” before “Groundhopper”.
L 232: Replace “bigger amount” by “larger amounts”.
L 233: Delete “the” before “both”.
L 233: Add “a” before “similar”.
L 234-235: Replace “in detritus, than in moss tissues” by “of detritus compared to moss tissue”.
L 235-236: Not clear what this sentence should mean.
L 236: Replace “We recorded, that” by “Based upon our results”
L 236: Add “a” before “better”.
L 236: Delete the comma.
L 237: Add “the” before “alimentary tract”.
L 238: Delete “Defaecated”.
L 238: Replace “, than other waste products” by “and contain only few other waste products”.
L 239-241: Not clear why this corresponds with the optimal foraging strategy. Why do they eat mosses at all?
L 241: Better suggest some alternative functions based upon the literature or own ideas.
L 244: Replace “moss tissues are” by “moss tissue is”.
L 244: Why “Another”? You didn’t present any other hypotheses before! Rewrite this sentence as follows: “It was hypothesized before that groundhoppers might consume mosses to obtain cryoprotectants (Cornelissen et al., 2007) before hibernation, but …
L 245: Replace “was possibility” by “is the possibility”.
L 248: Replace “the overwintering” by “hibernation”.
L 248-252: I think the sentences “We can summarize” to “unanswered” can be deleted. The authors should be aware of these findings after reading the text.
L 252: Replace “One explanation” by “Another explanation”.
L 253: Replace “can be matter for longer retention” by “facilitate a longer retention time”
L 253: Add “the” before “alimentary”.
L 254: Replace “whereby the digestions and efficiency of nutrient absorption could be more effective in these circumstances” by “improving digestion and efficiency of nutrient absorption”.
L 255: Replace “can” by “may” (good to see this hypothesis here, see my comment above).
L 257: Replace “It changing” by “It influences”.
L 258: Add “an” before “ancient group”.
L 258: Delete “own”.
L 259: Add “the” before “gut”
L 259: Replace “may be unable” by “may not be adapted“.
L 261-262: Replace this part by “which may compensate the lack of enzymes or an own gut flora of the groundhoppers”.
L 262: Add “probably” before “accessible”
L 264-272: This part is largely redundant and should be deleted. Instead, you might enter a short conclusion particularly focussing on what steps should be done next (e.g. analysing mixed diets).
Figure Heading to Figure 2: add “the” before “calorific values” and replace “by calorimeter method” by “using the calorimeter method”.


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: ENERGY BALANCE OF FOOD IN A DETRITO-BRYOPHAGOUS GROUNDHOPPER (ORTHOPTERA: TETRIGIDAE)
Review round: 1
Reviewer: 3

Output:
Basic reporting: Dear authors, dear Editor,

I like the chosen research topic. Unfortunately, the manuscript needs a major revision before it can be fully evaluated.
First, even if I am not a native speaker myself, I need to mention that the English is not very good. I was in some parts even unable to understand the meaning (e.g. lines 30-31). I encourage the authors to ask a native speaker to improve the writing.
Otherwise, I see some reasons in the structure and the presentation that a serious and very detailed revision can be done to improve this manuscript, see my suggestions in the next two sections and specifically in the attached PDF file.
Experimental design: The experimental design seems to be quite reasonable, however necessary information are omitted:
I) did you use only individuals of one out of two wing morphs?
II) please specify the types of balances
III) give at least a brief explanation of the elementary analysis
It is unclear to me why you used 240 individuals? You had six groups (3 different diets by 2 sexes) each with 20 animals, accounting for 120 individuals. You mentioned a replicate at least in the tables? This would explain the 240 individuals, but in that case, you need to explain this. I even thing that you need to include a factor replicate into your statistics, see also the next section.
Validity of the findings: It seems that most data have been provided, however the tables are presented in Czech! This does not allow me to check their validity.

More importantly, the statistics seems to be wrong: if three different feeding types were tested (moos species 1, moss species 2, detritus) in two different sexes (males, females) the statistical analysis is a three by two factor analysis, e.g. using ANOVA's. Instead of this, the authors split the statistics and test between sexes, see lines 168-169 for body masses, lines 173-174 for faeces, followed for a test between diets, see lines 170-172 for body masses and lines 174 to 176 for faeces. This is not appropriate and must be done in a single analysis.

We need to await the revision, before a full assessment of the validity of the presentation and the Interpretation can be conducted.
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: ENERGY BALANCE OF FOOD IN A DETRITO-BRYOPHAGOUS GROUNDHOPPER (ORTHOPTERA: TETRIGIDAE)
Review round: 2
Reviewer: 1

Output:
Basic reporting: Dear authors, dear Editor,

I still like the chosen research topic of this revised manuscript. However, after some hours of critical proofreading and commenting I still see severe problems. The manuscript needs a very major revision before it can be fully evaluated.
The English has increased since the initial submission, but some parts are still hard to understand. This starts with mixed usage of British or American English (in case of faeces or feces). I am unsure whether these problems are mere language problems or are indeed problems with the logic. However, the authors need to improve the representation in any part of the manuscript.
I have made many suggestions in the attached PDF file.

To pick just a few problems:
I) The writing is unspecific, not to the point. I have tried to make a huge amount of suggestions directly in the text.
Ia) To illustrate a very bad style:
The authors write in Line 189: “Calorific values of served food are shown in Table 1….”.
In a scientific text you never show something, you describe facts. The text should be specific, and the convention is that the text stands for its own and must be understandable without looking into a figure or a table. The same applies to figures or tables, they must be understandable without back and forth checking the text. This is no language problem but a poor style of presentation. The same issue in lines 186ff, 193ff

Ib) Mass and weigh are not used properly, see line 169.
Comment: Physically you weigh something to measure its mass

Ic) The season of the species does not continue from March to October as written in line 72. Comment: This is not exactly true: nymphs hatch in summer (August), molt to adulthood in autumn, hibernate and reproduce in spring. So the adult season is split into the autumn dispersal related cohort (Lehmann et al. 2018) and the reproducing spring cohort (Steenman et al. 2015).
Experimental design: II) There are problems with the data presentation:
IIa) The presentation is very hard to follow, sometimes data are presented in Joule (the SI unit), sometimes in calories, sometimes units are missing entirely (see Figure 2).
IIb) I was not able to find out, whether data are presented as group means or adjusted to individual data.
IIc) Data are sometimes separated for sex (see Table 4) or the same data lumped across sexes as in Table 1. The authors need to be consistent across the manuscript.

III) The experimental still lacks some necessary information:
IIIa) you use individuals of both wing morphs, in which composition?
IIIb) please specify the types of balances (see line 120)
IIIc) give a description of the “Real caloric value of assimilated food”

IV) Statistics
IVa) As you used a replicate you need to include a factor replicate into your statistics; this was not done even if I have asked for it in my first review.
IVb) Describe the multifactorial analysis, be specific and explain the main effects of the post-hoc analysis.
Validity of the findings: The validity of the findings can only be fully explored after we have a concise and specific presentation of the data.
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: ENERGY BALANCE OF FOOD IN A DETRITO-BRYOPHAGOUS GROUNDHOPPER (ORTHOPTERA: TETRIGIDAE)
Review round: 3
Reviewer: 1

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

I congratulate you on the excellent job to improve the manuscript!

