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: CBL UPREGULATES CYSH FOR HYDROGEN SULFIDE PRODUCTION IN AEROMONAS VERONII
Review round: 1
Reviewer: 1

Output:
Basic reporting: Although the manuscript is generally written in an understandable way I strongly recommend re-reading of the entire text (e.g. by someone other than the Authors, preferably a native English speaker) to help point out and improve the weaker sentences (and some grammar mistakes) that are used throughout the manuscript, and which make understanding of some ideas unnecessarily difficult. For example, some of the sentences are constructed in a too simplified way, to the point that they are lacking the crucial words: (line 68) “Hydrogen Sulfide (H2S) is an unpleasant smell with toxicity (Lindenmann et al., 2010).” I would expect the Authors to describe H2S as e.g. “gas”, “compound” etc.. Other examples: “the total amount of 1 OD600 cells were harvested”, “H2S is able to resist oxidative stress”, “many antibiotics also trigger the production of ROS when they targeting inhibit their targets”, etc. Such sentences should therefore be expanded to clarify the meaning, increase the accuracy, and make the text easier to read (fluency).

Moreover, in the example with H2S, in the aqueous environment (depending on the pH) HS- species may also be found [e.g., Hydrogen sulfide (H2S) is readily water soluble, and, at physiological pH, about two-thirds exists as hydrogen sulfide ion (HS—) and one-third as undissociated H2S; Reiffenstein et al. (1992).]. I would expect the Authors to provide more detail on the chemistry of H2S/HS- in the cells/culture fluids/culture gases, as this is vital to the understanding of the methodology used in the study. The potential presence of H2S/HS- and analysis of their concentration both as gas and in the aqueous phase should also be reflected in the Materials&Methods section.
The Authors provide some data on cys operon regulation based on the Salmonella typhimurium example (lines 73-74). Is there any data on Aeromonas species regarding this matter? If not, I would encourage Authors to indicate that, as this is likely to increase the novelty of the study.

Dividing the information provided in the Introduction section into (more) paragraphs is likely to be beneficial. For example, it is not clear whether the information about the function of Clb relates only to Salmonella typhimurium or it is a more general fact. If it is the latter case, the information about Clb should be provided in a new paragraph.

Figure 2 B  - negative values on y axis (concentration) should not be present

Figure 4 A – it is difficult to observe the differences in microbial growth between the plates (low resolution/contrast)
Experimental design: Generally, in the Materials and Methods section, please add information about the number of replicates and the controls used in each of the experiments.

“LB/M9 media” - although these are typical growth media, I suggest providing more detail on their exact composition, as this often differs between manufacturers.

As mentioned above - regarding H2S detection - there is always the problem of HS- remaining in the medium. This very much depends on the pH and as such, the pH should be monitored at all times (was it?).

Were the Pb(Ac)2 paper strips submerged in the culture for 48 h, or were they wetted with the culture fluids after 48 h? Paper strip-tests tend to be more qualitative or semi-quantitative than other analytical tests. Please provide more detail about the strips used, including clear information about interpretation of the results (especially about quantitative comparison of the H2S/HS- yields based on color changes).

More details about the Cbl protein complementation procedure should also be provided in the Materials&Methods section.

Tukey’s test (named in the Figures) should be mentioned in the statistical analysis section.
Validity of the findings: “the absence of SmpB enhanced the synthesis of H2S”
Have the Authors performed any experiments to further explain this phenomenon?
Potentially, SmpB is a repressor of the cys operon, which helps to limit the production of potentially toxic H2S. H2S may be a byproduct produced at stationary phase, due to the lack of down-regulation provided by SmpB. Although this is quite crucial, this matter is only vaguely explained.

Is cysB transcription also up-regulated as a result of smpB deletion? There is no information about the transcription level of this crucial gene in the text or Figures.

The conclusions should be more focused on the outcomes of the study, and the parts about pathogenic strains and antibiotic resistance, which are not directly the object of the study should be a part of the discussion.
Additional comments: The title is a little misleading, as the Cbl affects H2S (as presented in Figure 3) regardless of SmpB presence/deletion. Of course the deletion of SmpB is a special case in which the role of Cbl may be more prominent.


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: CBL UPREGULATES CYSH FOR HYDROGEN SULFIDE PRODUCTION IN AEROMONAS VERONII
Review round: 1
Reviewer: 2

Output:
Basic reporting: Abstract: Line 39- re-write second messager to second messenger.

Introduction: Hydrogen Sulfide (H2S) is an unpleasant smell with toxicity (Lindenmann et al., 2010). Rewrite to Hydrogen Sulfide (H2S) is a gaseous molecule with unpleasant smell with toxicity (Lindenmann et al., 2010).

The Introduction part should be improvised with adequate facts on how the potential findings of the study support as dug targets for  aquatic diseases, since the conclusion delivers such a statement.

Lines 66-67- In all, SmpB is essential for………adaptability to stress. To which organism you mention this to? Please re-write the sentence.
Discussion: Lines 234, 237- Staring a sentence with Really or Logical is not apt for a manuscript.
The manuscript needs language editing and require technical typo corrections like space (like line 82 bothcbl instad of both cbl; line 132, 7.Electrophoretic mobility shift assay-space)
Experimental design: Materials and method:
Line 128, under subheading 6- Please describe the procedure (Bykowski et al., 2002) briefly.

Experimental design is satisfactory with required methods.
Validity of the findings: Satisfactory
Additional comments: The manuscript need minor revisions stated 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: CBL UPREGULATES CYSH FOR HYDROGEN SULFIDE PRODUCTION IN AEROMONAS VERONII
Review round: 1
Reviewer: 3

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: The role of H2S in stress tolerance might be explained. Genes required for H2S synthesis might be explained in detail.  The statistical significance of P<0.01 found to be less significance. If we get P<0.001 or P<0.005 more satisfactory.
Additional comments: More explanation regarding stress tolerance might be encouraged.


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: CBL UPREGULATES CYSH FOR HYDROGEN SULFIDE PRODUCTION IN AEROMONAS VERONII
Review round: 2
Reviewer: 1

Output:
Basic reporting: The manuscript has been thoroughly improved. However re-reading of the improved manuscript by a native English speaker is still recommended.

Some of the remaining minor mistakes are listed below:
line 69/70 "that contribute to reduce oxidative state" (meaning unclear, English revision needed)
line 70 "hydrogen Sulfide" (S->s)
line 71 "H2S is a gaseous molecule with unpleasant smell with toxicity" (improve the "with toxicity" part; e.g. which at high concentrations is toxic to most living organisms/live cells etc.)
line 116 "as the water solubility of H2S. (?)" (unfinished sentence?)
line 122 "there is a little evidence" or rather "there is little evidence" (confirm, as these phrases have a slight difference in meaning)
line 122 "evidence regarding Cbl regulates to cysJIH in Aeromonas species" (English revision needed) --> e.g. /regarding the role of Cbl in regulation .../
line 138 "when facing to the adverse stress" (English revision needed)
line 193 "The production of H2S were increased " -> was
line 273 "Previous smpB deletion was reported to survives better than WT " (meaning unclear, English revision needed)
line 227 "explained the the "
line 276 "to compensate for tolerance defects caused by SmpB deficiency. " (unfinished sentence)
Experimental design: The M&M section has been considerably improved.
Some minor issues are listed below:
line 148 "contained 20% 5M9 salts, " (unclear notation, it would be better to list the salts or provide a suitable reference)
line 150 "was amplified by PCR and inserted" (too vague, please add the conditions for the reactions)
line 154 "The supernatant was collected by centrifuge " (lab jargon)
Validity of the findings: It is rather uncommon to provide literature references in the conclusions section (Line 274).
Additional comments: The improved title corresponds better to the findings presented in 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: APIS ANDRENIFORMIS ASSOCIATED ACTINOMYCETES SHOW ANTIMICROBIAL ACTIVITY AGAINST BLACK ROT PATHOGEN (XANTHOMONAS CAMPESTRIS PV. CAMPESTRIS)
Review round: 1
Reviewer: 1

Output:
Basic reporting: The authors have isolated and identified three Streptomyces strains associated with the black dwarf honey bee (Apis adreniformis) as well as several strains from underrepresented actinobacterial genera. The three Streptomyces strains were found to inhibit the growth of the Gram-negative plant pathogen Xanthomonas campestris pv. campestris in vitro. The authors also demonstrated that organic extracts prepared from the inhibitory Streptomyces strains were able to inhibit Xanthomonas campestris pv. campestris. The authors argue that the presence of inhibitory Streptomyces strains and rare actinobacterial genera isolated from this rare honey bee species may make it a valuable source for future natural product discovery efforts.

