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: NONSPECIFIC STRESS RESPONSE TO TEMPERATURE INCREASE IN GAMMARUS LACUSTRIS SARS WITH RESPECT TO OXYGEN-LIMITED THERMAL TOLERANCE CONCEPT
Review round: 1
Reviewer: 2

Output:
Basic reporting: The manuscript is well structured and conforms to PeerJ standards. The English language needs to be improved to ensure understanding of your text and findings. I have given line item suggestions in general comments below for the early sections of the manuscript. Grammar and sentence structure are an issue throughout and especially in the discussion. Please revise with this in mind, paying special attention to the discussion.

Line 23: Although you are indeed the first to assess the combination, anyone working on non model organisms can use a similar statement as the leading sentence in almost every study they do. I suggest modifying the organization of the introduction to draw in the reader (and also refocus the abstract). Are the amphipods themselves the unique and interesting thing that lead to this study, or was it driven by the question and they are an excellent study organism to address it? You could possibly lead the introduction with the paragraph at line 52 which is well written and indicates your research was question driven.

Figures are relevant and high quality. The figures could be improved by adding more details to the legends. Please include number of replicates and number of amphipods per replicate in figure legends: n for each bar and number of individual amphipods for each n.

I am glad to see the raw data is provided, however I think further organization would clarify how the study was conducted. The western blot gels do not provide what the ladder is to conclude the bands on the gel are actin and HSP70. Also the replicate structure is unclear (see additional questions pertaining to this in experimental design comments). Are the HSP 70 replicates additional measurements on the same initial samples, or repeat measurements that consist of new specimens from the same temperature treatment? No repeat measurements were done on the same samples for LDH, and lipids, but there were for HSP70, is that correct? Is what you call "replicates" in the HSP70 supplement repeat measurements of the same initial samples? My understanding is the Glk022s1 etc are your sample naming system, please label as such. What are the numbers 1-14 etc under the column with no heading in the HSP70 supplement? Why is there no background etc listed for the first HSP70 samples, but there are for all replicates? What are the numbers below the Glk022s1 numbers in the first block of HSP70 raw data?
Experimental design: This research is original primary research and within the scope of the Journal.

The research has been preformed to high technical and ethical standards. When the replicate and repeat measurements structure is better described it will become clear how rigorous the investigation is. Were the same samples used for all biochemical analysis, or unique organisms for each analysis? Based on the numbering in the supplement it appears that unique samples were used for each biochemical analysis, but that needs to be described more clearly.

The Methods description needs to be improved in several key areas to ensure sufficient detail is provided. How many individual organisms were in each 2L aquarium? How was the temperature of the aquarium controlled? Exactly what are the control treatments? Were those organisms kept at 7°C for the same amount of time as the longest temperature experiment? Please justify the 1 degree per hour increase. It is referred to as "gradual", but how does that compare to how fast the environmental temperature changes for this species in the location you collected it? Also it is unclear what you mean by the phrase  "ambient temperature". Is that the acclimation temperature in the laboratory? If so ambient is not an accurate description of it. It is stated in the abstract that 7°C is the mean annual temperature of the lake, but what was the temperature of the water when specimens were collected? Did you check the concentrations of all the biochemical markers you examined from specimens frozen in liquid nitrogen directly after collection? This would inform what there constitutive levels are. You state at line 292 that there is a "initially high pool of these proteins in cells" but it may be that HSP 70 was induced in response to the temperature change between environmental temperature and laboratory.

For all biochemical methods: HSP, LDH, lipid peroxidase: how many specimens were used for each measurement? Did you pool specimens, or are measurements on individuals? For example line 102- what you mean by samples? Is that a single individual from each temp treatment? I see in line 97 you say 2-3 specimens per sample. So only 2-3 individuals were exposed to each treatment? Were those 2-3 then combined for one measurement? At line 140 you state all experiments were carried out with 3-8 biological replicates- what does this mean?
Validity of the findings: It is difficult to assess the validity of the findings in the current version of the manuscript. This is due to the mentioned gaps in information on the experimental design, and grammatical and structural errors in the English language throughout the discussion. These issues need to be addressed before the findings can be properly considered.
Additional comments: Line 59 Poor readability. This sentence needs to be improved to ensure the research question is well defined, and the phrase " ambient temperature gradual increase" needs to be revised throughout the manuscript (for example also at line 153). It is not clear what you mean by ambient, and this phrase is awkward and too vague.

Line 61: This species is from a saltwater lake, but in line 65 you say lentic an steam ecosystems, implying freshwater. Does it occur in both fresh and salt water habitats? Line 69 you list as euryhaline. I suggest you move the sentence at line 69 up and either combine it with the one at line 65, or have it just before to clarify the habitat these amphipods live in.

Line 65: Add with: "species with a..."

Line 74: "plant food"- change to plant material

Line 84: Please clarify: the plankton tow was done bottom to surface, but you then list above 7 meters depth, do you mean 7 meters water depth? Or do you mean 7m to surface for the tows?

Line 85: Do you have data for temperature range in the natural habitat? Including daily temp fluctuation in the summer at this location, and day night change?

Line 96: Please add what the longest experimental duration was in hours.

Line 98: Why this reference? Are you citing that paper because you fixed in liquid nitrogen in a way suggested in that publication? That citation does not seem relevant here.

Line 112: Please check this antibody number, as the sigma produce #A266 is not for β-actin

Line 122: Please provide product number for LDH express kit, as I could not find it when searching the Vital-Diagnostics webpage.

Line 129-131: Poor wording, restructure as "To separate lipid peroxidase fractions, 1 ml of distilled water was added to samples, which were stirred by xx then incubated at 25°C for 30 min." Please also add how stirring was achieved (xx), and if you want to use intense, explain what you mean by that.

Line 152: Sentence structure issue: why is ambient in the sentence (similar issue with line 59 of the introduction)? You haven't stated what the ambient environmental temperature was when you collected specimens. Do you mean the acclimation temperature (laboratory)? Possibly change to "... during the entire exposure of acclimation at 7°C and gradual (one degree per hour) temperature increase to 33°c "

Line 153: What effect are you refereeing to it imposing? Inhibitory?

Lines 163-164: Sentence structure, try taking out the "in" after "obtained", and add "the" after "conjugates in".

Line 166: Change from till to until, which is formal English.

Lines 178-183: Poorly structured sentence, please revise.

Line 184: Is this according to early experimental data in this study? If so reference where that data is provided. if it is refereeing to data from another study please cite.

Line 191: What do you mean by earlier? Another study? If so please reference it.

Line 203: Provide a reference here.

Line 203: Please change  "evidences" to "demonstrates."

Line 242: Change "participates" to "catalyzes".

Line 249: Add s at the end of "concentration".


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: NONSPECIFIC STRESS RESPONSE TO TEMPERATURE INCREASE IN GAMMARUS LACUSTRIS SARS WITH RESPECT TO OXYGEN-LIMITED THERMAL TOLERANCE CONCEPT
Review round: 2
Reviewer: 1

Output:
Basic reporting: 'no comment'
Experimental design: 'no comment'
Validity of the findings: 'no comment'
Additional comments: The n should be specified specifically, not an interval
Should discuss with other bioindicators if there are studies related to the topic and otherwise expose it clearly in the discussion


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: NONSPECIFIC STRESS RESPONSE TO TEMPERATURE INCREASE IN GAMMARUS LACUSTRIS SARS WITH RESPECT TO OXYGEN-LIMITED THERMAL TOLERANCE CONCEPT
Review round: 2
Reviewer: 2

Output:
Basic reporting: There are still a few areas that need some clarification. Overall the basic reporting has been improved. I appreciate the author's effort reporting sample size, and number of individuals per replicate and for improving the raw data tables. With some more minor revisions the manuscript should be ready for publication.
Experimental design: The experimental design is clearly explained and justified. My only concern is in the western blot images for HSP 70: the ladder you provided information for has a 75 kda band at the first pink band. The images of the western blot appear to have that band at the top. Could you please mark the Kda band sizes for your ladder to demonstrate that the bands from the samples are at the correct size to interpret as HSP 70. Also you do not refer to a positive control in your methods though it is flagged on the gel. Is actin, what you call your "reference protein" your control? Please clarify and use a consistent term.
Validity of the findings: With some minor revisions to the discussion and conclusions the validity of the findings will be more evident. The data is robust, though the sample size is small due to limitations working on non-model organisms. Conclusions are generally well stated, and supported by the results. Although it is not clear to me that you demonstrate evidence for high constitutive levels of HSP 70. Please clarify, and include references to previous work if that is how you are interpreting this as high.
Additional comments: Line 34 and 35: But you didn’t measure lactate accumulation, only LDH activity. Please revise to either say LDH, or to clarify you are citing a previous study.
Line 74: Change “in gradual temperature” to “during gradual temperature…”
Line 77 Awkward sentence: maybe: In many organisms when exposed to stress conditions the amount of HSP 70 is elevated due to the increase in damaged proteins”
Line 140: Is this your biological replicate you refer to in the statistical analysis section? Need to clarify both sections.
Line 169 “optical density…” needs to be moved up to HSP section. Please provide spectrophotometer information in LDH section. What temperature was it measured at?
Line 227 on: This sentence needs to be rewritten, it doesn’t make sense, “will inhabit” what?
Line 236 Please change “freshwater” to “saltwater”
Line 239 Change “conservative” to “conserved”. “diply” maybe you meant in-depth?
Line 241: What do you mean by “molecular underpinnings”? Rephrase or clarify
Sentences from Line 242 -260, HSP section is repetitive, and should be rewritten to be more concise.
Line 274 Are you saying you consider the levels of constitutive HSP 70 high in this study? I do not see evidence for this, so please clarify and support this. (Also line 337)
Line 295 How instantaneously is LDH synthesized? Would an hour be fast enough for it to increase?
Line 324: “Increase the number” does not make sense to me. Clarify if you mean increase the use of anaerobic pathways, or increase to using more than one?
Line 331 change “this can evidence” to “this demonstrates”
Line 337: change “probably” to “possibly”. You will need to add more to the discussion to justify that you think there is high constitutive levels