I am happy with the new version and recommend its publication.

Greetings
Anonymous


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: TESTING PTEROSAUR INGROUP RELATIONSHIPS THROUGH BROADER SAMPLING OF AVEMETATARSALIAN TAXA AND CHARACTERS AND A RANGE OF PHYLOGENETIC ANALYSIS TECHNIQUES
Review round: 1
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: The MS by Baron brings interesting data and deserves to be published in PeerJ if the author manages to reply to (accepting or comprehensively contesting) the points raised below.

General (major) comments
Lines 170-175: after the first paragraphs of the “Introduction”, where the author discusses several pterosaur phylogenies, he focus on those based on Britt et al. (2018), questioning the choice of outgroups of that data-base. This is fine, but it would be nice to see here some comments on the outgroup choice of the other studies mentioned before. At least as a counterpoint to that of Britt et al. (2018). This would also give the reader the notion that, if the other data-matrices do not have that problem, but are still disagreeing with one another, the reason for that disagreement is probably not outgroup choice. Also, if the problems is the outgroup choice of Britt et al. (2018) one would expect the outcome of running that database with more outgroups would be closer to those of studies with adequate outgroup choice.
Connected to the comment above, I cannot see Herrerasaurus in the phylogeny of Figure 1a-b.
Lines 246-258: the author conducts a series of analyses with the dataset he modified (including both characters and outgroup taxa) from Dalla Vechia (2019), partially with the aim of finding if outgroup choice has an impact on that study. Therefore, in order to do that, it is chief to do a first run (and latter discuss the results) of the analysis with the added outgroup taxa, but without the added characters. In this way, the author can be sure that the new results he is getting is actually from the new outgroup choice, and not from the characters he added.
Lines 271-332: here the author compares his results of running a data matrix modified from Dalla Vechia (2019) with results of other pterosaur phylogenies. This causes confusion, because it is harder for one to see (among the various comparisons) the actual differences between the new results and those of Dalla Vechia (2019). The author should first discuss these differences (between his and Dalla Vechia’s studies) only, and after provide a more generalist comparison with other phylogenies.
Lines 364-366: as Pterosauria Kellner (2003) is node-based and Pterosauromorpha Padian (1997) is stem based. As such (and because the use different specifies), these can never take objective “precedence” over one another, only have the same inclusivity under a given phylogenetic hypothesis.
Lines 373-469: why are new names given to clades “7” and “8” and not to other unnamed clades. In fact, I would be more comfortable if a pterosaur specialist (which is not my case) also reviews this MS, particularly the adequateness of such new names. It is surprizing, for example, that one of the named clades “is the least consistently supported” (line 410)
Line 473: it is not clear to me what is this “resampling” about. Is that the Ezcurra (2016) protocol, right? He uses resampling for the bootstrap calculations. Are you using a bootstrap tree in Fig. 3a?
Lines 471-512: the author comments on the outcomes of two different analytical procedures applied to the same dataset. I see two problems here: (1) why are the results of the first analysis discussed in so much more detail (lines 264-469) that the others? (2) if the focus is on the different procedures of these two analyses, the discussion (here and in the “Discussion” section) also has to be focused on these procedures, not only in the results, e.g., why use implied weights? why the topology changes because of that? It is not enough to compare the results here, you have to investigate the reasons behind the different results. Another option, surely easier and with no significant loss, is to exclude the two last analyses (lines 471-512).

Specific (minor) comments
“Andres” is misspelled in parts.
 “but” (I guess) is misspelled as “bar” in parts.
All clades are monophyletic, please do not use “monophyletic clade”.
Line 415: “that” not “the”
Lines 418: choose another word than “subsequently”, because one of such works is older and the other of the same year.
Line 422: you use derived under ‘’ here. This is OK, and should also be used in other parts discussing the position of taxa in trees.
Lines 433-442: a ten-pages etymology is a bit too much.
Line 474: for the sake of completeness, you could mention the position of Dinosauriformes here as well.


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: TESTING PTEROSAUR INGROUP RELATIONSHIPS THROUGH BROADER SAMPLING OF AVEMETATARSALIAN TAXA AND CHARACTERS AND A RANGE OF PHYLOGENETIC ANALYSIS TECHNIQUES
Review round: 1
Reviewer: 2

Output:
Basic reporting: No particular issue here. The paper revisits the question of pterosaur relationships, with special attention to basal taxa, including new closely related species to Pterosauria.
Experimental design: The approach is correct. One always might have different ways, but there is no problem in the way that the authors delimited his study.
Validity of the findings: Conclusions are correct based on the method used.
Additional comments: The ms. by Baron essentially revisits the relationships of pterosaurs, including a several new taxa outside Pterosauria in order to test the results known to date. There are not that many papers that try to rigorously test the relationship in group relationship of basal Pterosauria and therefore I welcome and endorse the publication of this study.

There is are not specific problem with this analysis - one could include other data-matrices or discuss different characters and perhaps use other datasets. But this does not invalidate the interesring approach of the author to this complex problem that surely will have follow up that might show different results.

One point that the author should point out that dinosaurs were included in phylogenetic analysis before, even dinosauromorphs. The information is there in his ms, but I think that he should make this clearer, citing the appropriated contributions, including the first studies that included such taxa.

Another point that might be interesting to clarify is regarding supports. Baron made a comment about low support of Bremer support for some clades of previous studies. But is this not the rule?  The fact that there is little support does not mean that the retrieved relationship is not correct. Perhaps he would like to elaborate on that  (optional).

Lastly, it is hard to keep up with literature these days. Perhaps, just for the sake of completeness, the author might like to include in the introduction the new discoveries of pterosaurs in the Antarctic Peninsula (optional).


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: TESTING PTEROSAUR INGROUP RELATIONSHIPS THROUGH BROADER SAMPLING OF AVEMETATARSALIAN TAXA AND CHARACTERS AND A RANGE OF PHYLOGENETIC ANALYSIS TECHNIQUES
Review round: 2
Reviewer: 1

Output:
Basic reporting: OK
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: TOXICITY THRESHOLDS OF THREE INSECTICIDES AND TWO FUNGICIDES TO LARVAE OF THE CORAL ACROPORA TENUIS
Review round: 1
Reviewer: 1

Output:
Basic reporting: Very good English
Good ciations, some suggestions made
professional article.
Self contained
Experimental design: within aims and scope of Peer J
well defined and relevant research question
investigated with rigor
methods mostly well define some clarity requested
Validity of the findings: rational clearly stated
data seem robust
clear conclusions
Additional comments: PEER J 48551: Reviewers comments
This is a well written paper that provides timely and useful data.  The experimental process has been completed with rigour and is mostly clearly laid out. The results will be useful in setting future guideline values.  The graphs and tables are well presented and clear.  After reviewing the manuscript, I recommend it for publication after consideration of some minor changes to help clarify methodology, results, and context within existing literature.  Further details are below.

Line 75: suggest change ‘need to be included’ to ‘should be included’
Line 90: Suggest changing:
 “However, many of the recently detected pesticides are not included in the current Australian and New Zealand water quality guidelines (ANZG 2018).”

to

 “However, many of the recently detected pesticides have not been provided Default Guideline Values (DGVs) in the current Australian and New Zealand water quality guidelines (ANZG 2018).”