The English is mostly clear but should be improved in several sections throughout the paper prior to publication:
•	Line 24 and 25 – to be more consistent, the word “with” should be removed and 84.4% should be put in parentheses.
•	Line 31 – the word “a” should be added in “effect with a lower MIC…”
•	Line 48 – The sentence “It is the 5th honey bee…” should simply say that it is “one of the known seven honey bee species”, unless there is some numbering scheme to rank them.
•	Line 53 – The word “in” should be added in “distributed in nature both in terrestrial…”
•	Line 55 – The word “bees” is repeated between leafcutter bees and stingless bees
•	Line 58 – The word “belonged” should be changed to “belonging”
•	Line 69 – The word “been” should be removed or should be changed to “has been declining”
•	Line 70 – “search and discovery program” should be made plural “programs”
•	Line 70 – The word “is” should be changed to “has”
•	Line 78 – The word “problem” should be made plural; however, it would be more helpful to describe the actual problems to human health and the environment, such as antibiotic resistance and toxicity.
•	Line 80 – “chemical products” should be changed to a more accurate word such as “antibiotics”
•	Line 85 and 86 – I believe the sentence starting “Actinomycetes associated with…” is missing the words “with activity” or something similar before the word “against”
•	Line 94 – The article “The” in “The permission was received…” should be removed
•	Line 119 – The first “of” in the title should be changed to “for”, “Screening for antagonistic activity…”
•	Line 132 – The word “store” should be in the past tense as “stored”
•	Line 148 – “actinomycete” should be made plural
•	Line 157 – The year in “Yoon et al.” should be “2017” not “20017”
•	Line 171 – The word “actinomycete” should be plural
•	Line 180 – The article “a” needs to be added in “growth of a phytobacterial”
•	Line 196 – The word “program” can be removed
•	Line 198 – The word “these” should be removed
•	Line 198 – The word “their” should be changed to “the most”
•	Line 213 – “were blasted” should be changed to “were analyzed by BLAST…”
•	Line 217 – “shown” should be changed to “showed”
•	Line 222 – The word “the” in “first evidence of the rare…” should be removed
•	Line 228 – “belonged” should be changed to “belonging”
•	Line 233 – The word “common” should be changed to “commonly isolated from” to reflect that these studies have all been isolation based
•	Line 236 – “diverse actinomycetes population” should be “diverse actinomycete populations”
•	Line 241 – The words “antibacterial” and “antiviral” should be changed to “bacterial” and “viral” as the authors are describing the pathogenic agents
•	Line 244 – I just don’t know….
•	Line 251 – The word “a” should be added to “in a pot experiment…”
•	Line 257 – The sentence should be changed to say “are effective in the control of plant…”
•	Line 259 – The words “for screening” should be removed or the sentence should be restructured.
•	Line 286 – The sentence should say “obtained from an adult bee and is phylogenetically…”
•	Line 289 – The word “This” should be removed
•	Line 290 – There should be a comma between “usage” and “especially”

The much of the relevant research is cited; however, several of the papers that are cited are between twenty and thirty years old. In line 54, the findings are stated to be “recent”. There have been many more recent publications on the association of actinomycetes with fungus growing ants and leafcutter bees and they should be included. Additionally, in the discussion the authors state that there are several publications supporting their view that insects might be a source of new antimicrobials; however, they do not cite a recent publication documenting the antimicrobial potential of over 10,000 actinomycetes isolated from over 1,000 insect hosts that would greatly bolster their claim.
•	The paper is a 2019 paper from Chevrette, M.G. et al. in Nature Communications.

The article conforms to the structure suggested by PeerJ and the figures and tables are relevant to the text in the manuscript.

All 16S sequences appear to be submitted to publicly available databases.

The results presented are relevant to the hypothesis being tested.
Experimental design: The research presented in the manuscript is within the aims and scope of PeerJ.

The research question is provided in the introduction and the relevance of the research is made clear. The authors state that this is the first study to isolate Streptomyces from the black dwarf honey bee in the abstract and the introduction; however, they later cite a paper from Santisuk et al, that reports isolation of Streptomyces spp. from the black dwarf honey bee with anti-nematode activity.
•	The authors should make sure that they state that their study is the first to identify the antimicrobial potential of these Streptomyces, especially in the abstract.
•	Additionally, the authors should mention in the introduction, perhaps at the end of the paragraph ending at line 71, why “rare genera” actinomycetes are of interest for drug discovery programs as well.

Replication was performed in each of the experiments and extra efforts were performed to confirm the MIC level by streaking the cultures from the microtiter plates onto agar plates. Recent versions of all bioinformatic tools were used in the production of the sequence alignments and phylogenetic trees.
•	The MICs of the inhibitory Streptomyces organic extracts are all at the highest concentrations tested (128 and 64 mg/L). The MICs would be more accurate if the serial dilution was more evenly distributed around the MIC value.

The methods mostly rely on previously published protocols with minor adjustments that are described in the manuscript.
•	A brief description of the extraction method should be provided in the manuscript, as the authors later, in line 136, describe the extract as an ethyl acetate extract without providing any context to how it was produced.
•	It is unclear in line 100 if the five adult bees and five pupae were taken from each of the six collected combs or if it was five adult bees and five pupae total.
•	Additionally, was an equivalent number of pollen and honey collections also sampled for actinomycetes?
Validity of the findings: The authors provide a table with information on the number of strains isolated from each source. Later in line 286, the authors describe isolate ACZ2-27 as being isolated from an adult bee.
•	It would be very interesting to know where the other two inhibitory isolates (ASC3-2 and ASC5-7P) were isolated from, especially since the authors isolated from various sources in the honey bee environment.
•	Additionally, it would be interesting to know where the “rare genera” actinobacteria were isolated from, or at least the representative strains. Did they come from unique isolation sources compared to the Streptomyces strains?
•	The authors may also want to comment on any inhibitory activity observed in any of the “rare genera”.
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: APIS ANDRENIFORMIS ASSOCIATED ACTINOMYCETES SHOW ANTIMICROBIAL ACTIVITY AGAINST BLACK ROT PATHOGEN (XANTHOMONAS CAMPESTRIS PV. CAMPESTRIS)
Review round: 1
Reviewer: 2

Output:
Basic reporting: English needs a lot of improvemet.
Experimental design: No Comment
Validity of the findings: No Comment.
Additional comments: No comment


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

Input:
Title: APIS ANDRENIFORMIS ASSOCIATED ACTINOMYCETES SHOW ANTIMICROBIAL ACTIVITY AGAINST BLACK ROT PATHOGEN (XANTHOMONAS CAMPESTRIS PV. CAMPESTRIS)
Review round: 2
Reviewer: 1

Output:
Basic reporting: The authors have isolated and identified three Streptomyces strains associated with the black dwarf honey bee (Apis adreniformis) as well as several strains from underrepresented actinobacterial genera. The three Streptomyces strains were found to inhibit the growth of the Gram-negative plant pathogen Xanthomonas campestris pv. campestris in vitro. The authors also demonstrated that organic extracts prepared from the inhibitory Streptomyces strains were able to inhibit Xanthomonas campestris pv. campestris. The authors argue that the presence of inhibitory Streptomyces strains and rare actinobacterial genera isolated from this rare honey bee species may make it a valuable source for future natural product discovery efforts.

The English is mostly clear but should be improved in several sections throughout the paper prior to publication:
•	Line 49 – This sentence is repetitive and likely not necessary, since the other six species are never mentioned. The repetition should be removed or the sentence should be removed.
•	Line 93 – The word “the” should be removed from “with the antagonistic…”
•	Line 190 – The word “All” does not match with the percentages in line 186 and 187. Line 186 says that 65.6% of actinobacteria were isolated from adult bees, not all.
•	Line 240 – the word “was” should be “were”
•	Line 241 – the word should be “Actinomycete”, not plural
•	Line 248 – There should be a space between the words “from” and “at”
•	Line 250 – There should be a comma after “adreniformis”
•	Line 255 – The word “are” should be changed to “were”
•	Line 257 – The words should be “bacteria and fungi” not “bacterial and fungal agents” as you are describing what the compounds with activity are acting on.
•	Line 260 – This sentence is difficult to read, it might be easier to read if you change it to “variable antimicrobial activity” instead of “antimicrobial activity with a variable spectrum”
•	Line 268 and Line 270 – If all isolates in the study were determined to be Streptomyces, as it says in line 270, then “Actinomycetes” can simply be changed to “Streptomyces” and the sentence starting on 269 can be eliminated in order to simplify the sentence.
•	Line 300 – A space should be between “albicans.” and “Recently”
•	Line 310 – The “organic extract” of isolate ACZ2-27 had the lower MIC, not the isolate itself.

The much of the relevant research is cited and is relevant to the study.
The article conforms to the structure suggested by PeerJ and the figures and tables are relevant to the text in the manuscript.
All 16S sequences appear to be submitted to publicly available databases.
The results presented are relevant to the hypothesis being tested.
Experimental design: The research presented in the manuscript is within the aims and scope of PeerJ.

The research question is provided in the introduction and the relevance of the research is made clear. The authors state that this is the first study to isolate Streptomyces and other rare actinobacterial genera from the black dwarf honey bee and suggest these may be useful in future natural product studies.

Replication was performed in each of the experiments and extra efforts were performed to confirm the MIC level by streaking the cultures from the microtiter plates onto agar plates. Recent versions of all bioinformatic tools were used in the production of the sequence alignments and phylogenetic trees.
Validity of the findings: The conclusions are well stated, and the authors provide evidence for the novelty of their findings. The final sentence in the abstract; however, is not true.

•	The authors state at the end of the abstract that “This is the first report to identify the antimicrobial potential of Streptomyces especially the antibacterial activity against phytopathogenic bacteria.” This claim is shown to be untrue in lines 256-274 that detail many studies on the antimicrobial activity of Streptomyces against phytopathogens in the same genus as the one being studied here. The authors either need to qualify the statement or remove it from the manuscript.
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: APIS ANDRENIFORMIS ASSOCIATED ACTINOMYCETES SHOW ANTIMICROBIAL ACTIVITY AGAINST BLACK ROT PATHOGEN (XANTHOMONAS CAMPESTRIS PV. CAMPESTRIS)
Review round: 2
Reviewer: 2

Output:
Basic reporting: The article seems to be well written. However has major technical errors.
Experimental design: There are technical limitations.
Validity of the findings: -
Additional comments: The antibacterial assays are not understandable. The experiments need to be repeated.