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

Input:
Title: DRAMATIC DECLINE AND LIMITED RECOVERY OF A GREEN CRAB (CARCINUS MAENAS) POPULATION IN THE MINAS BASIN, CANADA AFTER THE SUMMER OF 2013
Review round: 1
Reviewer: 1

Output:
Basic reporting: This paper is extremely well written from a technical perspective with the author using very clear language throughout. There are no grammar issues, and the readability level is high. Literature is well-sourced, with a selection of the most current and seminal references on the topic of green crabs being cited. The figures and tables are well formatted, and the raw data, once re-analysed, confirm the results. The paper follows logically from hypotheses to relevant results and discussion.
Experimental design: I feel strongly that this research fits within the scope of PeerJ’s mandate. The research is topical, and with the regional population crash of 2013, is quite important to the region as it remains an understudied phenomenon. Having been involved in similar long-term studies investigation population fluctuations for this species, I can say definitely that this represents the most thorough attempt at describing the trends observed across the region.

I do however, question the suitability of using shoreline quadrat data as an appropriate methodology for assessing green crab populations in lieu of using more traditional trapping or aquatic survey data. Without traditional indices such as Fukui trapping CPUE, it is difficult to superimpose these results across the region. An more thorough emphasis on the potential drawbacks of using shoreline data should also be included. For example, while the author notes that green crabs may retreat deeper into the intertidal zone, the actual number of crabs that actually do complete this behaviour is unknown and could depend regionally due to many factors – one of which, tidal fluctuations, is quite important in the area sampled. This impact should not be understated. WE do not know how much of the population remains on shore between tidal flushes, and this ratio or number is essential in discussion overall population trends. While the author does include data collected from flounder fishers as an attempt to validate or control for the shoreline data, the potential for this fishing methodology to capture green crabs is not thoroughly explained. If the author were to elaborate on these methodologies, as well as to explain why trapping was not possible, I think the methodology explanation would be improved greatly.

Methodology descriptions aside, I feel that the investigation of these data was of a very high technical standard, with all appropriate statistical analyses performed. However, the author might consider a more robust multivariate methodology for the investigation of the potential biological characteristics that may affect the densities of crabs.
Validity of the findings: This paper presents its findings in a conclusive manner. The conclusions are well stated, and the derivations of said conclusions are not without merit. I find this paper to be a valuable addition to the green crab literature.
Additional comments: I enjoyed reviewing this paper as it attempts to answer a question that I have heard repeated around the region in regards to the population trends over the past few decades. While I do have reservations about the use of shoreline data for inferring population trends, I understand that trapping is not always feasible. However, I am confident that the comparison from year to year, at least for the Minas Basin, is entirely appropriate.

It would be great if you could find some water temperature data for the area. While air data are appropriate to use as a proxy, they tend to fluctuate much more broadly, and may represent a potential issue.


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: DRAMATIC DECLINE AND LIMITED RECOVERY OF A GREEN CRAB (CARCINUS MAENAS) POPULATION IN THE MINAS BASIN, CANADA AFTER THE SUMMER OF 2013
Review round: 1
Reviewer: 2

Output:
Basic reporting: This study is observational, with no experimental component, but the length of time that the study spanned makes it an interesting contribution, since ecological mechanisms can only be studied after patterns are carefully described.  However, the data analyses are weak in a number of ways, which requires further work.  Detailed comments follow below:
L119. Adding habitat photographs (never a limitation for online articles) would enhance the description of the studied habitats.
L120. This study was done in Clarke Head. How general are the patterns in this entire region? Is Clarke Head an anomaly? Or a good representative of the generality?
L160. At one same site on the Maine coast, I have seen no green crabs during the day, but many during the night. How different would results have been had the author made the surveys at night?
L168. What range of intertidal elevations (in meters above chart datum) were sampled? Intertidal species abundance and size depends heavily on intertidal elevation (see, for example, Scrosati, Grant & Brewster 2012. Density and size gradients across species distribution ranges: testing predictions from the abundant-centre model using the vertical distribution of intertidal barnacles. Vie et Milieu - Life and Environment 62: 197-202).
L197-198. In online articles, where space is not a limitation, there is no need to state “results not shown”. Results should be shown in one way or another. Otherwise, readers are being asked to believe in the text solely based on faith, which is not evidence-based science.
L219. This question seems particularly well suited to be analyzed through multiple linear (or nonlinear) regression, as opposed to separate correlation analyses for each independent variable. Models including all possible combinations of variables could then be compared using their AIC scores, in that way one being able to discern what variables were the most important.
L232-233. Could the specific months that affect larvae, juveniles, adults, etc, be selected on a case-by-case basis, instead of only using fixed “seasonal” groups of months? That approach could increase the chance of detecting significant relationships.
L240. Seawater temperature, air temperature, or both? Intertidal organisms are affected by seawater temperature during high tides and air temperature during low tides. This is addressed later in the text, but it should be explained here, as it is the first mention of temperature.
L261-263. Those values of air temperature were likely not unequivocally related to intertidal organisms, because only values of air temperature calculated during low-tide periods should be used. Matching the timing of low tides to air temperature values should therefore be done in order to use only the values important for intertidal organisms. Values of air temperature calculated during periods of high tide (for the sampled elevations) should be excluded from analyses concerned with air temperature as an ecological factor.
L270. That assumption is not reasonable, because crabs normally stay under macroalgal canopies during low tides to avoid heat stress. Air temperature values, therefore, will usually not tell what crabs are actually experiencing at low tide.
L282-283. This could be approached by comparing models using AIC, as I mention above.
Table 3 must provide a P value for each r coefficient. This is a cornerstone concept in statistics. There will always be an r value for any X-Y relationship, but concluding whether it matters or not can only be done after calculating its significance. In other words, speaking about “high” or “low” values of correlation without making the corresponding significance tests is meaningless.
In Table 3, it is also unclear what “SD”, “Min”, and “Max” represent.
L423. Again, such a conclusion can only be provided when any given r value is found to be significant (usually P < 0.05) after the corresponding statistical test.
L466. Chignecto
As this study is entirely observational, it is not possible to identify causal factors for the observed patterns. Therefore, the Discussion section can only offer speculations on why things happened the way they did. However, the provided speculations, although open to interpretation, are generally reasonable. Overall, because this paper identifies a major ecological change in 2013, it represents a useful contribution, although the statistical issues identified above must first be addressed to allow this paper to be based on sound data analyses.
Experimental design: See the "Basic reporting" section above.
Validity of the findings: See the "Basic reporting" section above.
Additional comments: See the "Basic reporting" section above.


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

Input:
Title: DRAMATIC DECLINE AND LIMITED RECOVERY OF A GREEN CRAB (CARCINUS MAENAS) POPULATION IN THE MINAS BASIN, CANADA AFTER THE SUMMER OF 2013
Review round: 2
Reviewer: 1

Output:
Basic reporting: I find that the author has addressed the concerns laid out in the previous review adequately. I find no other issues with this submission. I find it a valuable piece that contributes to Green crab and invasive species research both regionally, and locally.
Experimental design: No comment.
Validity of the findings: No comment.
Additional comments: No additional comments


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

Input:
Title: EXOME SEQUENCING STUDY OF 20 PATIENTS WITH HIGH MYOPIA
Review round: 1
Reviewer: 1

Output:
Basic reporting: This paper is well written and is easy to follow. In terms of writing, one minor suggestion is that the authors should provide some statistics or references to support their claim as "The high prevalence of high myopia in East Asian population" in the Introduction section.
Experimental design: The objective of this study is well defined and of high value. The experimental design was properly demonstrated and the proposed variant analysis approach was reasonable. The authors focus on rare variants uncovered by exome sequencing, so it is helpful to discuss the effect of common variants on high myopia and if any GWAS have been conducted to study this disease.
Validity of the findings: Several potential disease genes were identified in this study, and the functional relevance of these genes was validated by gene expression analysis and network enrichment analysis. The authors may discuss the potential clinical application of these findings.
Additional comments: In this paper, the authors presented a study to use exome sequencing for investigating the genetic basis underlying high myopia. The authors proposed a phenotype-driven approach for filtering and analyzing genetic variants, followed by expression analysis and network enrichment analysis, which provided relatively strong evidence for the functional roles of the uncovered disease genes. The experimental design was reasonable and the results were informative and valuable. Therefore, this paper was believed to make some contributions to the research community and should be accepted.