Line 150:  Along with later references to the age of the larvae (e.g. line 217).  It is unclear if separate experiments were run, one using 7 day old larvae and the other using 10 day old larvae of if the larvae that were 7 and 10 days old were combines for one experiment. Can this please be made clear.
Also worth noting is this is the age of the larvae at the start of the experiments but as the 48 processes to larvae do get older.  It would be good if this could be considered in the text.
Was just one single test completed for each pesticide can you clarify in the text. See also line 247.

Line 159: This section and in other places (e.g. line 175-178, 236) does not make clear the actual endpoint because the words ‘settlement’ and ‘metamorphosis’ are used interchangeable. I suggest a series of photographs which highlight when settlement success was deemed to be achieved and some photos that show when it wasn’t (e.g. loosely attached).  Given that there are a series of steps in the process of metamorphosis to settlement the actual ‘endpoint’ needs to be made clear.
Also how were larvae scored if they were not moving or disintegrated? Dead?
I also suggest using the term ‘metamorphosis to settlement’ rather than ‘settlement and metamorphosis’ (consider lines 107, 112, 116, 236, 254, 296, 390, figure 1 caption)

Line 162: Can you provide a table with the measured concentrations used in each experiment.

Line 171-172: Were larvae still being exposed to the contaminants when they were transferred to polystyrene culture plates? Or is this more like a recovery period.  Can you clarify the duration of exposure in the detail here.
Also note polystyrene is known to leach materials in the water, were your containers aged?

Line 197:  The replicates were in small 20mL vials which cannot fit common DO and conductivity probes.  Can the authors describe how (what samples) the physical chemical parameter were determined…e.g. in pooled replicate samples of each dose? And when… At 0 and at 48 hrs, after the additional 24hrs ?

Line 205:  it would be interesting to see where significant difference occurred between controls and dose concentrations.  Can these be calculated and presented.
Line 276: and further in this paragraph naled and Dibrom are the same pesticide compound so they should be consistently referred to in the same way.

Line 278 suggest rewording:
“The toxicity of diazinon to coral larvae adds to evidence that AChE inhibitors, including the organophosphate chlorpyrifos (Acevedo 1991; Markey et al. 2007) and naled (Ross et al. 2015), are toxic to corals.”
to
“The toxicity of diazinon to coral larvae adds to evidence that AChE inhibitors, including other organophosphate pesticides chlorpyrifos (Acevedo 1991; Markey et al. 2007) and Dibrom (Ross et al. 2015), are toxic to corals.”

Line 282: Change:
“Ross et al (2015) found that as low as 2.96 μg L-1 naled inhibited larval settlement of the coral Porites astreoides.”
to
“Ross et al (2015) found that concentrations as low as 2.96 μg L-1 Dibrom inhibited larval settlement of the coral Porites astreoides.”

Line 285: Provide references sources in text for the studies.

Line 312: References of use to support text and to consider for this section:
P Butcherine, BP Kelaher, MD Taylor, BJ Barkla… - 2020 - Impact of imidacloprid on the nutritional quality of adult black tiger shrimp (Penaeus monodon) Ecotoxicology and Environmental Safety  …,

Butcherine, P. Benkendorff, K., Kelaher, B., Barkla, B.J. (2019) The risk of neonicotinoid exposure to shrimp aquaculture. Chemosphere 217, 329-348.

Ewere, E., Reichelt-Brushett, A., Benkendorff, K. (2019) Imidacloprid and formulated product impacts the fatty acids and enzymatic activities in tissues of Sydney rock oysters, Saccostrea glomerata. Marine Environmental Research. 151: 104765.

Ewere, E.E., Powell, D., Rudd, D., Reichelt-Brushett, A., Mouatt, P., Voelcker, N.H., Benkendorff, K. (2019) Uptake, depuration and sublethal effects of the neonicotinoid, imidacloprid, exposure in Sydney rock oysters Chemosphere 23,0 1-13

Line 301 and 335: Clarity required:
See like there is only a fine subtlety between ‘insecticide’ and ‘pesticide’ in these two statements/claims but it is misleading and creates some confusion.  Can the text be reworded to provide better clarity?
“While A. tenuis larvae were not as sensitive to fipronil (NEC10: 12.3 μg L-1) as grass shrimp, this was the most toxic insecticide tested to corals in the present study.”
“Chlorothalonil was the most toxic pesticide tested in this study to coral larvae with a NEC of 2.42 μg L-1.”

Line 366-367: it would be good to say why it is inferred that the logkow value would more likely result in narcotic effects…and define ‘more’ that what….
Line 369: maybe ‘duration of exposure’ rather than ‘length of exposure’

Line 376: Reference to consider here and in previous detail relative to larval motility:
Reichelt-Brushett, A. J. and Harrison, P. L. (2004) Development of a sub-lethal test to determine the effects of copper and lead on scleractinian coral larvae. Archives of Environmental Contamination and Toxicology. 47 (1) 40-55.


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: TOXICITY THRESHOLDS OF THREE INSECTICIDES AND TWO FUNGICIDES TO LARVAE OF THE CORAL ACROPORA TENUIS
Review round: 1
Reviewer: 2

Output:
Basic reporting: Clear, unambiguous, and professional English language is used throughout the manuscript. Introduction is clear, with a great logical flow, and provides the necessary background to show the context, relevance and importance of this study. The literature is well referenced & relevant. The structure of the manuscript conforms to PeerJ standards (but see my comment on Introduction subsections). Figures are relevant, high quality, well labelled & described. The raw data are supplied, but not the associated R code to reproduce the analyses.
Experimental design: This study is original primary research, within Scope of the PeerJ journal. The research question is well defined, relevant & meaningful and it is stated how the research fills an identified knowledge gap. Rigorous investigation were performed to a high technical & ethical standard, and methods are described with sufficient detail & information to replicate.
Validity of the findings: All underlying data have been provided; they are robust, statistically sound, & controlled, but see my comment on the control values for the propiconazole, and my comment regarding the findings on the non-quantified swimming behavior. Speculation is present in the discussion and a greater emphasis but should put to identify it as such. Conclusions are well stated, linked to original research question & limited to supporting results.
Additional comments: Overall comment:

This study investigates the effects of 5 pesticides and copper on the settlement and metamorphosis success in Acropora tenuis. This is a very well written study, globally well-constructed (but see my comments regarding the discussion on the need for a better definition of some terms). The topic is well introduced (the sensitivity of coral early life stages to emerging waterborne pollutants), and the scope of the manuscript is well defined (providing baseline and robust data to improve water quality guidelines). The method and the data look robust and are well presented. More specifically, authors have properly controlled the nominal concentrations of the different pesticides that they have used at the beginning and near-end of the exposure experiment, and state to have use adapted and reproducible methods (but see my comment on the Bayesian or binomial models). This study is timely given the current context of coral reefs, and provide strong data and interesting discussion (but see my comment on their speculative aspect) that are currently missing in the literature. Overall, I recommend the acceptance for the publication of this study, if the following comments (see below) are addressed.