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

Input:
Title: APIS ANDRENIFORMIS ASSOCIATED ACTINOMYCETES SHOW ANTIMICROBIAL ACTIVITY AGAINST BLACK ROT PATHOGEN (XANTHOMONAS CAMPESTRIS PV. CAMPESTRIS)
Review round: 3
Reviewer: 1

Output:
Basic reporting: See below
Experimental design: See below
Validity of the findings: See below
Additional comments: I believe the changes to the manuscript are acceptable and that the manuscript, once edited for English language, should be accepted for publication.


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

Input:
Title: TACKLING THE PHYLOGENETIC CONUNDRUM OF HYDROIDOLINA (CNIDARIA: MEDUSOZOA: HYDROZOA) BY ASSESSING COMPETING TREE TOPOLOGIES WITH TARGETED HIGH-THROUGHPUT SEQUENCING
Review round: 1
Reviewer: 1

Output:
Basic reporting: The manuscript is well written and concise in its current form but the introduction could include more detail about cnidarian evolution and hydrozoan biology. At the moment, this is very briefly discussed in the first paragraph, with specific reference to hydrozoan traits on lines 39-40, but it could be expanded and placed in the context of cnidarian evolution more broadly. For instance, did hydrozoans diversify in a similar way (i.e. through bursts of diversification?) as other cnidarian classes? How do their life cycle strategies compare with other groups? I understand this is more of a methodological paper but I think it's important to frame this properly.
Figure 2 could be improved by colour coding branches within Hydroidolina rather than using more grey boxes to define each taxonomic group. This would help focus the reader's eye on the group of interest.
In the interest of data accessibility and transparency, it would be helpful to have the NCBI accessions included in Table 1. Accessions should also be provided in Table 2 for previously published data. It would be best to provide the subclass (or outgroup) label horizontally at the top of the first column rather than vertically.
Experimental design: The authors have significantly improved understanding of hydrozoan evolution and phylogeny by including many taxa that were omitted in previous studies. The laboratory and bioinformatic approaches are certainly sound and described with sufficient detail. The results are drawn from two methods of phylogenetic inference that largely agree with one another but both rely solely on concatenated data. We know that data concatenation can be problematic in the face of gene tree discordance, which isn't limited to just shallow relationships (i.e. deep coalescence and ancient introgression). In light of this, the authors should consider implementing a multispecies coalescent model for phylogenetic inference, even just a summary coalescent approach like ASTRAL. The paper by Liu et al. (2019) (https://www.nature.com/articles/s41467-019-09454-w#Sec1) uses a similar approach for resolving the phylogeny of mosses, a diverse group that includes 30 different orders.
Validity of the findings: As stated above, I would encourage the authors to implement a multispecies coalescent model for phylogenetic inference to compliment their current analyses and support the framework provided here.
On lines 345-346 the authors state that the Filifera clades remain without a clear definition. Does this simply reflect the fact that Filifera is a particularly understudied group within the subclass? Or have attempts been made to examine the morphology and anatomy of these groups and still no defining characters have been discovered? It might be worth clarifying this here.
Otherwise, the conclusions are well stated and linked to the author's original objectives.
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: TACKLING THE PHYLOGENETIC CONUNDRUM OF HYDROIDOLINA (CNIDARIA: MEDUSOZOA: HYDROZOA) BY ASSESSING COMPETING TREE TOPOLOGIES WITH TARGETED HIGH-THROUGHPUT SEQUENCING
Review round: 1
Reviewer: 2

Output:
Basic reporting: Lines 24-25: "The Bayesian posterior topology infers Aplanulata as earliest branching lineage of Hydroidolina" I suggest revising this to "The Bayesian posterior topology infers Aplanulata as the sister group to all other members of Hydroidolina". Neither of these two clades is earlier branching than the other. This applies to all occurrences of "earliest diverging" throughout the manuscript.

Line 54: Comma after parenthesis should be a period.

Line 182, 283: It isn't clear how or why the topologies that are examined in these section were selected. They aren't related to specific results in previous studies.

Line 197: Why weren't these topologies all compared to the results of the present study instead? It has much better sampling and is an exciting advance over these previous analyses.

Line 323: "far-reaching consequences" be more specific here. Especially after featuring it in the abstract, was surprised to not have more discussion of this. I think it is very interesting, but am not exactly sure what consequences the authors have in mind. It reads as if implying that the MRCA of Hydroidolina was solitary, but this is not clear. It could be useful to include a tree where these traits are explicitly mapped.

Lines 374-376: This is quite speculative and I think only captures part of the issue. Mitochondrial genomes are really small - there just aren't many data to work with. Rate can play a role too, but it is clear that many difficult challenges in cnidarian phylogeny require considerable more data than are available in mt genomes.

Line 403: Remove or reword. The maximum likelihood tree is the literally the most likely tree.
Experimental design: The only comment I have regarding experimental design is the topological investigation summarized in Figure 3. My major concern is that so many nodes are constrained in each topology is very difficult to understand what features of these topologies are driving the differences in likelihood. It is much more informative to constrain only one or maybe two nodes at a time to really understand the impact of topology on likelihood.

Rather than constrain many nodes to compare the entire topologies, I suggest identifying a list of specific relationships that have been variable across analyses, and then constraining those one by one.
Validity of the findings: This is a very sound body of work, and I have no concerns about validity apart from the issues noted above in experimental design.
Additional comments: This is a very interesting and exciting paper. It will be of great interest given the fascinating diversity of traits in these groups. It makes important advances on key challenges that have been highlighted over the last decade as many other relationships in the group firmed up.


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: TACKLING THE PHYLOGENETIC CONUNDRUM OF HYDROIDOLINA (CNIDARIA: MEDUSOZOA: HYDROZOA) BY ASSESSING COMPETING TREE TOPOLOGIES WITH TARGETED HIGH-THROUGHPUT SEQUENCING
Review round: 2
Reviewer: 1

Output:
Basic reporting: The authors have addressed my comment about hydrozoan biology by adding relevant text to the Discussion. The revised version of the manuscript is well written and makes a significant contribution to hydrozoan evolution and phylogeny.
Experimental design: The authors addressed my concern about data concatenation and have revised accordingly. The methods here are sound, described with sufficient detail and address the objectives set out by the authors.
Validity of the findings: The authors addressed my concern about data concatenation and have revised accordingly. Additionally, all underlying data have been provided.
Additional comments: This is a great piece of work and I look forward to seeing it 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: TACKLING THE PHYLOGENETIC CONUNDRUM OF HYDROIDOLINA (CNIDARIA: MEDUSOZOA: HYDROZOA) BY ASSESSING COMPETING TREE TOPOLOGIES WITH TARGETED HIGH-THROUGHPUT SEQUENCING
Review round: 2
Reviewer: 2

Output:
Basic reporting: Be sure that each phylogeny figure identifies whether the tree is a cladogram or phylogram, and if it is a phylogram that there is an indication of meaning of branch length.

On figure 4, I believe the constrained nodes are shown incorrectly. In all cases, the solid circle indicates Hydroidolina, which has the same composition in each analysis and so does not differ. The constraint should be on the in-group that excludes the tested sister. From left to right, that is the clade Capitata...Siphonophora, Filifera...Aplanulata, Capitata...Siphonophora.
Experimental design: No issues noted.
Validity of the findings: No issues noted.
Additional comments: The authors addressed all my previous concerns, and this is going to be a valuable contribution to the community.


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: AN ARTIFICIAL NEURAL NETWORK FOR AUTOMATED BEHAVIORAL STATE CLASSIFICATION IN RATS
Review round: 1
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: This study provides a novel artificial neural network that classifies behavioral states in freely behaving rats. The network receives in input electrophysiological features, previously extracted, that are subsequently processed by two fully connected hidden layers. The output of the network consists of 3 units, each representing the probability that the input data was recorded during the wake, REM, or non-REM state. The performance of the network in classifying the correct state is high and it was assessed after cross-validation. This tool is promising because it is not computational expensive, it is free, and open source. It is a helpful tool to speed up the time necessary to label the behavior state, particularly addressed to sleep researchers.

In my view the following points should be addressed in the revision to improve the manuscript further.

Major points:

1.	Even though the description of the architecture of the network is clear in terms of number of hidden layers and number of units per layer, it would be helpful for the reader if the authors could describe why and how they ended up in choosing this particular configuration of the network, i.e. 4 fully connected layers (256, 128, 32, and 3, line 136). Did the authors investigate any other configurations of a feedforward network, i.e. different number of hidden layers and/or different number of units per layer? Is the current architecture the best one in terms of performance and/or time consuming?

2.	It would be helpful if the authors could provide an additional figure with the training performance and the loss values as a function of the training epochs (maybe adding another panel into Figure 2 close to the architecture of the network).

3.	 In the ‘feature extraction’ section (line 147), the authors explain the input to the artificial neural network. According to the architecture of the network, the input consists of 256 units but still, it is not very clear to me what these 256 units are representative of. Different features from EEG-EMG signal are extracted but it is unclear how they sum up to 256 input units. I would suggest spending few more words describing the input structure, and why 256 units as input.