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: EXOME SEQUENCING STUDY OF 20 PATIENTS WITH HIGH MYOPIA
Review round: 1
Reviewer: 2

Output:
Basic reporting: The manuscript is well written and clear. This is an interesting genetic study in the clinical setting. The following are my comments:
1. Line 55, "disease-causing genes": in genetic studies, people usually use the "disease risk genes" or "disease-associated genes". Finding causal-relationship is challenging and usually needs detailed downstream functional experiments performed in addition to the genetic study. If the genes are merely associated with the disease by previous studies, I would suggest changing the wording to "disease risk genes" or "disease-associated genes".
2. Preprocessing the data: line 129, RPKM was transformed by log2. People usually use log2(RPKM + 1) to avoid negative values. Without adding the pseudo count, the numerical behavior for RPKM close to 0 will be problematic. Please use log2(RPKM + 1) and redo the analysis and figure 2.
3. Line 167, how was the 709 genes assembled? This should be described in detail in the methods section.
4. Details for some statistical analysis should be described. Line 174, what statistical test is performed to obtain the p-value? This should be described in detail in the methods section.
5. Line 147, "a total of 20 potential pathogenic (3 splicing, 1 frame-shift and 16 deleterious missense) heterozygous variants in 12 genes were identified in 16 patients". In line 116, "Pathogenicity of missense mutations was assumed if predicted pathogenic by at least 6 out of 8 computational methods". What happens if the criterion is relaxed (say 5 out of 8, 4 out of 8, ...)? How does that change the number of selected variants and downstream bioinformatics analysis?
Experimental design: Please see basic reporting
Validity of the findings: Please see basic reporting
Additional comments: Please see basic reporting


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: EXOME SEQUENCING STUDY OF 20 PATIENTS WITH HIGH MYOPIA
Review round: 1
Reviewer: 3

Output:
Basic reporting: In the manuscript, Wan et al performed whole exome sequencing analysis for 20 individuals with myopia to identify pathogenic gene variants. This resulted in the identification of 20 potential disease-associated variants from 18 out of the 20 patients. Following that bioinformatics analyses were performed to investigate the relevant functional subnetworks and pathways enriched for genes carrying these variants. Overall, the manuscript is clearly written, but some revisions are required before the manuscript can be published.
Experimental design: (1) Causality. Limited by the experiment design and small sample size, this study cannot provide strong evidences that support the identified rare variants are causal. It is also difficult to calculate statistical significance of association of these variants with the disease. As shown all variants except one was identified only once (I assume the variant indented twice are from patients of the same family). Thus, it would be needed to discuss these limitations in the “Discussion”.

 (2) Two of the patients are from the same family. It will be interesting to provide more information about their variants. I assume that these two patients are R0027 and R0029. Do the majority of variants identified shared by them? Genetically related individuals tend to share variants, but may inherit a different set of genetic variants. However, the variants causing the disease should be shared.
Validity of the findings: (1) Enrichment analysis. It is not clear what genes are used as background in the network and pathway enrichment analyses. Since these myopia-associated genes were selected by following a filtering protocol that focus on eye-disease related genes. It will certainly result in enrichment of related pathways and GO terms, if the background was not carefully selected (e.g., use all genes as the background for enrichment analysis). Thus the bioinformatics analyses reported in this study may not be valid.

(2) In the discussion, the authors described that many of the genes identified in this study have also been identified in previous studies. But it is not clear whether some of these 20 variants have also been reported in previous myopia sequencing studies.
Additional comments: Minor comments:
(1) In the Abstract, the meaning of “Our phenotype driven filtering method yielded much higher diagnostic rate than previous WES studies” is not clear. It should be removed from the abstract.

(2) Some sentences in the manuscript need to rephrased to avoid confusion. For example, in Line 100 “Variant calling was performed by a consensus call of at least 2 methods out of 4 haplotype-based calling algorithms”.

(3) Line 174, P< < 0.001 should be P<0.001.


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: EXOME SEQUENCING STUDY OF 20 PATIENTS WITH HIGH MYOPIA
Review round: 2
Reviewer: 1

Output:
Basic reporting: The revision has addressed all my comments to a reasonable extent.
Experimental design: The revision has addressed all my comments to a reasonable extent.
Validity of the findings: The revision has addressed all my comments to a reasonable extent.
Additional comments: The revision has addressed all my comments to a reasonable extent.


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: EXOME SEQUENCING STUDY OF 20 PATIENTS WITH HIGH MYOPIA
Review round: 2
Reviewer: 2

Output:
Basic reporting: All my comments have been addressed. I thank the authors for carefully revising the manuscript.
Experimental design: None.
Validity of the findings: None.
Additional comments: All my comments have been addressed. I thank the authors for carefully revising the manuscript.


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

Input:
Title: PHYLOGEOGRAPHY OF SWERTIA PERENNIS IN EUROPE BASED ON CPDNA MARKERS
Review round: 1
Reviewer: 1

Output:
Basic reporting: The documentation and  presentation is very good, most parts are well written, but there are many language errors (article use, singular/plural) and also typos.

Otherwise documentation and main parts are well written although there are some language issues with the grammar which would need attention

There are plenty of language problems – e.g.:
Line 55: level of gene flow in the recent past?, line 58: difficult to speculate about relations?? 118 Taberlet, 137: inhabits, 148 flourishEs.350 formation of divergent cpDNA?  355 specialist sp.?
Experimental design: The authors provide a phylogeographical study based on cpDNA variation of populations of Swertia perennis, a species of minerotrophic bogs and alpine wet grasslands widespread in Eurasia, but disjunctly distributed in Europe. The authors studied mainly populations of European mountains although the species is widely distributed in Eurasia. Despite haplotypic variation found the authors could not confirm any clear genetic and phylogeographical structure, which does not allow strong conclusions.
Validity of the findings: The authors evidenced that there is a lack of phylogeographical patterns and raise several possibilities (postglacial immigration, many refugia,…) as explanation. As the sampling is uneven I would recommend to provide the total distribution of the species in the study area. This should give additional support for the “lacking pattern”.
I can follow more or less that there is a (complete?) lack of phylogeographic structure, nevertheless they could test hypotheses. It might me worthwhile to test the hypothesis that (like in other alpine species) late/postglacial immigration from Central Asia might be a valid explanation for the pattern observed. Swertia perennis is a very common species in large parts of Eurasia. I am also not fully convinced that there is no phylogeographical structure, the three Alpine samples cluster in one clade.
I suggest that the authors provide at least summary statistics (e.g., using Arlequin) to show lack of pattern (e.g. using AMOVAS of regions). Also other statistical phylogeographical approaches could be taken into account. (testing for expansion, etc.).

The authors state that “clonality” could have an effect, were more than sample per population analyzed? This is unclear. I understood that only one sample per population was sequenced, this no conclusions on clonality could be drawn.
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: PHYLOGEOGRAPHY OF SWERTIA PERENNIS IN EUROPE BASED ON CPDNA MARKERS
Review round: 1
Reviewer: 2

Output:
Basic reporting: The presented manuscript of Urbaniak et al. addresses phylogeographic differentiation of Swertia perennis, the plant species occurred in peat bogs of northern hemisphere. The authors based their study on the cpDNA markers to elucidate the history of its nowadays distribution. As the information of the phylogeography of wetland species in Europe is still insufficient, the paper brought some news to our knowledge of the genetic differentiation of this group of species.  The methods used seems to be adequate and the manuscript seems to be well structured. However, the manuscript needs to be improved before an acceptance to the publishing. The authors used only one individual per population and they should be more aware of their hypotheses thorough the text. Every time they should use something like “the individuals represented populations” .. not only “populations”. Additionally,  they discuss the genetic position and potential history of lowland populations but they used only four populations - also here they should be more cautious. One part of the hypothesis is connected with isolated site or isolated population but we have no definition of these terms and no list of the individuals (populations) linked with these terms.
In some parts of the text the authors are not congruent. There is not clear what authors treat as one haplotype – sometimes they used parsimony network, sometimes phylogenetic tree. It could be explain and consistent. Maybe they should list somewhere in the results the numbers of localities shared the same haplotypes or put it in the Table 1.
The manuscript should be also linguistically and grammatically improved. As the English is not my mother language I do not feel good enough to improve it.
The authors should check carefully the German, Slovak, Czech last names and names of localities for diacritical marks – as there are many differences between references and the main text; in the table 1 there is any for Slovak localities.
Experimental design: Original primary research within Aims and Scope of the journal.

The submission should clearly define the research question, which must be relevant and meaningful - see the comment in the basic reporting section.

Rigorous investigation performed to a high technical & ethical standard.
Methods should be described with sufficient information to be reproducible by another investigator - see the comment in the basic reporting section.
Validity of the findings: Conclusion are well stated, linked to original research question & limited to supporting results, but see in the basic reporting.
Speculation is welcome, but should be identified as such, .
Additional comments: For the authors:
The presented manuscript of Urbaniak et al. addresses phylogeographic differentiation of Swertia perennis, the plant species occurred in peat bogs of northern hemisphere. The authors based their study on the cpDNA markers to elucidate the history of its nowadays distribution. As the information of the phylogeography of wetland species in Europe is still insufficient, the paper brought some news to our knowledge of the genetic differentiation of this group of species.  The methods used seems to be adequate and the manuscript seems to be well structured. However, the manuscript needs to be improved before an acceptance to the publishing. The authors used only one individual per population and they should be more aware of their hypotheses thorough the text. Every time they should use something like “the individuals represented populations” .. not only “populations”. Additionally,  they discuss the genetic position and potential history of lowland populations but they used only four populations - also here they should be more cautious. One part of the hypothesis is connected with isolated site or isolated population but we have no definition of these terms and no list of the individuals (populations) linked with these terms.
In some parts of the text the authors are not congruent. There is not clear what authors treat as one haplotype – sometimes they used parsimony network, sometimes phylogenetic tree. It could be explain and consistent. Maybe they should list somewhere in the results the numbers of localities shared the same haplotypes or put it in the Table 1.
The manuscript should be also linguistically and grammatically improved. As the English is not my mother language I do not feel good enough to improve it.
The authors should check carefully the German, Slovak, Czech last names and names of localities for diacritical marks – as there are many differences between references and the main text; in the table 1 there is any for Slovak localities.