Major comments:

a.	I recommend to clearly define settlement and metamorphosis, as these terms are used repeatedly, and sometimes interchangeably throughout the manuscript. Settlement and metamorphosis are most often synchronized and associated, in many species. However, in my opinion (and I have no issue with different definitions as long as they are clearly provided) settlement refers to the organism arrival and installation in its new environment (i.e. ecological process, here from planktonic to benthic life), while metamorphosis refers a developmental process (here of a planula into a polype). I suggest the authors to better define these terms and to check throughout the manuscript if the use of “settlement”, “metamorphose” or “settlement and metamorphosis” is best suited. For example, in the Materials and Methods “Settlement assays” subsections, authors write that they assessed metamorphosis after 24h in 6-well plates. So this brings a confusion to the reader whether settlement or metamorphosis was assessed. And actually, both settlement and metamorphosis were assessed as authors state that changing “from free swimming or casually attached pear-shaped forms to squat, firmly attached, disc-shaped structures with pronounced flattening of the oral–aboral axis and with septal mesenteries radiating from the central mouth region” were the parameters considered. I acknowledge that this is very “etymological”, but I believe this would strengthen the clarity of this manuscript to better define this terms and better define what was precisely examined.

b.	I recommend the authors to clarify their use of the terms “new”, “alternative”, “emerging”, “recently-detected” when referring to pesticides. From my understanding of the study, these terms are referring to the same thing throughout the manuscript and have been used interchangeably. If this is the case, I suggest choosing a single one of this term and then only use this term throughout the manuscript. Otherwise, I recommend the authors to specify/clarify this, as I acknowledge that it is fine to use multiple terms as long as their use is justified.

c.	I am not convinced that introducing studies that looked at the effects of insecticides on adult corals (Introduction, lines 127-133) adds important information to the readers, but rather dilutes the message and disperses my focus, as authors have already guided their introduction towards early-life stages earlier in the paragraph. I recommend the authors to better justify their use or maybe to remove these few lines.

d.	The Introduction is overall very clear, well referenced, and the logical flow is well adapted to the topic and very convincing. I am not familiar with subsection titles in the Introduction, and while they are fine to me, I can also say that their removal would not impair the flow of the Introduction given the great piece of writing that the Introduction is.

e.	It is not clear to me whether the larvae that the authors have used were 7-10 days old, or 10-13 days old at the time of metamorphosis (given the 3-day exposure duration, i.e. 2 days in glass vials and 1 day in 6-well PS culture plate). This is really not a big deal to me, but since the authors, in the Results section, mention the metamorphosis success for 7-day-old larvae vs. ten-day-old larvae, I guess this is important to fix. From my understanding, authors want here to say that whether larvae were exposed at 7 or 10 days old, their metamorphosis success after exposure (i.e. 3 days after) was similar. If this is the case, I recommend the authors to edit their results and materials and methods sections to make this clearer.

f.	I invite the authors to justify the great emphasis on larvae swimming behavior and disintegration observations, in both the abstract and results. The reason I say this is because 1/ these measures were not quantified as stated in the results section, 2/ there is no Materials and Methods about how these observations were made; and 3 / these observations apparently only concern copper and chlorothalonil, but none of the 4 other pesticides. I am not saying that these information/data are not valuable and I commend the authors for bringing such information that are interesting. However, in my opinion, NEC, EC10 and EC50 of the 6 chemicals are the main results of this study and this should be reflected in the abstract and results sections. I therefore suggest that authors put a greater emphasis at describing the NEC, EC10 and EC50 outputs and a lower emphasis at mentioning the swimming behavior and disintegration outputs, in both the abstract and results sections. I also recommend the authors to bring more information about how the swimming behavior and disintegration observations were made, and why only copper and chlorothalonil were concerned by these observations.

g.	The two first paragraphs of the Discussion, before the Diazinon subsection, would benefit from a rewording, as the critical information here but, in my opinion, is a bit dispersed and sometimes repetitive (see lines 246 and 260-261). Specifically, I recommend the authors to gather these two paragraphs in one “introductory” discussion paragraph, in which they 1/ briefly summarize their findings (all the tested pesticides affected coral larval settlement/metamorphosis, but with varying toxicity levels as evidenced by the great range of NEC and EC; 2/ introduce the value/importance of their of their study (importance of testing settlement/metamorphosis sensitivity given the critical role of this life-history transition for recruitment and replenishment of populations and the structure of the ecosystem they inhabit or even drive (in the case of corals), the well-known sensitivity of such early life stages to pollutants, in corals but also in all organisms that undergo metamorphosis (~80% of animal species), and value/novelty of their study in this respect and towards better guidelines); and 3/ introduce that the specificity of the mode of action of each pesticide could explain their results and will be discussed in followings subsections.

h.	The discussion on the effects and modes of action of the different pesticide is essentially speculative, which is not an issue to me as it is still valuable and overall the discussion is very interesting. However, in my opinion, the sentence used by authors “The following sections compare the mechanisms of action of the pesticides tested with the possibility of a specific response by the coral larvae” does not sufficiently acknowledge that this discussion is speculative, and this should be greater emphasized. Also, if being speculative, why not also going even further and discuss the potential for these pesticides to disrupt the signaling pathways that are responsible for coral metamorphosis? I acknowledge that this is all very speculative and most is unknown regarding these processes, but I found this study to be a great invitation towards a better understanding of the molecular determinants of coral metamorphosis, and the pollutants that could disrupt it.

i.	When looking at panel E) Propiconazole in Figure 1, I observe that the starting value (for herbicide concentration of 0 µg L-1), which corresponds to the solvent control if I understood well, are different from the starting values of all the other panels of this figure. When looking at the raw data, I observe that this is due to the fact that different controls were used for propiconazole. Can the authors explain the reason why different controls were used?

j.	In the Materials and Methods, authors state that they used “Bayesian or binomial segmented-regression models” but do not justify why one or the other method was applied, and to which dataset each of this method was applied to. I invite the authors to precise this.

Specific and/or minor comments:

- Adding page numbers would help the review.

- Abstract, line 20: I’m not sure that “alternative pesticides” are well known by non-specialists. I suggest rewording this sentence and the previous one to make it clearer to the readers what “alternative pesticides” are, and that they are the same as the “new pesticides” that you mentioned in the previous sentence.

- Introduction, lines 77-78: Is/Are there reference(s) for this or is it already a conclusion of this study?

- Introduction, line 87: There is probably a missing “,” after “Australia”.

- Introduction, lines 105-109: I suggest a slight rewording the second part of this sentence, as it is not “the effects” that are impacting the the photosynthetic capacity, but the herbicides. I also suggest to separate the last of this long sentence in a single sentence, to make this part about the subsequent “reproductive outputs” clearer.

- Introduction, line 107: Add “this” before “symbiosis”.

- Introduction, line 110: I would remove “potentially”.

- Introduction, line 112: Add “planulae” before “metamorphosis”.

- Materials and Methods, line 206: There is an extra “(“ here. I recommend rewording as follow: “No effect concentrations (NECs) and effect concentrations, i.e. concentrations of each pesticide…”

- Materials and Methods, line 221: Remove “statistical package” as “package” is already mentioned before “jagsNEC”.

- SD vs SE. Data +/- SD are used in the Materials and Methods section, while Data +/- SE are used in the Results section. I recommend the authors to be consistent throughout the entire manuscript regarding this.

- Results, line 228: I suggest to remove “inhibiting metamorphosis by 10%” as this is already what the EC10 means, as defined by the authors in their Materials and Methods. A potential rewording could be “Chlorothalonil was the most potent pesticide towards A. tenuis metamorphosis, with an EC10 of …”.