4.	The training set was oversampled, as stated in line 167. What is the procedure the authors adopt to oversample the training set?

5.	It is not clear what the ‘uncertain label’ defined in line 185 means and how it is further used. A better explanation of the use of the label would be very helpful.

Minor points:

1.	Line 163: what does pseudorandom mean in this context? I would say ’50 randomly chosen epochs’

2.	Line 205: ‘[…] quality metric is less than 1.67 […]’: why exactly 1.67? It would be helpful if the authors could provide a reason behind the choice of this value for the quality metric threshold.

3.	Line 161: ‘[…] initial training set (560, 4s epochs; 2.6% of daily total) […]’. Why did the    authors choose exactly 560 4s epochs? It would be helpful to explain in more detail the choice of the size of the training set.

4.	Line 227: ‘SWA’ should be defined.

5.	When reporting the p-values (for example line 300), if the p-value is significant (much smaller than 0.001), I would suggest writing only the order of magnitude like p=10^-7.

6.	Line 333: the following sentence ‘[…] High levels of certainty were associated with excellent model performance’ is not clear because high level is not quantified: is it 70%-80%-90%-100%? I think it is a very general statement that I would personally avoid.

7.	Figure 3 C-D: labels of x-axes (i.e., Acc-W-NR-R) should be defined in the caption. Moreover, in figure 3D I would specify the sensitivity-specificity color bar as done in figure 3C.

8.	Figure 4B-C-D. Please, provide the legend for the color bars for each figure.

9.	Figure 6A-B. Please define the labels of x-axes in the caption.
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: AN ARTIFICIAL NEURAL NETWORK FOR AUTOMATED BEHAVIORAL STATE CLASSIFICATION IN RATS
Review round: 1
Reviewer: 2

Output:
Basic reporting: //
Experimental design: //
Validity of the findings: //
Additional comments: Ms. Ref. No.: PeerJ (#61856)
Title: An artificial neural network for automated behavioral state classification in rats
Jacob Gould Ellen and Michael Bennet Dash

Overview and general recommendation:

The classification of an animal's behavioral state as a function of time is important in the study of neuroscience problems. Usually, this procedure is performed by experimenters in a manual way requiring a lot of time and energy. In this paper the authors propose the use of an artificial neural network to make this procedure semi-automatic (labels must still be manually assigned to a fraction of data for supervised training). In particular the authors test their algorithm considering as input of the network electrophysiology data in freely moving rats and as output the ability of the network to distinguish between awake, REM and NREM states. Although there are already several algorithms in the literature that can solve this task, the authors emphasize that their method is innovative because it is easy to use, open source and computationally inexpensive.

In terms of presentation, I find the article well written and easy to read and understand, the methods are well described so that the reader can reproduce the results themselves and the bibliography is more than comprehensive.

Nevertheless, I found some important (and some trivial) issues that I think are critical to address in order to proceed with an eventual publication in PeerJ that I list below.

Major comments:
1) The network used has 256 input units, but it is not at all clear to me what all these input features are since the authors define only 17 in the text. Which are the others?
2) How does the performance of the network vary when the architecture parameters vary, i.e., number of layers, number of units per layer, different nonlinearities?
3) It is a bit vague how the size of the training set is defined in line 161.
4) Among the features considered as input, which are the most relevant in terms of performance? The purpose of ANNs in general is to find from the learning process the features that are needed for example in a classification problem. Since you already have the relevant features, what is the use of a neural network with different layers? Isn't a perceptron or even a look up table sufficient?
5) I understand that the network has been tested on rat data but the features considered are general. What do you expect from the same ANN architecture on monkey or human data?
6) In line 388 you talk about supervised learning as an alternative approach to deep learning when actually the deep learning in this contest can be seen as supervised learning with number of hidden layers greater than one, then your case falls within the deep learning. In general, it is not clear to me the innovation of this work with the works cited in the literature.
Minor comments:
1) Typo in line 141: output->input.
2) Usually, the term epoch in machine learning is defined as the step in which weights are updated using a batch of data as training, here in this article the term epoch is used to say that different features of electrophysiology are studied on time windows of 4s. I would try to avoid this ambiguity of language replacing epochs with time interval for example.
3) In line 145 it is already mentioned probability, so it is redundant to specify that the sum is one because it must be one by definition of probability.
4) In line 160 it is written "ANN models operate as supervised learning algorithms" as if all the artificial neural networks followed a protocol of supervised learning, this obviously is not true. Change the sentence in "ANN models can operate as supervised learning algorithms".
5) Typo line 183: modal->model.
6) In Figure S1 the caption is missing.
7) In line 227 specify that SWA refers to slow wave activity.


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: AN ARTIFICIAL NEURAL NETWORK FOR AUTOMATED BEHAVIORAL STATE CLASSIFICATION IN RATS
Review round: 2
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: The authors addressed properly all the comments made by the reviewers. I think the paper is now ready 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: AN ARTIFICIAL NEURAL NETWORK FOR AUTOMATED BEHAVIORAL STATE CLASSIFICATION IN RATS
Review round: 2
Reviewer: 2

Output:
Basic reporting: Ms. Ref. No.: PeerJ (#61856)
Title: An artificial neural network for automated behavioral state classification in rats
Jacob Gould Ellen and Michael Bennet Dash

Overview and general recommendation:

I find the second version of the article improved and I think the explanations that have been added will be important for anyone who decides to use this algorithm for their research. I am glad that the authors have answered in detail both the major and minor issues I had raised. That said, I am confident that the work is now mature enough to finally be published in PeerJ.
Experimental design: //
Validity of the findings: //
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: PLASTIC RESPONSE OF MEDICAGO SATIVA L. ROOT SYSTEM TRAITS AND COLD RESISTANCE TO SIMULATED RAINFALL EVENTS
Review round: 1
Reviewer: 1

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


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

Input:
Title: PLASTIC RESPONSE OF MEDICAGO SATIVA L. ROOT SYSTEM TRAITS AND COLD RESISTANCE TO SIMULATED RAINFALL EVENTS
Review round: 1
Reviewer: 2

Output:
Basic reporting: 1.Basic reporting
	In my opinion taking into account that I myself am not a native speaker of English language, the linguistic quality of the manuscript (MS) meets the standards of the journal. There is only a small number of minor corrections that I have detected and pointed out for the authors to correct. Please find detailed comment below.
	Literature references are sufficient and cover the necessary background.
	The structure of the MS conforms to the journal’s format.
	Figures are relevant, however some improvements are necessary concerning figure resolution and legends for both figures and tables. Please find my specific comments below.
	Raw data of all traits examined have been made available, thank you. There is a minor spelling issue i.e. column title of the starch values is written as strach. I also urge the authors to add titles in the first 3 columns and include the units of measurement for each trait in order to facilitate future readers.
	The article is self-contained and the results could become (extensive improvements are needed) relevant to the hypothesis.
Experimental design: 2.Experimental design
	The work is within the Aims and Scope of PeerJ.
	The working hypothesis is clear and meaningful. The contribution that the MS is intended to have on filling knowledge gaps is clearly explained.
	The investigation performed is moderately rigorous (please see specific comments below). In general, technical and ethical standards are met. Statistical approach is the main issue.
	Methods description could be improved. Please find the relevant comments below.
Validity of the findings: 3.Validity of the Findings
Although the technical part of the work seems overall nicely done, there is a major issue with the statistical analysis and therefore the presented results and consequently their validity. Authors should incorporate at least two more statistical tests and reconsider their whole statistical analysis in order to use a method that is more appropriate for their experimental design. Authors should also include in the description of their Statistical Analyses section (Materials and Methods), all the appropriate information regarding the PC analysis, that is now missing. Please find more details in the comments below.
Additional comments: 4.General comments
In this MS, the authors aspire to provide further information on the role of mainly root morphological traits and their plasticity in cold tolerance of alfalfa. To do so, they investigated intraspecific variation and plasticity of root morphology and physiology among 3 cultivars by submitting them to 3 simulated rainfall treatments and examining their effects on selected root morphological and physiological indexes. They then tried to assess if these modifications result to an increase in cold tolerance of the cultivars using semi-lethal temperature as an indicator. The MS is of good quality and in general well-written (linguistically speaking), and could prove quite interesting for the readers of PeerJ.  However, authors should consider making extensive alterations and additions for the improvement of some of the quite weak (at the moment)  parts of the MS. Please find my specific comments per section below.

Title: Perhaps authors should consider using “plastic responses” instead of response, which is less informative.
The term cold resistance and tolerance seem to be used throughout the text interchangeably. Given the overall confusion around these two terms I would greatly appreciate it if authors could add a sentence for clarification purposes of their use of the terms. If resistance is used for describing the lack of adverse effects on the plant despite the existence of the stress factor, then cold tolerance is a better term for the title.

Abstract
Minor comments:
Line 19-22: I am not sure that the word phase clearly describes what the authors did. Of course there were two phases regarding the sequence of the work done, but if I understood correctly, actually these were not two phases but two different treatments: cold-treated plants and non-cold-treated plants.  I would suggest a rephrasing that clarifies that from early on.
Line 31: perhaps examining instead of analyzing

Introduction
This section is well written, provides all the necessary background information and clearly states the goals of the article.
Minor comments:
Line 83: Given the logic of the plant x environment interactions, this sentence should be rephrased.  Water management could prove important for increasing cold tolerance and not the other way around as implied.