Some detailed suggestions:
L45: The cpDNA were analysed to detect genetic relationship between…
L47: 20 haplotypes were revealed across the dataset. They  …..
L54-55: the sentence is not congruent with the text: see l.280-286, 301-303, 323– it should be deleted or written in another way.

Introduction:
L.63: ..are a consequence of..
l.66: Hewitt….; Taberlet et al....
l.67: ... in the front of…
l.69: Slovák ..
l.90: …potential refugia for …
l.90-91: …in fragmented subranges in several countries: - rewrite - it should be written regard to regions not countries.
l.106: Winkler et al., 2012;
l.105-107: the authors should mention here the article of Swertia by Lienert at al (2002) at some point.
l.111: The populations …
l.114: …populations which..
l.118: Taberlet …
l.125: .. for investigation the genetic..
l.131: this part of the hypothesis is not well constructed as the authors did not defined and did not listed what they considered as isolated population or isolated site for Swertia perennis in their study. They should explain it  and define or change (delete?) this question.

The species
l.136-137 – somewhere should be done the family of the species.
l.137: …is a highly morphologically variable taxon, …
l.140: ..but it is also..
l.147: ..cm
l.148: It is a diploid…
l.148-149: The information about pollination system, fruits and dispersal mode should be done.
l.149-150: I will advice at least to cite Lienert, Fischer, Diemer (2002: Local extinctions.. ) and Pawlikowski & Wołkowycki (2010) according to the disappearance of the species localities (or put some sentences with citation).

Study area & sampling
l.155-160. The authors have to directly write that it was one individual per population. They should also write about their idea of sampling – as they write in l.128 – 1300 km disjunctive distribution, some individuals were sampled in closed populations (see l.294-295) etc
l.159: tissue=leaves?, ……in to=into
l.160: persevered?

DNA isolation….
l.173: ….in these …, ….biogeographically…
l.176: trnLF?
l.179: The DNA extracts were used to …..
l.184: …45 s min?
l.192: analyses

cpDNA data…
l.199: should be here explain why both cpDNA regions (see l.170: three chloroplast…)
l.220: this sentence should be rewrite to be more informative (e.g. In total, 28 sequence of S. perennis individuals and sequences of five other species used as an outgroup……)

Results:
The spaces between numbers – be consistent with the journal requirements
l.228: …populations.
l.236: …varied from?
l.245: dataset
l.246-247: rephrase the sentence
l.250: ..populations
l.250. …One of them represents a common haplotype … There is not exact as we see on the Fig. 3 two haplotypes: 16,25,27 and 3,4. I think that the authors should be more consistent with using “haplotype” in this paragraph and use word clade or another word for the clade of the tree (eg. L.251-253: Other clade combines two haplotypes: ….). Maybe the authors should rewrite this paragraph to be more clear for readers.  I am not sure from the text and figures what authors treat as type of haplotype (what is the primary analysis?).
I will also advice to put the names/localities or numbers of some samples.
l.256: rewrite sentence: not populations but individual represented population.
l.256/7: most of them (how many?)
l.258: ..of the same and/or other …. – what does it mean? If the authors have more than one specimen per population the results will be more convinced. They also should put this information in the method section.
l. 259 ..across the studied part of the European range (or something like that)

Discussion
l.256: analyses
l.267-270 – I am not convinced if the example of R. pygmaeus is good here as the authors in the next sentence write of the origin of Swertia perennis from Central Asia. Next sentences (l.270) do not prove this hypothesis, rather more complicate it. Maybe the authors should use better explanation of their hypothesis (like diversity of genus Swertia or better explanation of distribution of S.perennis).
l.272: across its studied part of ….
l.276: .. based on cpDNA from populations existed in ….(geographical regions)
l.276-277:  the authors did not studied polymorphism within populations! – this sentence should be rewritten or delete. Surely, they should refer to the paper of Groff et al but be aware of the dataset (geographical regions, number of samples per population)
l.277-280: delete – it is written l.258..
l.279: analyses
l.282-286: this part of the text should be clarified; it should be clearly written that this are the results of Groff et al. In first reading it seems that the authors studied polymorphism within populations (more than one individual per population) and they did not observe seedlings.
l.289: please change ..the whole of Europe ..
l.290: ..cpDNA of …
l.293: ..among close (neighbouring) geographically?
l.294: Hala Cebulowa … should be in the new sentence with region listed.
l.293-300: should be rewritten to clarify – it is very interesting that close localities are so differentiated
l.311: (any citation?)
l.318: (17,18,19,26,28)
l.319 – delete “the Alps” – none of the listed samples d not originate from the Alps.
l.320-322: the authors should be aware here of the glaciations history of the polish lowlands. Maybe the 25 could be the older then 26-28. Maybe the authors will find the palynological papers from these part of Poland with the history based on peat bogs. If so, it could bring a new insight.
l.324-326. I am not sure if the example of Quercus species is here good – there are several species and they are woods.
l. 326: Dumoulin-Lapègue
l.335-337: these sentences should have placement (detailed information of subspecies) in The species section or should be deleted.

Conclusion
l.341-355: some of the sentences should be rewritten according to changes in the main text.
l.341: in Europe: in studied part of European range
l.346: other northern taxon types?= other plant taxa with northern distribution?
l.347: populations
l.348: Alsos et al., 2012).
l.350: cpDNA
l.355 sp.??

References
The authors should carefully check the cited positions in accordance to: diacritical marks, letters of names, letters in titles (small/capital), dots after journal types, comma after last name, spaces.
Here are some examples that I noticed:
l.369, 372, 373, 376, 408, 430, 435, 465, 497, 502, 518, 565, 568, 571.

Figures
Fig. 1: Distribution of studied populations of Swertia perennis…. …..analyses.
(it would be nice to have distribution range shaded)
Check the color for 3, 4 – according to Fig. 3 it should be different and, of course, consistent through all figures.

Fig. 2 …Swertia….

Fig. 3. ….of Swertia perennis.
The authors should be consistent with the size of the circles: too large for 1,23, 3,4, 7. Be aware of the colors as it was mentions above.

Table 1. Sampling localities for the studied individuals of Swertia perennis across the European range of the species.
The authors should use the diacritical markers for the localities names. The columns 2, 3 should be left aligned.
27: Kotlina??

Table 2 and 3: Variable positions in the cpDNA t….region within Swertia perennis individuals. ….The number and locality names correspond to Table 1.


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: TH2 CYTOKINE BIAS INDUCED BY SILVER NANOPARTICLES IN PERIPHERAL BLOOD MONONUCLEAR CELLS OF COMMON BOTTLENOSE DOLPHINS (TURSIOPS TRUNCATUS)
Review round: 1
Reviewer: 1

Output:
Basic reporting: The paper is well written and even though is not totally ground-breaking, it is an important contribution to the nanotoxicity field. It provides new insight on how the nanomaterials can affect different organisms, and those differences can be seen even at cellular level.
The conclusions are well supported by the results.
Experimental design: The methods used are appropiate for the study.
Validity of the findings: Again, the conclusions are well supported by the data presented in the results section. No invalid extrapolations or specutalions were done (as tempting as it can be).
Additional comments: The paper is well written and even though is not totally ground-breaking, it is an important contribution to the nanotoxicity field. It provides new insight on how the nanomaterials can affect different organisms, and those differences can be seen even at cellular level.

The discussion can be improved. The information presented is all relevant, but sometimes it seems repetitive, making this section unnecessarily long. In general, try to be more concise, to the point.

I would also add some discussion on the effects of nanomaterials on the immune system of other marine organisms. For example, it has been demonstrated that nanomaterials can negatively affect the function of hemocytes in mussels (Canesi et al, 2012 Mar.Env. Res., 76:16, and related publications). Eventhough the organisms are very different and their immune systems have different modes of action, it would be illustrative to discuss how nanomaterials affect the immune system, making aquatic orgnisms more susceptible to infections.

Specific questions, suggestions and corrections are described below:

Line 130 - Please define ConA and briefly introduce the concept.

Line 158 - ug/ml, change the micro symbol.

Line 312-313 10 ug/l is not higher than 0.1 ug/l; please rephrase what you mean. It is well-known that the toxicity of nanomaterials depends on multiple factors, such as the properties of the nanomaterials (including the chemical composition) and the conditions of the experiment. It is briefly mentioned that the studies cited used a "variety of AgNPs". Though the specifics are discussed later in the paper, please briefly mention what was different: was it the composition? was it size? Also, briefly discuss if the experimental conditions were different and how that might influence the results.

Line 316-323 Please be more concise. For example: "It has been demonstrated that ConA induces proliferative activity and gene expression of cytokines in bottlenose dolphins, but no information is available regarding the time course."

Line 332 - What do you mean by "escalating trend"? In Figure 2 I do not see significant differences between time 4, 16 and 24.

Line 395 - Change "inhibition" to "inhibitory".

Line 416-417 - Change "have been" to "be". The likelyhood of exposure to AgNPs might increase in the coming years as its use in commercial products keeps increasing.