- Discussion, lines 250-252: I would remove this sentence, which is ‘out-of-topic’ in my opinion and thus breaks the logical flow of this paragraph on the importance of sensitivity of settlement/metamorphosis.

- Discussion, line 267: I suggest the authors to be a bit more specific here, e.g. by rewording this sentence by something similar to: “Organophosphate pesticides, like the diazinon, can inhibit acetylcholinesterase …”. This sentence, and the following one, would also benefit from reference(s), there are plenty on coral reef fishes for example.

- Discussion, lines 293-294: This sentence would benefit from reference(s).

- Discussion, lines 317 and 322: “GBRMPA MMP” has been defined in the Introduction section, but is the MMP mentioned at these lines the MMP from the GBRMPA MMP?

- WQGV is defined in Table 4, but not in the discussion text.

- Discussion, line 321: This sentence would benefit from introducing what K0w refers to.

- Discussion, lines 336-338: This sentence would benefit from reference(s).

- Discussion, line 345: There is probably a missing “concentration” word after “at”.


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

Input:
Title: IDENTIFICATION OF 10 HUB GENES RELATED TO THE PROGRESSION OF COLORECTAL CANCER BY CO-EXPRESSION ANALYSIS
Review round: 1
Reviewer: 1

Output:
Basic reporting: Meng et al.performed WGCNA to identify hub genes in the tumourigenesis of CRC. By combining data from TCGA and GEO, they identified ten hub genes (MT1X, MT1G, MT2A, CXCL8, IL1B, CXCL5, CXCL11, IL10RA, GZMB, KIT) and closely related to the progression of CRC. They performed a lot of computational analysis to validate their findings.
Experimental design: The experimental design is reasonable
Validity of the findings: 1. In the result "GO functional and KEGG pathway analyses of mRNAs", could the author show some specific biological processes or pathways that are involved in the progression of CRC?
2. Please clarify your reasoning for selecting GSE28000 and GSE42284 for analysis from the GEO database.
3. This article identifies 10 related genes (MT1X, MT1G, MT2A, CXCL8, IL1B, CXCL5, CXCL11, IL10RA, GZMB, KIT) closely related to the progression of CRC were identified. Why IL10RA and KIT related to overall survival time?
Additional comments: 1. The author's full text is too simple, and it is suggested to elaborate in detail, for example: introduction part (Please describe your rationale for selecting your candidate genes in the Introduction), result expression part.
2. I would suggest the authors to increase the figure resolution. It's hard to see clearly the texts in most figures.
3. In the result "GO functional and KEGG pathway analyses of mRNAs", could the author show some specific biological processes or pathways that are involved in the progression of CRC?
4. Please clarify your reasoning for selecting GSE28000 and GSE42284 for analysis from the GEO database.
5. There are some format errors in this article, which need to be modified.
6. This article identifies 10 related genes (MT1X, MT1G, MT2A, CXCL8, IL1B, CXCL5, CXCL11, IL10RA, GZMB, KIT) closely related to the progression of CRC were identified. Why IL10RA and KIT related to overall survival time?


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

Input:
Title: IDENTIFICATION OF 10 HUB GENES RELATED TO THE PROGRESSION OF COLORECTAL CANCER BY CO-EXPRESSION ANALYSIS
Review round: 1
Reviewer: 2

Output:
Basic reporting: The authors constructed a prognostic model based on the candidate ten genes by using the data set GSE28000 and GSE42284. WGCNA，co-expression network and the PPI network were used to screen hub genes. Survival analysis suggested that IL10RA and KIT were significantly associated with the prognosis of patients.Overall, the authors performed a lot of validation work for CRC.I still have some concerns.
Experimental design: The experimental design is reasonable
Validity of the findings: 1.Why did the authors select GSE28000 and GSE42284 for further analysis?
2.The pre-processing procedures of the TCGA and GEO RNA-Seq datasets should be explained in detail.
3.The title of this article is“Identification of 10 Hub genes related to the
progression and prognosis of colorectal cancer by co-expression analysis”.Why survival analysis suggested that only IL10RA and KIT were significantly associated with the prognosis of patients, how about survival analysis of other eight genes?
4.I would suggest the authors to increase the figure resolution. It's hard to see clearly the texts in most figures.
Additional comments: 1. Authors have done a sloppy job in writing the entire manuscript. English check/edits are required to make the article easy to understand.
2.The authors should re-check of language mistakes in the revised 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: IDENTIFICATION OF 10 HUB GENES RELATED TO THE PROGRESSION OF COLORECTAL CANCER BY CO-EXPRESSION ANALYSIS
Review round: 1
Reviewer: 3

Output:
Basic reporting: I think the article is well written, the logic is clear, the research content is reliable, and the science is good, which basically meets the publication requirements of the magazine.
Experimental design: It's a very good study. And the aim of this study is clear.   Methods described sufficient detail & information to replicate. Some researchers may interest in it.
Validity of the findings: This manuscript is well written and novely, I think that it has the potential for acceptance of peer j.
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: EXCESSIVE G–U TRANSVERSIONS IN NOVEL ALLELE VARIANTS IN SARS-COV-2 GENOMES
Review round: 1
Reviewer: 1

Output:
Basic reporting: Overall, the manuscript is concise and has an easy to read narrative. A few comments:
Lines 116-121: It would be better to use percentages as well as the raw number ratios, as is done later in the text. Comparing the raw ratios is not very intuitive for the reader.
The phrasing ‘remarkable feature’ in lines 142 and 146 is a bit unclear. Maybe change to ‘observations’?
Line 148: change ‘plus’ to ‘positive’ strand.

The introduction is short and to the point. One misunderstanding of the current literature is that the virus RaTG13 is not technically the closest known relative of SARS-CoV-2 (lines 21-22 & 47-48). That would be the virus RmYN02 (Zhou et al., 2020, Current Biology 30, 1–8; https://doi.org/10.1016/j.cub.2020.05.023). This should be clarified in the introduction.

Mutations and substitutions calculated through both methods used by the authors are presented in tables 1 and 2, and figure 1 clearly showcases the data.
Experimental design: The methodology used by the authors seems reasonable, although some parts are unclear in the manuscript. In lines 66-67 the authors do not explain how they have derived the consensus sequences. They should cite any existing methods they have used, or if inferred with their own code mentioned that they have done so. In the latter case, if the authors believe their method diverges from conventional ways of inferring consensus sequences it might be worth making the code public with this publication.

In lines 82-88 the authors describe the bat virus relatives to the human coronaviruses used in their analysis. I would urge the authors to include a phylogeny of how these viruses are related in their manuscript. That would make the analysis much clearer to audience that is not familiar with the coronavirus phylogeny.