Materials & Methods
This section needs some improvement for the better clarification of the experimental processes and data analysis.
Experiment Designs and treatments
Lines 107-116. It is not very clear which was the WHC throughout the experimental period for each of the SRE. What WHC did the D8 plants reach on the 8th day before the irrigation? In D4 plants, 4 days after the initial irrigation WHC reached 40%? Rewatering was done till WHC 60-65% for both D4 and D8 plants? Please explain this procedure in a more helpful for the reader way especially taking into account that rehydration affects the branching pattern.
Line 116: phase 1, please see comments above (abstract). Sampling was done on half of the total experimental plants i.e. 3x3x6? Please clarify.
Line 125: Do the authors mean the remainder plants i.e. the rest half of the experimental plants? Please clarify.
Authors should also explain why they chose to measure the non-cold-acclimated plants 3 days before the cold-acclimated plants, which means that there is a small but significant difference of the root system development that should have been taken into account for reasons of comparability. Especially considering that root structural modifications are quite fast and can become obvious after about 1 day, a phenomenon that the authors exploited for investigating the plasticity.
Authors should also make an effort to justify the choice of the timing (i.e. why 3 days of acclimation?) of the second sampling. How was the WHC change taken into account? Please justify.
Line 129: I understand the logic behind the light intensity reduction but this change adds another environmental factor that may affect the results. Has this been taken into account?

Measurements
Line 132: How much was WHC when plants were moved to the incubator? Were they rewatered before moving them? Were they rewatered during cold-accclimation?
Line 138: It would be very helpful if authors provided some visual material e.g a photograph of the root system.
Line 157-159: Please add a phrase explaining why the authors chose different temperatures for the two treatments.
Line 174: Please rephrase this sentence because it sounds like the material used for the measurement of LT was grounded and then used for the biochemical measurements which I guess and strongly hope is not the case.
Line 192: The incubation time is quite lower than the one proposed in Bates, 1973. If authors have modified the protocol, please mention and explain.

Statistical analyses
Line 225-227: Please clarify if the criteria for conducting a parametric test i.e. normality and homogeneity of variance were met. The tests done should also be mentioned. This would also justify the use of Pearson correlations instead of Spearman.
Line 229: According to the experimental design followed, a simple effect analysis is not the appropriate statistical test for comparing the variance. There are at least 3 factors i.e cultivar (3 levels), irrigation treatment (3 levels) and cold acclimation treatment or not (2 levels). If taking account temperature difference between the last treatments it would add 1 more factor with 2 levels. I suggest the authors should conduct a 3- way ANNOVA, and try to explain as suggested above (comments for Line 125) why temperature was not taken into account as an extra factor of possible variance.
Given that the authors have also conducted another statistical analysis i.e. PCA, the relevant information should be added at this section. Does the PCA include both the data from cold acclimated and non acclimated plants? Because the points in the biplot are only about 54 where it should be 18 treatments x 6 replicates =108 as mentioned in Line 112-113.
However, given that PCA is very useful as an ordination method I think that the authors should try exploit the very interesting information their analysis is providing them, instead of using it only as a correlation method. If so, a pairwise correlation matrix for all traits would be more suitable and just enough.
According to the PCA biplot, the grouping of the water treatments along the first axis provides a clearer picture of the interactions and the trade-offs among the examined traits and reinforces the results of the paper. Furthermore, a very useful and enlightening addition in the biplot would be to make a distinction among cultivars (eg by different shapes of the markers) and phase 1 and 2 data (eg by different shape outlines or even by circling them) and see how the cultivars, SREs and “phases” affect the ordination along the two axes. This would help explaining what each component represents in a more meaningful way. Perhaps authors should consider taking advantage of the different groupings/ordination on the biplot and try explain them in a more constructive way.

Results
Given that the statistical analysis is not appropriate for this kind of set of data, the presentation of the results is also not appropriate for understanding the intraspecific variation, the SRE effect, the cold effect and their interactions effect.  The whole section needs reconstruction after the appropriate statistical analysis is done.
It would be more meaningful to compare the plasticity of each trait induced by the 3 SREs among the 3 cultivars and then check for probable links of such differences (either due to cultivar or SRE) with the cold acclimation response.
The comparison of the mean trait value among D2, D4 and D8 irrespective of cultivar and ”phase”, or among cultivars irrespective of SRE and ”phase”  as presented in Tables 1,2,3,4,  Figure 1,2, 3, 4, 5, 6   is not very helpful. Why compare the mean for each SRE/cultivar without taking into account the possible effects of the rest factors that the experiment includes? Authors need to follow a different statistical approach that is suitable for their experimental design.
Under each of the above mentioned tables and figure, the sample size is written as n=6.  If I understood correctly, the mean for e.g. D2 derives from: either the mean of the mean values of the 3 cultivars therefore n=3, or the raw data i.e n=6 replicates x3 cultivars =18. Is that true?  The same then goes for the rest…Please check that sample size is correctly written.
Another possible improvement concerns the table labels. It is not very helpful for the reader to see all these abbreviations in the label. I would suggest an approach like e.g.: Comparison of the biomass / morphological responses/ architecture/ physiological indexes among the three cultivars, SREs etc., namely a category/ grouping of the traits instead of abbreviations, that are correctly referred under the table.
Effect analysis should include all traits and treatments. So Table 2 is not very representative at the moment.
Table 5 is supposed to help elucidate which factor had the main effect? I do not get the point of this Table that it is not properly discussed, too. Please present these results appropriately and comment on them in a meaningful way.
And why are only morphological traits used and not physiological indexes?  This is the case for the PCA, too. Why were only these included when it is expected that there are interactions among all traits,  interactions that need to be investigated? Please justify the trait exclusion or adjust your analysis.
Even if Table 5 is kept, some more information is needed  i.e. the sample size n=? is it the r or r2 presented. I am also wondering what kind of data SRE refers to.  There is no numerical trait referred as so. Please explain.
Line 3: represent statistically significant
Figures seem to have a low resolution but perhaps it is a matter of the pdf file. Maybe authors should check that their figures format complies with the journals instructions.

Discussion
Overall the quality and structure of this section are the MS best attributes. However, this is not the case for the scientific arguments that need confirmation. Considering the extensive changes that need to be done for a more suitable statistical analysis and the consequences this will have on the results, the authors will probably have to confirm their results before discussing them. A good example of the problem that the present statistical approach creates is the results and therefore the discussion for AGB, RGB and R/S.
Given that Table 1 does not give any information about the comparison of the traits of the same cultivar at the different SREs (which is the approach followed for all traits), no safe conclusion can be extracted regarding the changes of AGB, BGB, R/S because the plasticity within cultivar under the different SREs has not been examined.  It may prove that there is no difference at all but at the moment there is no evidence that can support that. Also one cultivar may be more plastic than the other ones and this may eliminate the variation when not addressing the plasticity of each cultivar separately. And unfortunately this is the basic weakness of all results discussed.
I strongly suggest that after the reconsideration of the statistical approach, authors need to confirm the validity of their arguments and conclusions.


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: PLASTIC RESPONSE OF MEDICAGO SATIVA L. ROOT SYSTEM TRAITS AND COLD RESISTANCE TO SIMULATED RAINFALL EVENTS
Review round: 1
Reviewer: 3

Output:
Basic reporting: This manuscript present results on the impact of different rainfall intervals on alfalfa freezing tolerance in relation with root traits and the concentration of stress-regulated metabolites under controlled conditions. The authors found that longer irrigation interval was beneficial in improving the cold resistance of alfalfa. While this paper adds new knowledge in the field, particularly on the impact of rainfall patterns on root traits, major improvements are needed to the manuscript to better explain what was done.