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: TH2 CYTOKINE BIAS INDUCED BY SILVER NANOPARTICLES IN PERIPHERAL BLOOD MONONUCLEAR CELLS OF COMMON BOTTLENOSE DOLPHINS (TURSIOPS TRUNCATUS)
Review round: 1
Reviewer: 2

Output:
Basic reporting: 1. Clear English language is used throughout with a few minor exceptions.
There are some issues of redundancy within sentences throughout the paper.
Line 394-395: major… mainly… use of both of these terms to describe the effect of C-AgNP20 in the same sentence is redundant
Line 416: “may” used twice in sentence
In line 111, “Homology” rather than “coverage” would be more appropriate here.
In line 332, authors used the word “escalating” which means increasing. However, the cytokines are decreasing over the time periods mentioned.
2. It is not necessary to present the abbreviation for silver nanoparticles or peripheral blood mononuclear cells in the article title.
3. There are some instances of non-English characters used in the supplemental materials. If possible, these should be changed to English to correspond to the text of the manuscript.
4. Indent line 381.
5. Intro and background sufficiently show context and literature comprehension.
6. Structure follows PeerJ standards.
7. Figures are clear and well-described.
8. All raw data has been provided.
Experimental design: 1. Original research falls within the scope of the journal.
2. Important research question addresses impact of silver nanoparticles on cPBMCs using an ex vivo system previously defined by other research groups.
3. While there have been several studies examining the effect of C-AgNP20 on human blood cells, Poirier et al. 2016 actually noted differences between AgNP20 and AgNP70 as regards their biological properties in modulating functions of human neutrophils. As it is not clear how different sized NPs may impact biological function of dolphin cells, this should be examined at some point.
4. What was the free Ag concentration of the AgNPs (see for example Kwok et al. 2012. Aquatic Toxicology 120-121:59-66)
5. There is a primary concern regarding the use of GAPDH as a housekeeping gene. The authors have referenced Chen et al (2015) that found that GAPDH was a fairly poor housekeeping gene, ranking 10th among 15 tested. It is also likely to vary with cells’ metabolic state, which is likely to be affected by mitogenic stimulation. How were your housekeeping genes selected and validated?
Validity of the findings: 1. Statistics were performed with appropriate rigor.
2. Conclusions are clear and do not, in general, overstate the findings.
Additional comments: 1. Line 44: Add the word “ratio” after “IFN-γ/IL-4” or state “Ratio of IFN-γ/IL-4”
2. Line 187: “(n=8)” seems out of place here.
3. Line 312-313: the range of doses given is from 0.1 μg/mL to 50 μg/mL, but the sentence says the lethal dose is generally higher than 10 μg/mL. This implies that some studies have shown lethality at lower doses. Then how can the conclusion be made in lines 314-315.
4. Line 320-321 state that previous studies have only presented a single timepoint. One of the papers you cited, Hofstetter et al., presented data from both 24 h and 48 h.


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: TH2 CYTOKINE BIAS INDUCED BY SILVER NANOPARTICLES IN PERIPHERAL BLOOD MONONUCLEAR CELLS OF COMMON BOTTLENOSE DOLPHINS (TURSIOPS TRUNCATUS)
Review round: 1
Reviewer: 3

Output:
Basic reporting: The paper looks clear and unambiguous, and the references are updated
Experimental design: The paper submitted can fit with the scope of the journal
Validity of the findings: This paper shows novelty that could be used to get information about silver nanoparticles presence in active stranding cetaceans. Additionally, conclusions are well stated and limited to supporting results.
Additional comments: This paper evaluates the cytokine responses of cPBMCs to citrate silver nanoparticles by quantitative RT-PCR. This is a nice study and give new elements in the knowledge of the posible impact of AgNPs  in marine mammals. However, there are some considerations before acceptance.
General Comments
Some sections of the manuscript are not well written due to unclear sentence structure or missing words. Therefore, introduction section is too long and there are some explanations that could be avoided. Important care has been taken in describing the methodologies accurately.  Conclusions and perspectives are well stated, linked to original research question and limited to supporting results. Nonetheless, there are some speculations in the discussion section that must be better explained or deleted. Figures show excellent information that is very helpful to the readers.
Some specific comments have been included below to aid the authors and explain the statements above.
In introduction section,
Line 67-71: Please shorten and link the two sentences.
Line 75: Please add “Nevertheless, to date the potencial toxicity…”
Line 87-95: Please redone to  avoid some repetitions.
Line 130-134: Please redone and move the entire paragraph to discussion section
Line 137: This paragraph must be redone  and last sentence moved to material and methods section

Materials and Methods,
Line 161-170: please shorten. Do not repeat the protocol twice
In results and discussion section,
Line 299: in relation to diminish mRNA expression levels of cytokines at different time (4 h vs 24 h culture). It would be very useful to know the precise time between the interval (4-24H) when it happens.
Line 314: Please explain better this sentence since you do not give enough information to demonstrate that cPBMC in dolphins  are weaker than human PBMCs to the toxic effect of c-AgNP.
Line 366-368: Please change “TNF-alpha, a central inducer …(Owen et al., 2013) by “ TNF-alpha is a cytokine specifically useful to measure the inflammatory state of an animal and it is primarily produced by macrophages and both Th1 and Th2 cells in response to both acute and chronic conditions (Eberle et al. 2018. Development and testing of species- specific ELISA to measure IFN-gamma and TNF-alpha in botlenose dolphins. PLOS one , volume 13, issue 1).
Line 373: after “organism and virus.” please add “Similar Th2 immune response was observed in other studies that evaluated the expression of cytokines  in different cetacean tissues (Jaber et al. 2010.  Cross-reactivity of Anti-human …Journal of Comparative Pathology, volume 143, 45-51).
Line 375: Please “Dubay et al. 2007, after Cvetnic et al 2016). In addition, you must include important mass stranding  by Morbillivirus infection (Domingo et al. 1990. Morbillivirus in dolphins. Nature, 348, pp21; Domingo et al. 1992. Pathologic and immunocytochemical studies of morbillivirus infection in striped dolphins. Veterinary Pathology, 29: 1-10).
Line 376-379: please add some references that show other locations, no just in the Pacific Ocean.


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: LESSEPSIAN MIGRATION AND PARASITISM: RICHNESS, PREVALENCE AND INTENSITY OF PARASITES IN THE INVASIVE FISH SPHYRAENA CHRYSOTAENIA COMPARED TO ITS NATIVE CONGENER SPHYRAENA SPHYRAENA IN TUNISIAN COASTAL WATERS
Review round: 1
Reviewer: 1

Output:
Basic reporting: English must be improved.
Lack of a number of literature references.
Not always self-contained with relevant results to hypotheses, often verbose with inappropriate interpretations.
For details see attached pdf.
Experimental design: no comment, apart from few notes in the attached pdf.
Validity of the findings: The results paragraph, altough shows data of a certain relevance, is verbose, ad often speculative, appearing more a discussion than the results paragraph.
For details see attached pdf.
Additional comments: This is a potentially interesting study, because few data are available on the parasites of this fish in both natural habitat and invaded range, and also because there is a general lack of information on the parasites of Lessepsian fish.
Given the relevance of the parasitological data, results and the whole discussion are quite weak, and do not exploit the potential of the data.
Few comparisons with other studies on the parasites Lessepsian fish are done, and several references on are not included.
I recommend to rewrite the whole paper under the light of these considerations, taking care to write in a correct English and in the third person.
Further comments in the attached pdf.


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

Input:
Title: LESSEPSIAN MIGRATION AND PARASITISM: RICHNESS, PREVALENCE AND INTENSITY OF PARASITES IN THE INVASIVE FISH SPHYRAENA CHRYSOTAENIA COMPARED TO ITS NATIVE CONGENER SPHYRAENA SPHYRAENA IN TUNISIAN COASTAL WATERS
Review round: 1
Reviewer: 2

Output:
Basic reporting: The English is clear and unambiguous.
Literature references require some revision; see my comments to the authors.
The article structure is good and conforms to the usual format.
The results directly address the hypotheses to be tested.
Experimental design: The research is original, the research questions are well defined and the authors state how it fills a gap in our knowledge of the topic addressed. The study was performed to a high standard and the methods are described in detail.
Validity of the findings: The findings are valid, the data are robust and statistically sound, and the conclusions clearly stated.
Additional comments: This study makes a useful contribution to our knowledge of the role of parasites in an ecosystem affected by invasions of non-native host species. It is well structured and directly addresses the questions posed in the Introduction. The only (relatively minor) criticisms I have concern the references; these are detailed below.
1. Line 146. How could the papers by Kuchta et al. (2008) and Køie (2001) possibly help with the identifications of the cestodes and nematodes found? These papers deal with particular taxonomic groups different to those found in this study. There are other more general textbooks and reviews that would have been much more useful, especially as the identifications are only to higher taxonomic groups (an order and a family).
2. Lines 251-252. The reference to Smit & Davies (2004) is missing from the reference list.
3. Lines 275-276. Add “see” before “Diamant, 1989)” and “Merella et al., 2016)” As it stands, this implies that these are authorities for the fish names and not references.
4. I could find no text reference to the following publications given in the reference list.
Bernardi et al. (2010); El-Rashidy and Boxshall (2009, 2011 and 2014); Innocenti and Galil (2007); Innocenti et al. (2009).

Otherwise, congratulations to the authors on a useful contribution and a well-written manuscript.


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

Input:
Title: LESSEPSIAN MIGRATION AND PARASITISM: RICHNESS, PREVALENCE AND INTENSITY OF PARASITES IN THE INVASIVE FISH SPHYRAENA CHRYSOTAENIA COMPARED TO ITS NATIVE CONGENER SPHYRAENA SPHYRAENA IN TUNISIAN COASTAL WATERS
Review round: 2
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: I am glad to see that the Authors have improved the quality of the article.
I attach a pdf file with the following content:
1) comments to your response
2) few (8) notes in the MS


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: LESSEPSIAN MIGRATION AND PARASITISM: RICHNESS, PREVALENCE AND INTENSITY OF PARASITES IN THE INVASIVE FISH SPHYRAENA CHRYSOTAENIA COMPARED TO ITS NATIVE CONGENER SPHYRAENA SPHYRAENA IN TUNISIAN COASTAL WATERS
Review round: 2
Reviewer: 2

Output:
Basic reporting: .
Experimental design: .
Validity of the findings: .
Additional comments: I believe that this revised manuscript is now acceptable for publication in PeerJ without the need for a re-review.