As I point out above RmYN02 is the closest known relative to SARS-CoV-2. The authors might decide to also use this virus in their analysis. In case they do they will have to account for some distinct recombination patterns in RmYN02 described in Zhou et al. (2020). Still, I believe that only using RaTG13 should not make much difference in the findings.
Validity of the findings: The findings seem valid and the increased G to U rate observation in SARS-CoV-2 should prove useful to other researchers exploring similar aspects of the virus. In their discussion the authors mention some rather speculative explanations of their findings (e.g. lines 178-181), nonetheless the phrasing makes it clear that these are speculations and the authors rightfully point out that further research on the topic needs to be conducted to support such hypotheses.
Additional comments: In this paper the authors quantify and compare the single nucleotide mutation rates and substitution rates between different members of the Coronaviridae family, focusing on SARS-CoV-2, a pathogen that is currently of paramount interest. The authors highlight the interesting observation that G to U changes tend to be more frequent in SARS-CoV-2 after its emergence in humans compared to its bat relative and other coronaviruses that have previously crossed to humans.
I believe that this observations could be of interest to the scientific community working on similar aspects of the SARS-CoV-2 virus.


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: EXCESSIVE G–U TRANSVERSIONS IN NOVEL ALLELE VARIANTS IN SARS-COV-2 GENOMES
Review round: 1
Reviewer: 2

Output:
Basic reporting: - References are provided.
- Relevant figures and tables are provided.
Experimental design: - Research questions are will defined.
- Methods are appropriately described.
Validity of the findings: - Conclusions are appropriately stated.
Additional comments: The manuscript of Panchin A and Panchin Y "Excessive G to T transversions in novel allele variants in SARS-CoV-2 genomes" is devoted to the analysis of the mutation and substitution spectrum in new SARC-CoV-2 coronavirus. Unprecedented attention to this virus causing the serious infection diseases COVID-19 leads to a large number of the sequenced genomes allowing to track the evolution of the virus in human. Authors investigated the mutations patterns of the SARS-CoV-2 and found the elevated number of the G to T mutations possibly reflecting the adaptation of the virus in human. Taking into account the urgent need for rapid publications of the studies of the SARS-CoV-2 the quality of the manuscript in general satisfactory, although I would suggest some improvements.
To clarify the nature of the G>T transversions I would suggest to answer in the manuscript the following questions:
1. What is the number of G>T transversions are located in coding and non-coding regions?
2. Among the G>T transversions in coding regions what is the number of synonymous and non-synonymous mutations?
3. Among the non-synonymous G>T transversions what is the number of missense and nonsense mutations.
4. What is the nucleotide context (5' and 3' nucleotide) of the observed G>T transversions?
5. The multiple alignment should be attached as the supplemental file.
6. Authors should also support the manuscript with the supplemental file containing the list of the observed G>T transversions and its genome positions.


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: EXCESSIVE G–U TRANSVERSIONS IN NOVEL ALLELE VARIANTS IN SARS-COV-2 GENOMES
Review round: 1
Reviewer: 3

Output:
Basic reporting: Panchin & Panchin present a comparison of nucleotide variation patterns in SARS-CoV-2 publicly available genomes. They key finding is a 9-fold excess of G to T (I personally prefer U) transversion. Not a lot of discussion is given about the mechanism for this excess, only speculating a role of the reactive oxygen species mutagenic base 8-oxoguanine (8-oxoG).

Please revise the literature for preprint already published. Like in the case of ref.18: https://advances.sciencemag.org/content/early/2020/05/15/sciadv.abb5813

I want to encourage the authors to use U instead of T across this manusctipt: title, text, tables and figure.
Experimental design: Overall, is a consice work that could be expanded. One major comment is whether the methodology is adequate. I believe that determining the ancestral state as the consensus sequence lacks biological correction. Why don’t the authors use the date of the samples to estimate these ancestral states? Sequences from december 2019-early january 2020 will certainly be a better proxy to estimate these N1 states (N1s) than the consensus. Tables 1 and 2 remain similar when looking at only at the first sequences availabable to determine N1s?

Another thing to take into account is how N2 changes are calculated. Every time in every genome that have a base different to N1 is counted? If thats is the case, it is likely that the authors are counting the same change many times. If that is the case, this big excess in G to U could be an artefact. An elegant way to adress this issue is to build a philogeny and count the changes that occur in wranches throughout the tree. This is a much better way of represent what is happening in reality. These viruses are from different time points and this temporality must be taken into account.

If this patterns are robust after the considerations given above, the next big step is to show where in the genome those mutations occur. They occur across the whole genome or are prevalent in some regions?
Validity of the findings: Statistical analyses are lacking. I suggest Chi squared analysis or Fisher’s exact test between groups to see if there are any significant differences, but another test could also be used.

About the 8-oxoG: A more extensive review of the molecular mechanism of this base modification and the machinery involved is very wellcome. More details about how and in what degree this mechanism can affect viral transcription and viral replication.
Additional comments: Another comment is that the data and code used in this analysis must be presented. A list of accessions is missing and the code is a good way of check the preocedure, besides the one described in Methods section. The alignments also must be presented. Given the big size of this genomes, I suggest the use more suitable algorithms than Clustal Omega (i.e.; MAFFT, BAli-Phy).

In lines 103 and 107-108, the authors mention reference genomes. Please specify the accession number of these reference genomes. Is it the RefSeq genome or the more similar to N1s? Again, if a list of accession is given (along with the code), these methodological questions will be more easy to be responded directly.


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: EXCESSIVE G–U TRANSVERSIONS IN NOVEL ALLELE VARIANTS IN SARS-COV-2 GENOMES
Review round: 2
Reviewer: 1

Output:
Basic reporting: The authors have addressed all changes requested, clarifying the relation between SARS-CoV-2, RaTG13 and RmYN02 and revising few minor phrasing and reporting suggestions.
Experimental design: In this version of the submission the authors have adequately described the experimental design and methodology and provided extra supplementary material essential for transparency and reproducibility of their research.
Validity of the findings: As mentioned in the first round of review the findings are interesting and valid in the way they are presented and discussed.
Additional comments: I believe that the authors have addressed all comments successfully and the manuscript now looks very presentable.


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: EXCESSIVE G–U TRANSVERSIONS IN NOVEL ALLELE VARIANTS IN SARS-COV-2 GENOMES
Review round: 2
Reviewer: 2

Output:
Basic reporting: I would like to highlight that all reviewers' comments have been properly addressed. I would also like to mention that there was considerable harmony in these comments, which clearly facilitates this process.
Experimental design: Again, I think the comments have been answered correctly.

As an extra comment (no response is needed), I want to add that the 193 positions reported on Table S2 also correspond to a G on the SARS-CoV2 reference sequence (NC_045512).
Validity of the findings: The G to U pattern observed in this virus is interesting. In addition to its magnitude, the authors also present statistics.

The authors add a few more references on 8-oxoguanine. I agree that it is a topic that is not very well known, let alone related to viruses.
Additional comments: This version, although it has small changes, is sufficiently improved. It is also better supported by supplementary material.