Abstract: The authors use freezing tolerance and cold resistance to express similar concepts. They should explain the difference between the terms and choose the one that relates to the concept they want to express throughout the manuscript.
Same observation for ability to survive winter and/or winter survival: these parameters were not measured in this study and winter survival depends on many factors, not only freezing tolerance.
Experimental design: My comments are regrouped in the section on General comments to the author
Validity of the findings: My comments are regrouped in the section on General comments to the author
Additional comments: This manuscript present results on the impact of different rainfall intervals on alfalfa freezing tolerance in relation with root traits and the concentration of stress-regulated metabolites under controlled conditions. The authors found that longer irrigation interval was beneficial in improving the cold resistance of alfalfa. While this paper adds new knowledge in the field, particularly on the impact of rainfall patterns on root traits, major improvements are needed to the manuscript to better explain what was done.
Abstract: The authors use freezing tolerance and cold resistance to express similar concepts. They should explain the difference between the terms and choose the one that relates to the concept they want to express throughout the manuscript.
Same observation for ability to survive winter and/or winter survival: these parameters were not measured in this study and winter survival depends on many factors, not only freezing tolerance.
Abstract L. 30. Contrary to the abstract, Fig. 6 shows higher concentration of starch in D8 than in D2 and D4. Rewrite.
L42 : Replace winter failure by crop failure due to winter conditions
L49: Change ‘There are two main impacts of climate change on plant overwintering: precipitation and temperature’ by ‘Two major factors linked to climate change are likely to affect plant winter survival: changes in precipitation and temperatures (Bélanger, 2002; CCAF, 2001).
L.59: Water management plays an important role in the winter hardiness of alfalfa because freezing injury is mainly caused by cell dehydration: The logic behind this sentence is not true: freezing injury is caused by cell dehydration because water freezes in extracellular space, which is not directly linked to water management. Rewrite.
L. 57 and L.58 The authors use freezing tolerance and cold resistance to express similar concepts. They should explain the difference between the two terms and choose the one that relates to the concept they want to express throughout the manuscript.
L.66 …were found to have a greater impact on herbaceous plants. As compared to what? Please complete the sentence.
L. 76-78: There is a very brief introduction on malondialdehyde (MDA), proline, soluble sugars and starch related to their antioxidant capacity and osmotic regulation in plants. The authors state that these compounds are closely related to cold resistance of alfalfa. It should be specified if the relationship is positive or negative and the role of each compound should be briefly explained in relation to freezing tolerance in this section.
L.80 and 82. On cold resistance or freezing tolerance? It is not the same thing. What did you measure in your experiment?
L.96: It is stated that a ratio of 4:1 sandy soil and nutrient soil mixture was used. What is a nutrient soil mixture? This needs clarification.
L114-131. This part is not easy to follow. If I understand, the plants were three weeks old when the treatments started. Plants received the different irrigation treatments during 8 weeks, followed by a first sampling. Then the remaining plants were cold acclimated 72 hours followed by a second sampling. A table or Figure explaining clearly the treatments and sampling should be provided.
L 131. If the acclimation treatment lasted 72 hours, how could the plants be watered as in the previous phase? Does it means that only D2 plants were watered during this period? It should be clarified.
L. 152-153. To assess electrolyte leakage, the authors sampled the crowns that were sliced into 9 pieces about 2-3 mm and put into 2-mL centrifuge tubes. It should be explained how were divided the crowns into 9 pieces of 2-3 mm since Table 3 reports crown diameters of 3 to 4 mm. Were several plants pooled together? Furthermore, I am not confident that actual plant damages could be assessed by using such a small quantity of materials (2mm). Did you validate your method with other plant survival measurements? Crowns are very small organs at the intersection of roots and stems so please explain how it was sampled.
L.173. Since the roots were used for the analysis of morphological traits, it is not clear on which plant material the root physiological indexes were measured. I understand that 0.2 g were used for MDA, 0.2 g for proline, and 0.2 g for soluble sugars, and a part was used for LT50 while the dry weight of the belowground biomass was around 0.6 and 0.9 g (Table 1). Were several plants pooled together to perform all these analysis? This part needs clarification. How big were the plants?
L. 174. After measuring electrical conductivity the remaining samples were ground into powder and freeze-dried to determine their SS: is it root samples or crown samples ( it is under the subsection on root physiological index). This should be clarified since crowns and roots biochemistry highly differ.
To avoid the degradation of the samples, it is recommended to freeze-dry the samples before grinding. Is it what was done? Please specify.
L.236. How was measured the biomass? It is not in the M&M section. The following abbreviation BGB, AGB, C, R and S should be spelled out on their first appearance in the manuscript.
L.272 and Fig. 1. How could you determine that damages were observed in alfalfa exposed for 1.5 h at 4C. The damages could be due to the protocol described since tubes were placed at 4°C for 2 h and then transferred into ice for 2 h before their transfer in a ZX-5C for the freezing test at temperature varying from 8, 6, 4, 2, 0, −2, −4, −6. The damages could be due to the two hours into ice. This should be clarified since alfalfa is usually not damaged by a short exposure at 4C.
L.293. What do you mean by growth rate (The MDA growth rate)? The increase from phase 1 to phase 2? It is not a growth rate.
The discussion is good.


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: RISK ASSESSMENT OF FREEZING INJURY DURING OVERWINTERING OF WHEAT IN THE NORTHERN BOUNDARY OF THE WINTER WHEAT REGION IN CHINA
Review round: 1
Reviewer: 1

Output:
Basic reporting: This manuscript is well written; however, some language mistakes still exist, it should be checked throughout the whole text. The manuscript structure is clearly arranged and meets the requirements of the Journal. And the figures and tables are related to the content. The manuscript can be accepted after some minor revisions.
Experimental design: The research on the climatic conditions of wheat freezing injury in the past is helpful to assess the risk of winter wheat freezing injury and increase grain yield. The research in this manuscript is based on 60 years of meteorological data in Zunhua and historical records of wheat freezing injury. A freezing injury prediction model was established and validated. The research question is well defined, which is within the aims and scope of the journal.
Validity of the findings: The research proved that this model is suitable for winter wheat-freezing injury prediction in different years, and provided a new reference for risk assessment of wheat freezing injury. The results are well presented and well supported by the measurement.
Additional comments: Abstract
1. Line 28: The abbreviation of "freezing inventory index" should be "FII" instead of "FI". After the abbreviation appears for the first time, the full name does not need to appear in subsequent places. Please make a unified expression.
2.Line 33: The expression "in the future" is not accurate and should be "in the short term".
3.Line 39: The expression of "freezing injury" or "frost damage" in the full manuscript should be unified.
Main part
1. The introduction needs revisions. The newest reference is from 2019, making the manuscript not up to date with the current literature. I strongly suggest a thoroughly review and update of the literature. Not only for the introduction but also for the discussion.
2. Line 56: The expression of "the variety structure of the northern spring wheat-planting southern area gradually transformed into the winter wheat" is unclear, and the first letter of wheat region name should be capitalized.
3. Line 82: The expression "in this study" is incorrect. Here is the information in the quoted literature, not in this research.
4. Line 91: The word "postdisaster" should be "post-disaster".
5. Line 107: In the title of this section, "research site" should be changed to " research region"
6. Line 119: Using "tiller" instead of "stem" can obtain better expression accuracy.
7. Line 127: The expression "passing through 0 ℃" is inappropriate and should be "stably lower than 0 °C".
8. Line 135: The expression of "freezing disaster-causing factors" should be unified in the full manuscript.
9. Line 168: The expression of "the death rate at the seedling stage" is incorrect and should be "the dead plant rate".
10. Line 181: "Morlet" is a proper noun and should be preceded by "the".
11. Line 190: When referring to the software used, it should not be divided into separate sections, but should be displayed in combination with the method.
12. Line 198: Note that the use of words should be consistent before and after the full manuscript, such as "starting and ending dates" or "starting and ending times".
13. Line 241: If the two variables pass the correlation significance test in univariate linear regression, it indicates that there is a credible linear relationship between the two variables, rather than significant correlation.
14. Line 277: The "time scale" should be replaced by "timescale".
15. Line 385: "Conclusions" instead of "conclusion".
16. Line 386: The conclusions are a concise part of the full manuscript, which needs to be further modified.
17. The authors should carefully examine the grammatical errors in the manuscript.
18. In Figure 7C of wavelet analysis, what do the red and blue curves represent respectively? Therefore, for each picture, valid information should be displayed in the annotation.
19. In the title of Table 1, "disaster causing factors of freezing damage" should be replaced by "freezing disaster-causing factors".


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: RISK ASSESSMENT OF FREEZING INJURY DURING OVERWINTERING OF WHEAT IN THE NORTHERN BOUNDARY OF THE WINTER WHEAT REGION IN CHINA
Review round: 1
Reviewer: 2

Output:
Basic reporting: Currently, a brief introduction to the research is sufficient. The content of the manuscript is clearly arranged and meets the requirements of peer J. However, there are still some problems that should be corrected; hence a minor revision is suggested for this manuscript.
Experimental design: Wheat freezing injury is often a restriction for winter wheat production. Understanding how climatic conditions impact wheat freezing injury in the past is of great importance for assessing the risk of winter wheat freezing injury. The freezing injury prediction model established in this research is useful for avoiding freezing injury in the research area.
Validity of the findings: This research established a freezing injury prediction model and proved that this model is suitable for injury prediction in different years. I think the result is valid.
Additional comments: 1. The literature review and discussion in this MS should keep pace with the latest literature published, while currently the latest literature was published in 2019.
2. "freezing inventory index" should be "FII" rather than "FI". Please revise it in the whole MS.
3. Line 71: The content description of the cited literature should be concise.
4. Line 114: The title of this section should be changed to "Data sources of meteorological and freezing investigation information" to make the information more detailed.
5. Line 157: The expression "were greater than one" is inappropriate and should be expressed directly with "> 1.0".
6. Line 204: Reference should be made when describing the pictures in the group diagram separately, such as (Fig. 2A).
7. Line 246: The expression of "FII index" is inappropriate, "FII value" should be used instead.
8. Line 270: "a" should be replaced by "an".
9. Line 324: The description of " under the premise of ensuring yield, quality, and disease resistance " is unnecessary.
10. Line 333: "It was observed from the results that" is redundant with the description in the previous sentence and should be removed.


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: CBF TRANSCRIPTION FACTORS INVOLVED IN THE COLD RESPONSE OF CAMELLIA JAPONICA (NAIDONG)
Review round: 1
Reviewer: 1

Output:
Basic reporting: The manuscript presents the characterization of four CBF genes identified in Camellia japonica and their functional analyses on different stress responses. The authors provide relevant study to explore the mechanisms of this species to adapt to stress conditions however, the manuscript lacks a large number of information that sometimes, but not always, they are in different parts of the text. There are statements missing references and the legends of the figures are totally incomplete making difficult to understand what the authors want to show. The manuscript requires a deep english review.
Experimental design: The manuscript lacks information on the methodologies and data of the experiments. Sometimes we find some information on the figures but they need to be provided in material and methods. In the discussion session, the authors describe information on the promoter region of the genes without any result be provided previously.
Validity of the findings: The results show novelties to the Camellia japonica species to explore the adaptation of plants to cold temperature.
Additional comments: I have some comments and suggestions to the authors as follow:

Abstract
line 13: review sentence: "..distributed in the northernmost edge of Camellia species in China, ..."