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

Input:
Title: URBAN WASTE DISPOSAL EXPLAINS THE DISTRIBUTION OF BLACK VULTURES (CORAGYPS ATRATUS) IN AN AMAZONIAN METROPOLIS: MANAGEMENT IMPLICATIONS FOR BIRDSTRIKES AND URBAN PLANNING
Review round: 1
Reviewer: 1

Output:
Basic reporting: Please see 'general comments' below.
Experimental design: Please see 'general comments' below.
Validity of the findings: Please see 'general comments' below.
Additional comments: This is a worthy topic for research.  The writing is reasonably good, although a few small corrections are noted.  Likewise, most of the relevant references are included, but a few are missing and suggested.  My main criticism is that more detail is needed in parts of the Methods, because I don’t think another researcher could reproduce your methods exactly with only the information presented here.  Please see below for specific concerns.

•	Abstract and elsewhere:  Please change ‘aircrafts’ to ‘aircraft’.
•	Line 57:  The term ‘spatialized’ very odd; in fact I have not hear that word before.  Please change to a more standard term.
•	Lines 80-82:  There are standard references you may want to cite regarding factors relating to hazard level: Dolbeer et al. (2000) Wildl Soc Bull 28:372-378; DeVault et al. (2011) Wildl Soc Bull 35:394-402.
•	Line 97:  The choice of references here is strange.  Instead of Dolbeer & Wright (2008), you should cite the peer reviewed article Dolbeer (2006) J Wildl Manage 70:1345-1350.  Also, the Dolbeer et al. (2011) strike report is quite old—these reports are produced annually and the most recent report should be cited instead.
•	Line 135:  Please clarify whether these 20 markets chosen were the only ones registered by the Manaus urban administration, or were they a sample of all those registered?  If the latter, how did you choose the 20 markets to study?
•	Methods—Bird Counts:  This section needs much more detail.  For example, how were the markets delineated?  Did you record all vultures seen, or just those over or in the market?  How did you avoid double counting?  Were all areas where vultures could be visible from the car?
•	Methods—Waste Storage:  I would also like to see more detail here.  It appears you recorded the proportion of waste type, but not the total amount of waste.  So was a situation with 6 kg red meat out of 10 kg total waste categorized the same as a situation with 60 kg red meat out of 100 total kg waste?  If so, that seems to be a poor way to score the amount of waste available.
•	Methods—Historical Birdstrike Records and Sighting Reports:  I understand how you gathered this information, but in the Methods you do not say why you gathered this information.  What did you do with all the data you gathered?
•	Lines 196-197:  Please rephrase this sentence.  I think you meant that all models with AIC < 2 were considered equally valid, but that is not what this sentence says.
•	Results—I found myself looking for a table of the results (models), but this was not present.  Please consider including this.
•	Lines 271-272:  Here you mention the ‘amount of fresh or decomposing animal waste available’, but I don’t think that is what you measured.  See my comment above on proportion vs. amount.
•	Line 333:  Change ‘amble’ to ‘ample’.
•	Conclusion—last two sentences are speculation and not warranted based on the findings from this paper.
•	Figure 1:  Where are the red squares?  I couldn’t find any.


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: URBAN WASTE DISPOSAL EXPLAINS THE DISTRIBUTION OF BLACK VULTURES (CORAGYPS ATRATUS) IN AN AMAZONIAN METROPOLIS: MANAGEMENT IMPLICATIONS FOR BIRDSTRIKES AND URBAN PLANNING
Review round: 1
Reviewer: 2

Output:
Basic reporting: Although English is not my first language, I believe the English used is quite adequate. The reading is clear, objective, succinct, fluid and technical.
References are adequate and mostly current. About 65% of references have less than 10 years, most of them less than 5 years. The oldest ones are either classic or indispensable references. Pay attention to the last listed reference (lines 521-522) - that is improperly positioned.
The manuscript is well structured. All divisions and subdivisions are adequate and the latter facilitate understanding. Figures and tables are appropriate and pertinent.
The results were well analyzed and support the conclusions presented.
The theme and objectives of the manuscript are consistent with the scope of the journal.
Experimental design: The purpose of the manuscript has scientific importance, both in terms of aircraft accidents involving birds such as public health.
Although it has been conducted in a large city in the Amazon region, it provides concrete, measurable and replicable data that can be extended to other urban centers, since the mentioned problems are recurrent in many urban centers around the world.
The methodology is well delineated and replicable.
.
Validity of the findings: The discussion is based on robust data, properly analyzed.
Additional comments: Congratulations. It was the best manuscript I have reviewed lately. I saw no problem whatsoever, which means that there was a great effort by you in preparing this manuscript.


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

Input:
Title: URBAN WASTE DISPOSAL EXPLAINS THE DISTRIBUTION OF BLACK VULTURES (CORAGYPS ATRATUS) IN AN AMAZONIAN METROPOLIS: MANAGEMENT IMPLICATIONS FOR BIRDSTRIKES AND URBAN PLANNING
Review round: 1
Reviewer: 3

Output:
Basic reporting: Basic Reporting
In general the manuscript is well written, but there are a few specific areas where this needs to be improved see below:
Line 57: Change ‘spatialized’ to ‘visual examined’.
Line 92: Remove ‘resting’.
Line 109: Change ‘in’ from before large Amazonian cities to ‘at’.
Line 112: Change ‘spatialized’ to ‘visual examined’.
Line 144: Change ‘modal’ to ‘main’.
Line 232: Remove ‘Delta’ and add the symbol for delta Δ.

Most of the citations are adequate, but here are a few suggestions:
Line 49: Vulture management is still an issue in cities that have stopped growing with adequate urban planning, so update the wording of this sentence.
Line 70: Dolbeer 2011 is specific to off-airport strikes, which often involve vultures. This should be updated to include information that bird strikes off airport property are increasing.
Line 72: Number of air carrier movements is not necessarily increasing in the states, so remove the Dolbeer 2005 reference.
Line 82: You should cite DeVault et al 2011 Interspecific Variation in Wildlife Hazards to Aircraft: Implications for Airport Wildlife Management to support this statement.
Line 93: The Novaes & Cintra 2013 reference deals with roosting sites not nesting, perhaps update this in the sentence.
Line 129: Add information on how much the city has grown over this time period.
Line 154: Can you add photographs of the storage containers?
Line 296: Are there any references about cultural notions of waste management in Brazil?
Figure 1 is not labeled properly. Please place the legend on the map itself (explaining red and blue squares). Why are there no red squares? Black arrows are not presence on the map, but white arrows are. Add an overview map of the cities location in Brazil. In the legend, add definitions to red circle symbols (sizes that correspond with how many vultures).
Experimental design: There are some fundamental flaws with the methodology.
First, how were the markets chosen? This is not stated. Were they randomly selected?
Next, I am concerned that vultures that were flying over the site within a 15 min survey period were counted. How far above the market were they? Were they low enough to be considered using the market, or were they just flying over. Since this is your response variable, I think it is important to make sure the reader is not confused. You could also subset observations to those vultures that were feeding or on the ground. For your surveys, how far was your radius of detection?
The composite index (DI) is not a useful addition to this manuscript. We do not know if markets located closer to airports are more hazardous than airports located further away. As vulture collisions occur at higher altitudes, perhaps markets that are further away from airports are considered more of a risk. I think this whole section should be removed.
The number of collisions reported in Table 2 are not specific to vultures, or other species that use markets, so the usefulness in the paper is limited.
Line 245: Can you add information about the height of the strikes? This would justify looking at vultures.
Line 258: What was the standard deviation of number of vultures observed?
Validity of the findings: There is some confusion on how data was collected, this should be fixed.
The conclusion section is a little misleading in that it does not discuss much about vulture abundance, and focuses on vultures as umbrella species. However, no other species were recorded in this study, so this statement is not appropriate.
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: URBAN WASTE DISPOSAL EXPLAINS THE DISTRIBUTION OF BLACK VULTURES (CORAGYPS ATRATUS) IN AN AMAZONIAN METROPOLIS: MANAGEMENT IMPLICATIONS FOR BIRDSTRIKES AND URBAN PLANNING
Review round: 2
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: Nice job with the revision.


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: ECONOMIC VALUE OF TREES IN THE ESTATE OF THE HAREWOOD HOUSE STATELY HOME IN THE UNITED KINGDOM
Review round: 1
Reviewer: 1

Output:
Basic reporting: English used throughout the manuscript is clear, unambiguous and professional allowing easy and smooth reading. Introduction shows context and understanding of the topic and leads logically to the aims of this manuscript.
Literature used is relevant and covers the topic broadly. Precision of referencing is, however, poor. Already in the first section of the introduction, there are three mistakes: L 50 Hunhammer should be Hunhammar, L 54 Nowak is listed as Norwak and Luttik 2010 is listed as Luttik 2000 in the reference list. There are further referencing mistakes throughout the manuscript. The authors should thoroughly check the references.
Figures attached to the current file are of poor quality. Species names on Figure 1 are almost impossible to read. Caption of Figure 3 does not match to what is actually presented. Furthermore, there is a general problem with differences between the data and results presented in the text and on the figures/tables. For example, Table 3: Whittaker’s Beta W reported 0.33 in Table 1 and 0.4 on line 130; about 35% of relative abundance of Quercus robur on Figure 1, more than 50% on line 133 etc.
The general structure of the manuscript follows the PeerJ standards, with raw data supplied in a readable and understandable format.
Experimental design: The aims of the study are well defined, relevant and meaningful. It is stated how the current manuscript will fills an identified knowledge gap. Although the manuscript is focused on one single estate in United Kingdom, the results are discussed in much broader context, making it interesting for wide variety of readers.
Methods used in this paper could be described with bit more detail. For example, the measured light exposure of the trees is only explained in the result section on line 150. Although I understand that the iTrees software methods have been described elsewhere, it would be helpful to have some basic principles explained in the current manuscript as well. That would save the reader from having to scan through long reports or download user-manuals to get an idea of the methods used in the software.
Validity of the findings: Data collected for this manuscript is robust and statistically sound. However, discrepancies between the figures/tables and text raise some concerns. Results in the text do seem to match the raw data supplied.
The conclusions are linked to the original aims, limited to the results, yet discussed in a broader context
Additional comments: L 145 - 154: Something has happened there, as the same results are presented three times. Please edit this section.
Also section Ecosystem services (L 162 - 171) is bit difficult to read.