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: EPITOPE-BASED CHIMERIC PEPTIDE VACCINE DESIGN AGAINST S, M AND E PROTEINS OF SARS-COV-2, THE ETIOLOGIC AGENT OF COVID-19 PANDEMIC: AN IN SILICO APPROACH
Review round: 1
Reviewer: 1

Output:
Basic reporting: The article has lost its flow and presentation. It’s written so elaborately that following the analysis and results is difficult. Authors need to considerably shorten the article and make it to the point.
Experimental design: There are several flaws in methods, probably due to the description of the methods. It’s evident in each method they have described and their corresponding results. For example, It is unclear which virus spike structure was used to predict the epitopes and authors have taken SARS-CoV-2, SARS-CoV and MERS-CoV. For sequence base prediction also, how they have selected the specific sequence and why for epitope prediction is unclear. Is the sequence unique from SARS-CoV and MERS-CoV?? And so on.
Validity of the findings: Authors need reanalysis with proper biological objectives
Additional comments: Complete rewriting the article to make it short and understandable is required


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: EPITOPE-BASED CHIMERIC PEPTIDE VACCINE DESIGN AGAINST S, M AND E PROTEINS OF SARS-COV-2, THE ETIOLOGIC AGENT OF COVID-19 PANDEMIC: AN IN SILICO APPROACH
Review round: 1
Reviewer: 2

Output:
Basic reporting: The manuscript seemingly sounds timely or opportunistic given the crowd sourcing attached to current Covid-19 pandemic.

Several preprint servers are flooded with this kind of computational studies on the novel coronavirus proteins and epitope binding analyses. Further, a number of candidate vaccines have already entered in clinical trials. These efforts need to be covered in literature survey. And also the authors need to be precise as to how their efforts are uniquely posited in comparison to the other efforts such that they be published in Peer J.

The language and presentation is poor and the manuscript could have been copy edited by a professional editing agency prior to its submission.
Experimental design: The design is of simplistic homology modeling, docking and simulation.

This reviewer would have liked a molecular dynamic simulation at femtosecond resolution scale.

Experimental approaches at high level expression and recombinant protein production are necessary to prove if the modeled molecules are indeed expressable or biologically active or stable.
Validity of the findings: Computational models could be misleading unless they are validated through molecular dynamic simulation and lab based verification.
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: EPITOPE-BASED CHIMERIC PEPTIDE VACCINE DESIGN AGAINST S, M AND E PROTEINS OF SARS-COV-2, THE ETIOLOGIC AGENT OF COVID-19 PANDEMIC: AN IN SILICO APPROACH
Review round: 1
Reviewer: 3

Output:
Basic reporting: The article is well structured and written in clear, unambiguous and technically correct English. It is easy and pleasant to read.

Literature references, sufficient field background/context provided.

The article perfectly contextualizes the research and fully justifies it, using very appropriate and up-to-date literature.

Professional article structure, Figures, Tables. Raw data shared.

Regarding Figures, both, those in the main text and those included in the Supplementary material, are adequate and help the understanding of the text. They are well numbered and correlate with the text. However, they should be of a higher quality due to the amount of material included in each Figure. In this way, Figures such as 4, 5 or 7 may be difficult to see if the quality is not improved. In any case, a higher quality of all the Figures would be necessary.

1.	In Figure 1, line 2, change "MERS-CoV is a trimeric conformation" by "MERS-CoV are trimers".
2.	In Figure 1, line 3, delete the space after "C".
3.	In Figure 1, line 3, change "Respective sequences of these three chains were aligned and visualized using Pymol" by "Structural alignment of these three chains using Pymol".
4.	In Figure 1, line 5, change "of the chain A and C" by "of the chains A and C".
5.	In Figure 2, line 4, change "Elipro" by "Ellipro".
6.	In Figure 3, line 4, delete "While green areas are not".
7.	Figure 4 needs some more discussion in the main text about docking and the presence of glycosylations. Additionally, higher quality is required in order to see all the details in the Figure.
8.	In Figure 5, higher quality is required.
9.	In Figure 6, line 4, delete the space after "server".
10.	In Figure 7, panel f, change placement of the legend.
11.	In Figure 8, panel d, align this panel with the rest of the panels (e and f).
12.	In Supplementary Figure 8, change “Envelop” by “envelope”.

Regarding the Tables, they are also suitable for the understanding of the text, however, the numbering of the Supplementary tables does not correspond to the numbering in the main text.

Regarding Tables and Figures, it would be helpful if the numbering of the S protein sequences is maintained throughout the text, Figures and Tables. For example, while in Table 1, the protein S sequence is the normative one, in the rest of the Tables, Figures and text, it is numbered differently (considering as amino acid 1 the beginning of both NTD and RBD), which leads to confusion when locating the different epitopes in the full length sequence of S protein. In this way, is recommended to clearly indicate this issue somewhere in the text to avoid confusion. Also as an alternative I would propose a Figure showing the full sequence of the S protein showing both the actual numbering and the numbering used by the authors.

Self-contained with relevant results to hypotheses

The article is self-contained, and is organized as a publishing unit, with its respective hypothesis and corroborating results. There are a lot of good looking and organized results that together form an interesting article.
Experimental design: Original primary research within Aims and Scope of the journal.

The article by Rahman et al., meets the Aims and Scope of the journal and I truly think it could be suitable for publication in PeerJ.

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

The article pursues an ambitious objective which is very relevant in the current health emergency situation by COVID-19. The development of a vaccine against the SARS-CoV-2 virus is of vital importance and I therefore believe that the article can give some clues on how to tackle this issue. The issue is therefore clearly defined, as is the objective of the paper and, as I mentioned above, it is extremely relevant due to the lack of information on the subject.

Rigorous investigation performed to a high technical & ethical standard.

The research has been rigorously performed following high technical standards although I have some concerns that are exposed as Major points in this document.

Methods described with sufficient detail & information to replicate.

The methodology set out in the article is clear, comprehensive and well explained, which suggests to me that the results of the article would be replicable.
Validity of the findings: Impact and novelty not assessed. Negative/inconclusive results accepted. Meaningful replication encouraged where rationale & benefit to literature is clearly stated.

The impact of the article is evident in the current situation and therefore the research is clearly relevant. As for the novelty, the use of multi-epitope approaches in vaccine design is quite novel and the literature on this topic has been growing significantly in the recent years. In fact, as discussed in the article, the presence of several epitopes in a single peptide avoids the need to use attenuated viruses and in turn allows the generation of an effective immune response against various viral protein epitopes. The results obtained are promising although I have some concerns regarding the role that glycosylations present in the viral protein and that are not considered in the design and elaboration of the experimental research, may have on the final result (See major points).

All underlying data have been provided; they are robust, statistically sound, & controlled.

All the data are provided both in the main Figures and Tables and in Supplementary material.

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

Conclusions clearly match the obtained results and answer the original research question initially proposed. My only doubt refers to the absence of glycosylations of the S protein throughout the paper and the role these may have in shielding the viral S protein from the antibodies generated as a result of the immunization with the proposed CoV-RMEN peptide. Additionally, if the glycosylated version is required, then, vaccine production will be much more expensive and not relatively cheap as exposed by the authors in the final conclusions.
Additional comments: Major and minor points to be addressed

The article by Rahman et al., is of great interest and in fact the proposed approach is attractive and feasible at a time. The article is well written, clear, and despite the large number of results, it is well organized. In general I think the approach is correct, but I have some comments that may be relevant and need author’s attention.