Introduction
Lines 50-51: requires reference. Perhaps this sentence of line 50-51 should be a new paragraph including the continuation with line 52, continuing.

Line 54: Requires a reference

Material and methods
Line 80: How many plants used in the experimental group and control group

Line 82: which method used to RNA extraction

Line 86: Confirm if the low temperature full-length transcriptome and drought full-length transcriptome database were both used. No information about the drought transcriptome were provided.

Line 87: Which technology was used to sequence the transcriptome? Is perhaps Illumina? (line 255 in discussion) please include also here.

Line 93: Include the references of the tools used for the analysis

Lines 95-102: Please, introduce the data that the phylogentic tree was prepared using 424 CBFs from 19 species.  Also confirm if it was 424 or 324 as in Figure 3.

Line 100:  Provide information of how the phylogenetic tree was constructed. It was on the basis of the similarities of the protein sequence of CBFs or by their conservative domains?

Line 107: Complete which plasmid was used to transform Y2HGold Yeast.

Lines 113-115: I am not convinced that transient expression is enough to show subcellular localization, provide references.

Line116-128: The description of the experiments and treatments should be described completely: temperatures used, treatments of drought stress, and which intensity of light.  It is very critical how drought stress was measured in the experiments.

Results:
Lines 134-135: The CBFs were named to which related plants? Are they CjCBF1, CjCBF2, CjCBF3 and CjCBF5? we only see the names in figure 1.

Line 135: Please provide which supplementary file.

Line 141: The analysis shown on Figure 1 is not clear to demonstrate 73.96% of similarity between the 4 CjCBFs identified. Also, the legend of Figure 1 is not clear. I am not sure if the colors red, green, blue and purple represent the bases A, G, C , T or the 4 CBFs.  Fig.1D shows CBF2, CBF3, CBF5 but in the legend also has CBF1. Please, be clear on that you want to show in the figure 1.

Figure 2. Please confirm if the colors represent amino acids or base? What do you mean of "same sequence of 5 or more"? "white" is the opposite to what? The legend of this figure is very confused.

Line 147: motif lengths between 6 and 50 amino acids? Please complete.

Line 156: The results related to the Phylogenetic tree are very confused. They genes were separated in clusters but the authors also mention about subclusters  not shown in the figure. Please review.

Figure 3: The name of the 19 species used is missing in the legend. Confirm if it was used 424 or 324 amino acid sequences of CBF in the phylogenetic tree. I am not convinced that the R package ggtree was used to beautify the tree. Please clarify your statement.

Lines 159-160: How monocotyledons and dicotyledons were clearly separated from dicotyledons? I believe you want to say that monocotyledons and dicotyledons were clearly separated. And dycotyledons were divided into two subclasses. I see only Clade I, II and III in the figure 3, it is not shown the subcluster A-II in the Clade I.

Line 163:  Could you please confirm if Clade III represents the large class of monocotyledons?

Line 166:  Cluster A-I and B-II  are in the clade I, II or III?

Line 170-177. I am not sure what you want to show in this analysis, the results in Figure 4 are not clear, what the different colors means?

The analysis of expression based on RNA seq is described based on figure 5. No in material and methods neither in this session, the treatments used were not described.

The reference Xie et al., 2010 describes the KOBAS 2.0: a web server for annotation and identification of enriched pathways and diseases. This tool was used to identify lncRNA?  Please explain your goal in this analysis.

Line 200: AbA ou ABA?
Please provide description of these experiments.

Lines 214-225: The expression analyses in different tissues are shown in figure 7. Please describe group CK? as in the legend of Figure 7, the expression of CjDBF1 of root in CK group was set as 1, only for root?  The relative expression of CjCBFs in Figure 7-E, F, G, H, I were related to CfCBF1? I believe the legend of this figure is completely nonsense and incomplete.

Lines 226-237: The expression of CjCBFs
Also the experiments were not described in the manuscript. I can not say if the treatment of high temperature was used

Supplementary materials: File S1, S2 and S3 listed on lines 318-321 do not correspond to the material sent. As supplementary materials there are 6 files:
the file S1: DjCBFs sequencing (xlsx);
file S2 shows the nucleotide sequence of the promoter sequence
File S3 shows the same file S2.
Files S4, S5, S6 are raw data for the qRT-PCR (xlsx).
Only in the discussion there is the analyes of those sequence related to the promoter region in the regulation of CBF expression. Base on which studies the authors claim they are promoter region?

Discussion
Line 248: Separate the words "brassinosteroid signaling"

Lines 249-250: "and the number of CBFs is often related to the size of the genome"  could you please give references and examples about this statement?

Line 254:  "the complex and large genome of Camellia" requires a reference

Line 283: The Supplementary table S2 shows the nucleotide sequence of the authors defined as promoter sequence of CBF1-C.japonica. These results may be important to be shown in the manuscript.

Line 285: The supplementary table S3 is missing

Lines 297-300: None of these studies were shown in 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: CBF TRANSCRIPTION FACTORS INVOLVED IN THE COLD RESPONSE OF CAMELLIA JAPONICA (NAIDONG)
Review round: 1
Reviewer: 2

Output:
Basic reporting: Language needs to be improved.
Experimental design: Some data are confused and the experiments are not well designed.
Validity of the findings: no comment
Additional comments: In this study, the authors analyzed CBF genes in Camellia japonica. They identified four CjCBF genes, and all these genes are responsive to low temperature. The authors also analyzed the expression of CjCBF genes in different tissues after cold stress. Although the authors demonstrated the roles of CjCBF genes in cold stress response, some results in this manuscript are quite confused.

1.	Line 132, what“11 transcripts”represents?
2.	It is difficult to understand the pictures in Figure 1?
3.	Figure 2B, how authors define different motifs? Are they known motifs?
4.	Figure 5, what T1, T2, T3 mean?
5.	It is not clear what Figure 5B means?
6.	AbA is usually used for yeast-one hybrid, and 3-AT is used in yeast-two hybrid to inhibit autoactivation. Why authors used AbA in yeast-one hybrid assay?
7.	Figure 6C, how can authors conclude that the CjCBFs are localized at plasma membrane?


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: CBF TRANSCRIPTION FACTORS INVOLVED IN THE COLD RESPONSE OF CAMELLIA JAPONICA (NAIDONG)
Review round: 1
Reviewer: 3

Output:
Basic reporting: Naidong is a geographical type of Camellia japonica distributed in the northernmost part in China, which cold tolerance is much higher than that of other C. japonica cultivars. Investigation on Its cold tolerance is of great significance to breeding of cold tolerant cultivars of C. japonica. The authors indentified four CBF genes in Naidong and analyzed these genes. The results provided new insight into the role of CBFs in the cold tolerance in C. japonica.
Experimental design: Naidong is one type of Camellia japonica that is distributed in the northernmost part of China. I think the expression of this material (Camellia japonica (Naidong)) in manuscript is not appropriate. Is it a geographical variation or a cultivar? There is no proper expression. Suggest to consider modification.
The description of experimental materials is too simple, and some information presented in the results is not expressed in this part, such as "40°C treatment" in LINE 229, "light stress treatment" in LINE 230, and "T1, T2, T3" in LINE 183.
The Latin names of some plants in manuscript are not italicized, so it is suggested to check and revise.
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: CBF TRANSCRIPTION FACTORS INVOLVED IN THE COLD RESPONSE OF CAMELLIA JAPONICA (NAIDONG)
Review round: 2
Reviewer: 1

Output:
Basic reporting: The manuscript was improved and my questions answered. I agree to the publication.
Experimental design: The complementation of the experiments was introduced into the manuscript.
Validity of the findings: The research is interesting for publication.
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: CBF TRANSCRIPTION FACTORS INVOLVED IN THE COLD RESPONSE OF CAMELLIA JAPONICA (NAIDONG)
Review round: 2
Reviewer: 2

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: The authors have addressed all my concerns.


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

Input:
Title: CBF TRANSCRIPTION FACTORS INVOLVED IN THE COLD RESPONSE OF CAMELLIA JAPONICA (NAIDONG)
Review round: 2
Reviewer: 3

Output:
Basic reporting: No comment
Experimental design: No comment
Validity of the findings: No comment
Additional comments: No comment


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

Input:
Title: HIGH BYCATCH RATES OF MANTA AND DEVIL RAYS IN THE “SMALL-SCALE” ARTISANAL FISHERIES OF SRI LANKA
Review round: 1
Reviewer: 1

Output:
Basic reporting: Please see general comments
Experimental design: Please see general comments
Validity of the findings: Please see general comments
Additional comments: This is an important study that provides an overall profile of mobulid bycatch in Sri Lankan artisanal fisheries over an 8-year period, as well as vital information on catch rates, annual landings and trends in body sizes for a number of mobulid species.

The manuscript is well-written and draws from an extensive long-term dataset to address knowledge gaps for threatened and understudied species, highlighting declines in catch rates and overfishing of vulnerable populations in the northern Indian Ocean. The findings provide a strong case for Sri Lanka and surrounding nations to improve management measures for these species in order to mitigate further declines.