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: ECONOMIC VALUE OF TREES IN THE ESTATE OF THE HAREWOOD HOUSE STATELY HOME IN THE UNITED KINGDOM
Review round: 1
Reviewer: 2

Output:
Basic reporting: The paper is written by professional English.
The paper as well as literature references deal mainly with Anglo-Saxon context. I suggest use more papers mainly from Germany (e.g. research of I. Kowarik and his team).
The paper has professional and clear structure.
Figures and tables are relevant.
Fig.1: The hight of columns does not correspond with information in text (lines 132 - 140).
Fig. 3: I suggest omit the column with London data that are not gained by authors´ own research. The caption of axis x should show the DBH interval in each columns.
Experimental design: The topic of the paper fits to scope of the journal.
The presented paper is a case study based on detailed enhancement of gardens in one estate. It is relevant for introduction of methodology how economic values of ecosystem services provided by gardens could be measured. Deep research of economic values of gardens should be based on more extent set of model areas.
Validity of the findings: Discussion and Conclusion are well stated and linked with research questions. However, the estimation of the value provided by trees to the estate (lines 199 - 202) should be more specified. It could be supposed that not only number of trees but also their composition (species richness, health conditions) will differ (be higher) in gardens than in common agrucultural and/or periurban landscape. How the presented estimation was made?
Additional comments: The paper is based on one case study, nevetheless, it brings important findings and is worth to be published.


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: MAXENT ESTIMATION OF AQUATIC ESCHERICHIA COLI STREAM IMPAIRMENT
Review round: 1
Reviewer: 1

Output:
Basic reporting: The paper is interesting but it suffers from a lack of clarity. Even in the abstract I find it hard to identify the main purpose and finding of the research. I recommend using Active voice rather than Passive.

For example in the abstract the authors write: Univariate models showed Water temperature as the best performing individual predictor, but models showed lower accuracy than conductivity, dissolved oxygen, or alkalinity. Accuracy was found to
be highest for alkalinity, and was found to be lowest for pH. - Does this mean accuracy of the models was highest for alkalinity? Is accuracy  important? If not, don't mention it. If it is then don't mention the other variables because you're loosing your message. I encourage you to use clear statements. What are environmental controls? Are these modifiable factors implicated in stream impairment? Is temperature an environmental control? can you control that?
Experimental design: The methods and results are sufficient but again they suffer from a lack of clarity and an abundance of jargon. MaxEnt has been described in detail elsewhere. It wasn't even entirely clear why you were using MaxEnt if you have data from all these sites, are trying to predict outside of these sites or find a relationship between the variables and the outcome?

A clear statement such as (not sure if this even the case) We used MaxEnt to identify the probability of stream impairment given a set of environmental variables would go along way to improving readability.
Validity of the findings: It is difficult to interpret figure 4, what are the black and white circles and why are some of the grids black?

You have a testing AUC around .70 which is pretty for machine learning algorithms using environmental data low but don't discuss the implications of this in the context of policy.
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: MAXENT ESTIMATION OF AQUATIC ESCHERICHIA COLI STREAM IMPAIRMENT
Review round: 1
Reviewer: 2

Output:
Basic reporting: This is a useful paper that demonstrates the opportunities in site-specific data analysis of E. coli concentrations in streams. Should such analysis be repeated for other sites, substantial progress could be achieved in understanding and mitigating surface water impairment. However, the manuscript has weaknesses in that the presentation is very general, and therefore targets a limited audience with the previous background in machine learning techniques. Moreover, the manuscript is also lacking any insightful discussion. For example, why different models (e.g., four variables vs. eight variables) do differently? Is that because of different capabilities of models or some physical understanding? Lastly, it is not clear how these models do in extreme situations. This might be useful for regulatory bodies and recreational activities. I also suggest bringing forth novelty in this manuscript as most of the points shown in the manuscript are well established (see some articles given below).  These limitations preclude publication of this manuscript.
Experimental design: Experimental design is appropriate, but some more details are required as indicated in the attached PDF file.
Validity of the findings: Findings are valid, but most of the findings are well known. So authors need to bring forth the novelty of their work.
Additional comments: Additional comments are as follows:
1.	Your introduction needs more detail. I suggest that you improve the description at lines 85- 98 to provide more justification for your study. Specifically, authors should include previous studies (Vidon et al. 2008, McKergow and Davies-Colley 2009, Money et al. 2009; Dwivedi et al. (2013) - just to name a few recent papers). There is a rich body of literature that this manuscript does not acknowledge or incorporate into the discussion, which causes the reader to question the diligence of the authors.

•	McKergow, L. A., and R. J. Davies-Colley. 2009. Stormflow dynamics and loads of Escherichia coli in a large mixed land use catchment. Hydrological Processes:doi: 10.1002/hyp.7480.

•	Money, E. S., G. P. Carter, and M. L. Serre. 2009. Modern Space/Time Geostatistics Using River Distances: Data Integration of Turbidity and E. coli Measurements to Assess Fecal Contamination Along the Raritan River in New Jersey. Environmental Science & Technology 43:3736-3742. doi:10.1021/es803236j.

•	D Dwivedi, BP Mohanty, BJ Lesikar, Estimating Escherichia coli loads in streams based on various physical, chemical, and biological factors - Water resources research, 2013

•	D Dwivedi, BP Mohanty, BJ Lesikar, Impact of the linked surface water-soil water-groundwater system on transport of E. coli in the subsurface - Water, Air, & Soil Pollution, 2016

2.	L100 – provide reference of the Maxent model.
3.	L 100-106 – it does not sound like a new approach, but Dwivedi et al. (2013) used water quality data to model E. coli in streams using a machine learning approach. Here, authors are just using a different machine learning technique.  Authors need to strengthen the rationale why to use a different model here.
4.	L115 – the goal of the present work should not to show whether a model works or not. The goal should be to fill knowledge gaps and connect to a bigger picture.
5.	L125-171 Sampling sites and data collection – provide more details such as the number of samples, the frequency for each water quality parameter. Maybe, a table describing these details would be a better choice.
6.	L177—why do you maximize the entropy of the raw distribution of impairments to get the optimum weights? Provide justification for that in a natural system like yours.
7.	L211 – Authors need to provide details about which samples were deleted. If deleted samples were associated with extreme values, then how good your bootstrap samples are? This would lower the usefulness of the model. Maybe, authors could include synthetic data to analyze extreme cases to show the validity of their model across the spectrum.


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: MAXENT ESTIMATION OF AQUATIC ESCHERICHIA COLI STREAM IMPAIRMENT
Review round: 2
Reviewer: 1

Output:
Basic reporting: Authors have done an excellent revision.
Experimental design: Same
Validity of the findings: -- same --
Additional comments: Authors have done an excellent job of revising the paper.  I agree that parsimonious models are preferable. However, I am not convinced that it is not possible to provide a discussion on model performance differences based on the selection of variables. As such the artificial intelligence (AI) based approaches are nothing but black boxes, so efforts should be made to enhance process understanding.


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: ASSESSMENT OF THE BACTERIAL COMMUNITY STRUCTURE IN SHALLOW AND DEEP SEDIMENTS OF THE PERDIDO FOLD BELT REGION IN THE GULF OF MEXICO
Review round: 1
Reviewer: 1

Output:
Basic reporting: The manuscript presented by Sánchez-Soto and collaborators is a 16S rRNA massive sequencing survey for a depth and longitudinal transect (20-3700 m) in waters in the northwestern Gulf of Mexico within the Mexican region.  The supplied background is enough although it would be enriched if detailing findings from other similar works like the related from the deep-horizon spill.

The manuscript presentation is overall good. However, it needs some extra work for some vague phrases all around the text that could be fulfilled with references and declaring missing values.

Authors make big assumptions about the predicted metabolic profiles and they are not taking into account the pitfalls of their chosen method (PICRUST), they are placing this as the main result but is a weak one, with any experimental support validating their assumption. I suggest to tone down the conclusions related to the metabolic predictions, which are just an educated guess.
Experimental design: The authors are short with methodological descriptions, which make the work impossible to be replicated by independent groups.

Experimentally, I have important concerns about your sampling methods where you state that you freeze at -20ºC the samples (L78). How much time left between the sampling, freezing, and DNA extraction? This storage temperature could modify your community profiles as microbial metabolism could keep on going at this temperature (Price & Sowers 2004; Panikov et al., 2006).

Is there any chance that you have water/sediment temperature to be added to your Table 1?

DNA extraction: How much sediment are you using for each extraction?

Did you perform MiSeq sequencing on your own or are you paying for the service? Please declare who did the sequencing.