Major points

1.	The main point against this work is the lack of evidences about the existing glycosylations in the Spike protein as a parameter that can affect the antigenicity of the proposed construction. In fact, this has considerable implications throughout the article. Thus, for example, in Figure 4 where the interaction of various NTD epitopes is modelled. 7 of the 10 epitopes shown in this Figure have some glycosylation (which have been previously published in Site-specific glycan analysis of the SARS-CoV-2 spike. Watanabe et al., 2020), which may affect their recognition, since glycosylations may hide certain epitopes. In fact, the proposed final construction (CoV-RMEN) has 139 amino acids from the NTD, which contains 5 asparagines that are glycosylated in the viral protein (Watanabe et al., 2020). In fact, some of the residues proposed as relevant in the interaction of CoV-RMEN with TLR2-4 are actually close to glycosylated residues in the viral protein and therefore, it would not be unreasonable to think that such epitopes, which would be functional in the proposed construct, would not be accessible in the viral protein. Examples of this are N122 (according to the complete sequence of the S protein of the virus), which is glycosylated (Watanabe et al, 2020), is located between the amino acids L101, I103 and I112, which are relevant in the interaction of CoV-RMEN with TLR3. Another example would be N165 (according to complete sequence of the virus S protein), which is glycosylated (Watanabe et al., 2020) and is the previous amino acid to C150 in the CoV-RMEN construct, which interacts with TLR3.

2.	The effect of glycosylations also has a side effect in the selected construct expression system. If what we are interested in is a construct as similar as possible to the real viral protein, we could consider the 5 glycosylations present in the proposed construct as important. If so, the expression system could no longer be E.coli, since it is not capable of reproducing such post-translational modifications. In this case, a HEK-type expression system, for example, would be more appropriate. I think these observations should be added and discussed in the article.

3.	In the 3D model proposed for CoV-RMEN, neither the SUMO tag nor the histidine tag are included, right? How would the presence of both tags affect the performed dockings? has the possibility of adding a site for a protease (for example 3C) considered for a cleaner and more efficient purification and thus avoid the possible effect of the tags?.

Minor points

1.	Lines 7, 11 and 15. The dash between the city and the postal code is missing.
2.	Line 61. Change “April 15” to “April 15th”.
3.	Lines 71 and 82. “Genus” and “Scientists” in lowecase.
4.	Line 101. Change “stimulates” by “stimulating”.
5.	Lines 122-124. Change the sentence "Multi-epitope vaccine candidates have already been designed..... and their efficacies have been further reported in MERS-CoV and SARS-CoV". In fact, the cited reports are just bioinformatic approaches like the one we are dealing with here and I sincerely do not think that the authors can say that these types of vaccines are effective with the references they provide.
6.	Lines 138, 200, 207, 446, 470, 572, 648 and legends for Figures 3, 6, Supplementary Table 2, 3 and Supplementary Figure 8. Change “envelop” by “envelope”.
7.	Line 141. It is not clear to me why the 3D conformation of S protein is predicted, when S protein 3D structure itself is used as a model, with the same starting sequence as that used by the authors. If the model has been made to complete the regions not modelled in the 6vsb structure, this should be indicated at some point, otherwise it is difficult to understand.
8.	Lines 144-145. Remove one line.
9.	Lines 155-157. Is the high degree of structural divergence referred to the SARS-CoV2, SARS-CoV and MERS-CoV S proteins? If so, it should be explained a little better and include the reference (Walls et al., 2020, Cell) both in Results and Discussion sections, explaining why this divergence is so high. Another option is to remove any reference to SARS-CoV or MERS-CoV as they do not ultimately contribute to anything essential in the article.
10.	Line 162. Add Fig.1 after “MERS-CoV” to clarify the sentence.
11.	Lines 169-171. Would it be possible to re-order the epitopes according to their position in the sequence and not according to their score? I say this because it is more intuitive and easier to compare the same epitopes among proteins.
12.	Line 174. Change “Elipro” to “Ellipro”.
13.	Line 180. Add "prediction" after “B-cell epitopes”.
14.	Lines 188, 205, 208 and 252. Supplementary Tables 1, 2, 3 and 4 are not properly labelled.
15.	Line 224. Add a space between "13-mer" and "124".
16.	Line 238. Define "aa".
17.	Line 246. Add "membrane protein" before "B-cell linear epitope".
18.	Line 309. Remove "(0.0%)".
19.	Lines 327-330. Remove 3 lines.
20.	Lines 353 and 353. Remove 1 line.
21.	Lines 354-367. In Table 3, TLR2 is also shown. Nevertheless, not a single mention is done along the paragraph.
22.	Line 399. Remove one of the parentheses from "((Biswal et al., 2015).
23.	Lines 401 and 402. Remove 1 line.
24.	Discussion section should be shorter. We can find several parts of the Discussion section where results are repeated and could be removed (for example lines 474-480). It would be interesting to make the discussion a little more concise and in any case include some of the comparison between SARS-CoV-2, SARS-CoV and MERS-CoV to ultimately meet the interest of comparing the physicochemical parameters of the S proteins of the three virus species (point 8). In addition, in lines 494-496 the different antigenicity of CoV-RMEN is mentioned depending on the program used but nothing is discussed about this fact.
25.	Line 519. Change "enhance" by "enhances".
26.	Line 549-553. Delete 4 lines.
27.	Line 571. "involved in".
28.	Line 660. " Glutamate should be in lowecase.
29.	Add one space line between lines 665 and 666.
30.	Lines 745-762. Take care about the quantity of empty lines between the two sections.
31.	Lines 773-774. Delete 1 line.
32.	Reference section. Please check the reference style as there are some errors (for example, some volume numers are in bold and some other are not).


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: EPITOPE-BASED CHIMERIC PEPTIDE VACCINE DESIGN AGAINST S, M AND E PROTEINS OF SARS-COV-2, THE ETIOLOGIC AGENT OF COVID-19 PANDEMIC: AN IN SILICO APPROACH
Review round: 2
Reviewer: 1

Output:
Basic reporting: The authors have answered efficiently to all my queries, generating a sufficiently improved version to be considered for publication in PeerJ.

However, there are still some minor points that need to be addressed before publication:

Minor points:

- Line 179: remove the hyphen between amino-acids
- Line 182: In vivo should be in italics
- Line 292: Remove a space between "et al., 2020). and Although".
- Lines 346, 353, 356, 407, 431 and line 3 in figure 5 footnote: Change CovRMEN to CoV-RMEN
Experimental design: No comment
Validity of the findings: No comment
Additional comments: No comment


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

Input:
Title: EXPLORING THE SOCIO-ECONOMIC AND ENVIRONMENTAL COMPONENTS OF INFECTIOUS DISEASES USING MULTIVARIATE GEOVISUALIZATION: WEST NILE VIRUS
Review round: 1
Reviewer: 1

Output:
Basic reporting: Manuscript is well written with introduction and background.
Figures are relevant, good resolution and properly labeled.

References are sufficient and relatively recent.

Structure, figures and tables are per the journal’s specification. Raw dataset is also provided.
Experimental design: Research question is well defined, relevant and meaningful. In order to improve the predictability of the model, authors accounted for spatial autocorrelation and non-stationarity using GWR.

While the SOM is explained, its algorithm is not provided. Authors simply introduce the mechanism of reducing the dimensionality of data, without providing the detail of the cluster process. Therefore, the cluster algorithm remains unknown.
Validity of the findings: Authors indicated in the introduction that they included demographic data in the GWR model to improve the model fitting. But I am not sure how the authors can conclude that the demographic data are enhancing the model performance without demonstrating the process. Also, only two clusters are analyzed which may not be adequate to represent the pattern of WNV infection.

The conclusions could have been stronger if the results were validated.
Additional comments: No additional comments