I commend the authors on the amount of work and dedication that has gone into data collection for this study. The statistical methods used are sound and thorough and I cannot find fault with them. I do, however, find that the introduction and discussion sections are quite long and could be improved by reducing the amount of text and re-focusing to be more concise.

I therefore recommend minor revisions of the manuscript prior to acceptance. Please also see my specific comments below.

Line 76-77. Suggest removing “at present” to improve sentence flow. It is also unclear which fisheries are reliant on small-scale and artisanal fisheries, please re-word for clarity.

Line 106-107. “Studies on the oceanic manta ray (M. birostris) have also expanded
in recent years, resulting in important baseline data on movements and feeding ecology” please include the relevant citations here.

Line 165. Please include the definition for single-day and multi-day vessels here. I see this is explained in more detail further on in the manuscript but it would be useful here upon first mention.

Line 317. In which program/package did you implement the Bayesian Linear Regression? Please include this here.

Line 567. change ‘examination’ to ‘examinations’

Line 568. “. and” - full stop in the middle of the sentence. I also suggest reviewing the term “bycatch fishery”, perhaps change to “…provide an understanding of the scale of bycatch from this fishery/fisheries” or similar.

Line 612-613. With regard to the birostris catch numbers in Indonesia, over how long a time period were these numbers reported? Please add this.

Line 720. de Vos et al - is this the correct citation here? In fact, the citations in this paragraph seem to be mixed up (line 718-727), please review.

Line 727. Rohner et al 2017: there is no reference to match this is the reference list, is this supposed to be Stewart et al 2017?

Line 756-758. this information is a repeat from the introduction, consider re-wording to avoid repetition.


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

Input:
Title: HIGH BYCATCH RATES OF MANTA AND DEVIL RAYS IN THE “SMALL-SCALE” ARTISANAL FISHERIES OF SRI LANKA
Review round: 1
Reviewer: 2

Output:
Basic reporting: Meets basic reporting criteria.
Experimental design: Meets experimental design criteria.
Validity of the findings: Meets validity of findings criteria.
Additional comments: The Authors present a comprehensive paper on the mobulid fisheries in Sri Lankan artisanal fisheries. The findings are novel, well-presented and alarming. The methods employed and results obtained clearly indicate an unsustainable level of take of these species, albeit limited data for M. kuhlii and M. thurstoni. It also interesting that no M. alfredi were recorded given the wide-ranging nature of the fishers and their well documented occurrence in the general area (e.g. Maldives, Seychelles, Chagos). M. kuhlii's inferred coastal habitat use (like M. alfredi's) probably accounts for the small landings (or none) of these species. I wonder if for the state-space models it'd be worth focusing on pelagic-only fishery data, and on the three species with the larger sample sizes? This might remove some biases from the models, as some of the numbers seem a bit extreme. I am not 100% familiar with the methods employed, but the extrapolated numbers seem large.

The paper is robust and the fishery numbers are indicative of pervasive lack of enforcement and management for these species. This is difficult to implement, especially in cases where decision-makers and managers are often politically appointed and have political agendas to fulfil. I commend the authors for the work, and would encourage that this paper is presented to said managers and decision-makers in a simplified form that highlights the ongoing level of take and known unsustainable fishing trends. Perhaps the authors can add to the discussion, if there is, a more 'absolute' approach to indicate the level of take or the N value for the population at large. This would help get a better perspective on what the fishery numbers mean in terms of sustainability and conservation. Possibly SNPs or other genomic tools to indicate adult females at large within the subpopulation (n, fished), especially given their 1-2 pup strategy.

Some specific minor comments below:
Abstract
Line 20-21: consider not using ‘such as’ twice in the same sentence

Introduction
Line 44-45: use a ‘review’ reference here, or a few e.g. references
Line 67-68: if ‘significant’ give numbers. Can use considerable, and would still use a number (e.g. 50%).
Line 113: mobulid specific? State if so.
Line 115: ‘these’ species

Discussion
Line 568: punctuation typo here
Line 647: remove ‘with the exception of M. birostris’ as not being addressed here
Line 653: give species examples e.g. 10% size reduction for Carcharhinus falciformis etc
Line 724: >300 m

Figures
Table 1: consider this as a Supplementary Table as this is discussed/informed in the main text already (not specific numbers, but clear nonetheless)
Table 6: need maturity in the title “Size at maturity (DW) at which 50% of males…” or similar. Consider single decimal unit for consistency in the table.
Fig 1. Any reason no coverage in the southeast? Just curious.
Fig. 7. N values here would be good, perhaps in brackets after species name


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: NO ADVERSE EFFECT OF A MATERNAL HIGH CARBOHYDRATE DIET ON THEIR OFFSPRING, IN RAINBOW TROUT (ONCORHYNCHUS MYKISS)
Review round: 1
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: no comments
Validity of the findings: no comment
Additional comments: In manuscript entitled ’ No adverse effect of a maternal high carbohydrate
diet on their offspring, in rainbow trout (Oncorhynchus mykiss)’, the authors investigated the long-term effect of parental high carbohydrate-low protein dietary exposure on the outcomes of their offspring using a carnivorous fish model. The research topic is quite interesting, and it is surprised to see that such ‘metabolic programming effects’ are very different compared to those in mammals. In general, the experimental design and statistical method are appropriate, results can support the conclusion, the manuscript is well organized and the text is well written and self-explanatory. I only have a few comments for the authors.
1.	In terms of the diet composition, the No carbohydrate (NC) diet was used as the control diet. However, actually NC might not be appropriate as a ‘real’ control diet. Because even in the commercial diet there should contain a certain percentage of carbohydrates. And the carbohydrate requirements during gametogenesis or reproduction cycle might be different. The authors may need to clarify this.
2.	The authors discussed some sex different effects in male and female offspring and put some metabolic markers in supplemental data. How about the growth parameter? Is there any sex-dependent effect?
3.	Line98-Line104, please provide the full name of abbreviations (like SGR, FE…) in the formula. For the survival index, should it be Survival rate (SR)?
4.	In Figure 1, please keep the diet name constant in the black box. In the ‘fecondation’ phase, the diet name should also be "HC/LP". And ‘fecondation’ is probably not an English word, please revise it to ‘fertilization’.
5.	In Figure 2B, please delete the "in" before the unit in the brackets.
6.	In Figure 2C, please provide the unit of "K" in the Y-axis.
7.	Line 232, please replace the word "that" with "than".


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: NO ADVERSE EFFECT OF A MATERNAL HIGH CARBOHYDRATE DIET ON THEIR OFFSPRING, IN RAINBOW TROUT (ONCORHYNCHUS MYKISS)
Review round: 1
Reviewer: 2

Output:
Basic reporting: The basic reporting is mostly fine. Here are a few instances where the English language needs to be corrected slightly:

line 36: change to "able to grow"

line 81: change "was" to "were"

line 189: change "choose" to "chosen"

line 221: change "was" to "were".

line 304: change to "compromise"
Experimental design: It is unfortunate that some unexpected problems with the water caused one NH treatment tank to be compromised, as described lines 89-90. Overall, there has been an effect on mortality as described line 90, but there may be potential effects on other variables as well that will affect the mean of the whole treatment in a way that other treatments were not affected (e.g. maybe a stress event like that would have long lasting effects on condition factor of survivors? etc.). I think there could be more discussion about other potential impacts from this compromise of the water, to reassure the reader that any effects seen in the data involving the NH treatment really were caused by parental dietary differences, and not the compromised water.
Validity of the findings: line 23 and 351: is this range actually provided somewhere? The only reference to the normal range of a phenotype variable I could find was about plasmatic concentrations and hepatic free glucose content on line 293-294. I think it would be useful to add some more references and discussion to show the reader that the many other variables (e.g. cholesterol, lipids etc.) for offspring of the fish fed more carbohydrates are also within the normal range for salmonids, in order to make sure the findings mentioned in the abstract (line 23) and discussion (line 351) are valid.
Additional comments: line 81-82: please provide a reference for this statement

line 215: I think the first "HH" here is meant to be "NN". Also on line 216 I think "HH" is meant to be "NN". Please double check all other times the combinations of "H" and "N" acronyms have been used to make sure there are no other errors like this.

line 251: It is being repeatedly mentioned how there have been important findings about this in mammals, but the difference between mammals and fish make this comparison possibly have limited usefulness. It should be discussed somewhere how species with internal development should be affected much more than species that broadcast spawn.

line 257-262: this section is confusing. This seems to be the first mention of a Saprolegnia infection, I think this should be clearly mentioned earlier, e.g. the methods or results. Also, was this infection only in one treatment/tank, or spread evenly among the treatments? If it was spread evenly, then it shouldn't affect the experimental comparisons. It may, however, affect the inferences possible from the study (i.e. the results are only applicable to trout that have undergone such an infection, and different results may occur in an aquaculture system free from this infection). There should also be discussion about this possibility.

line 305-306: which particular stress are you talking about here?

line 322: it seems an interesting result that if you reduce the protein content of the diet of the parents, then this will cause a relative increase in tissue protein content of the offspring, and this result I think may be of significance for the value of the aquaculture product. Maybe there should be some more discussion about the potential reasons and implications for this result? The abstract is relatively short and could probably be expanded with some more details of specific results; maybe a potentially important result such as this could be one such addition to the abstract.

line 337: I guess it means here that if the mother and father are both from a given high/low protein treatment, then effects will be greater than if only one was. This could be made more clear in this sentence.