For the data analysis methodology, there are several missing descriptions, and I recommend to the authors to prepare supplementary information material regarding your bioinformatic and statistical procedures. You should consider using jupyter notebooks, or any markdown log available and upload your parameters and scripts to GitHub, figshare or any other public repository.

Price, P. B., & Sowers, T. (2004). Temperature dependence of metabolic rates for microbial growth, maintenance, and survival. Proceedings of the National Academy of Sciences of the United States of America, 101(13), 4631–6. http://doi.org/10.1073/pnas.0400522101
Panikov, N. S., Flanagan, P. W., Oechel, W. C., Mastepanov, M. A., & Christensen, T. R. (2006). Microbial activity in soils frozen to below −39 °C. Soil Biology and Biochemistry, 38(4), 785–794. http://doi.org/10.1016/J.SOILBIO.2005.07.004
Validity of the findings: Findings seem legit. Metabolic predictions should be toned down and identify it as speculative in the discussion, not as hard result.

Could not assess the validity of the findings because of the lack of the raw data, specifically OTU tables with their assigned taxonomy, metadata files, and the metabolic predictions. This should be added as supplemental information for the manuscript and publicly available. This could be overridden with a detailed methodologic description as suggested before.
Additional comments: L29 What is statistically more abundant?
L41 Wrong citation? What in the world is chemical homeostasis? Leloup et al. 2009 do not even say homeostasis in his manuscript!
L45 microbial (?) community
L59 Missing reference (oil spill 2010).
L70-71 You are just predicting, guessing to close related sequenced strains, what do you know about pan-genomic variability?
L102 16F and 16R? please write 5'-ATCG-3' pair of primers used (complete).
L114-115 Where did you sequence your samples?
L121 What are q, r and p parameters?
L126 What parameters did you use to compare against Greengenes DB?
L132 Normalized? Do you mean rarified? Subsampled?
L145-154 NOT reproducible without further details about the parameters used. How many enzymes are involved in hydrocarbon and xenobiotic degradation within PICRUST/KEGG? Did you just perform this analysis?
L169 What is clearly distinguished?
L173 NMDS  what is the stress value? Is it a robust ordination?
L261 incomplete comparison, sentence (differentially abundant)
L263-L265 Incomplete idea, check sentence structure. Missing references?
L268-272 I don't get your idea is quite confusing
L309 References missing
L309-312 I don't understand your sentence. Rhizobiales (in your samples?) what is a complex diversity?
L319-323 Did you found any correlation between your physicochemical variables with shallow samples? Have you tired Mantel tests?
BASELINE. You are giving one snapshot, I think it is pretty presumptuous to establish a baseline with 11 samples in just one season. Tone down baseline term use of the manuscript.


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

Input:
Title: ASSESSMENT OF THE BACTERIAL COMMUNITY STRUCTURE IN SHALLOW AND DEEP SEDIMENTS OF THE PERDIDO FOLD BELT REGION IN THE GULF OF MEXICO
Review round: 1
Reviewer: 2

Output:
Basic reporting: 1. the English is not bad (certainly better than my Spanish), but could use help from an editor at PeerJ or a professional English language editor.  there are places thorughout with usage patterns that are just a little different than what i would expect in a scientific publication, and this is beyond the duties a reviewer should be addressing

2. there are issues throughout with references cited that do not pertain to the preceding statement.  i list specific examples in the General Comments below, but this was very frustrating, especially since i know many of the papers cited and was confused to see them cited where they were.  this will need to be fixed, all references should be checked and verified by the authors to be necessary and accurate.

3. some of the figures can be improved, comments are below.  this is not a fatal flaw, but i feel that improvement will really enhance the manuscript.
Experimental design: generally acceptable, however please add references for the TOC and TOM methods described, there is a brief summary of the method, but no citation for what is a standard method that should be cited.
Validity of the findings: this is the strongest area of the manuscript - this is a baseline study for an underexplored region of the Gulf of Mexico, and i believe that has merit.  the sampling strategy is well designed, and the authors treat the data well.
Additional comments: Summary:
the authors here present microbial diversity and correlations to some environmental variable for surface sediments collected along a transect from shelf to deep waters int he Gulf of Mexico (for which the acronym used in the manuscript should be GoM or GOM, not GM).  they analyzed 11 samples for basic chemistry and 16S rRNA diversity.  this is a small dataset, but baseline datasets are valuable and data from this region of the Gulf is rare.  i appreciate that the authors generally do not try to overstate what data they have or what it can say, but i think there could be more work put into the deep versus shallow comparison, and especially comparison with any previous data.  in addition, i encourage them to look deeper into PICRUSt to determine if it can really be used with this dataset.  at this stage, i believe the manuscript requires major revision before it can be considered for publication, but i also believe that the authors have a publishable dataset that can be appropriate for publication with the appropriate revisions.

Specific comments:

Abstract:
please remove the sentence: "These microbial communities were composed of more than 20 phyla, being Proteobacteria the dominating phylotypes in all the analyzed samples."  this is overly broad and unsurprising enough to not warrant mention in the abstract.  in general, the language in the abstract is poor and needs to be revised.  the remainder of the manuscript is better, but also needs review from an English language expert/native speaker.

Introduction:
Line 45: the Santelli reference is not appropriate here, this is a study of microbiology on seafloor exposed basalts, it doesn't get much into environmental factors regulating community structure

Line 49: here and forward (i will not continue to edit), you must use "Alpha-".  Also, it should be Gammaproteobacteria (or whichever class you are identifying), not Gamma-proteobacteria

Line 56: there are NO hydrothermal vents in the Gulf of Mexico - the vents discussed in this reference are from the Galapagos spreading center.  remove this citation.

Line 58: there is SO MUCH work related to the DwH spill, you should cite some of these papers.  anything from Mandy Joye and Joel Kostka's groups will suffice, i know you cite a review from them later but they have much more and some should be included here.

Line 71:
change "explored" to "predicted based on trait modeling software"

Methods:
Line 82: must add citations for methods used in TOM and TOC analysis

Results:
Line 182: first of all, you are referencing phyla for a figure that shows class, although the key gives phyla and class, so maybe this is ok. second, there are too many classes displayed and several have the same colors - change this figure to display only classes >1% relative abundance or some other cutoff that allows you to discuss the groups you want but reduces the number in the plot.  you can (and should) include a supplementary table of the OTU distribution per sample with OTU taxonomy indicated and readers that want finer details can go there to learn about their favorite minor community members, some of which is already in your first supplementary figure

Line 204: which hydrocarbonoclastic communities?  this comes out of nowhere, with no details on how you determined which communities are hydrocarbonoclastic, and which members of the communities putatively are associated with this metabolism.  you need a few sentences or a whole paragraph explaining why you care about hydrocarbonoclastic microbes in this region, and then identifying which of your iTags may be indicative of that metabolism.  then you can back it up with the PICRUSt analysis (assuming low NSTI values)

Line 204: PICRUSt works best for human microbiome studies  - report your weighted NSTI values, and if they are close to or above 0.20, then these calculations are not meaningful.  also, why did you pick those putative metabolisms?  was this a fishing trip or do you have hypotheses you are trying to test?

Discussion:
Line 244: diversity indices are HIGHLY influenced by sampling depth - you can only make this comparison if you have reanalyzed the data from the other papers, and i do not believe you did that here

Line 246: th Kimes citation is incorrectly formatted in the reference list

Line 261 - this sentence should read "The detection of high abundance of lineages known to be anaerobic..."

Line 277 - change to "The phylotypes detected chich have been previously described as aerobic include Flavobacteriaceae..."

Line 290: no idea why you are citing Redmond and Valentine here, this is not appropriate for what you are looking for (your deep sites much deeper than the DwH hydrocarbon plume they were studying)

Line 292: the study of hydrocarbonoclastic microorganisms has gained in relevance, the organisms themselves are not aware of their own relevance.  this is nitpicky, but will improve the language

Line 298: the Math 2012 and Fuhrman 2015 references are incorrect for this statement and need to be removed here.  throughout the paper, your references are frustratingly inaccurate, a reference must refer to the statement it is cited after, Fuhrman 2015 is a review paper about microbial ecology and network methods, which has nothing to do with GoM hydrocarbons.  and the Math 2012 paper describes a bug from KOREA, yet you reference it to illustrate GoM work.  frustrating.  at least the Math 2012 paper belongs cited the next time it appears

Line 309: about aromatic compound degradation - need a citation for this.  again, you are not pushing your PiCRUST interpretation too far, but be sure the NSTI scores are appropriate enough to use with your samples.  i have had to abandon a PiCRUST analysis in one of my own papers because the scores were too high and therefore the bugs in the PiCRUST database do not well represent those in my samples (possibly true for you as well)

Figures 3 and 4 - the color schemes are very difficult to interpret, i think these figures should be redesigned to display ONLY what you want the reader to know.  with so many colors, and the keys so (so) small, i cannot figure out which color bars refer to which groups

Figure 4, only - i'm not sure panel A is informative since there's no indication of the OTU taxonomy beyond class.  maybe you can remove this one, or move to the supplement.  i actually think this aspect (differential abundance for deep vs shallow samples) is probably most interesting and would like to see it discussed a bit more in the text.

while the transition in microbial diversity with depth from shelf to deep sea may not be well documented in this area, i bet there have been other studies looking at that question from other regions.  i can't name them off the top of my head, but encourage the authors to seek them out and cite them in their intro, and also make comparisons of their data to any previous data in the discussion.  additionally, there is a well established and reviewed literature on this transition in macrofauna - see chapter 13 in Miller and Wheeler 2012, Biological Oceanography (textbook), for a discussion and many references.  please cite some of these classic papers, it gives context to your own work.