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: CLINICAL OUTCOME OF ENDONASAL ENDOSCOPIC PRELACRIMAL APPROACH IN MANAGING DIFFERENT MAXILLARY PATHOLOGIES
Review round: 1
Reviewer: 2

Output:
Basic reporting: This is an interesting study on 21 patients submitted to endoscopic prelacrimal recess approach to treat different pathologies of the maxillary sinus. Post-treatment quality-of-life has been properly analyzed together with general outcomes of surgery.
The manuscript should be revised by an English native speaker in order to improve grammar, spelling (e.g. "infratempora" instead of "infratemporal", line 111) and punctuation.
Experimental design: The study is well conducted. Minor suggestions to improve the manuscript are:
1) The CT-based measurements of the prelacrimal recess (AP diameter and height) should be correlated to the post-operative sequelae encountered, especially those related to sensory nerves injury (transient dental/cheek numbness). A possibile result might be an inversely proportional relationship between the dimension of the prelacrimal recess and the risk to have post-operative neural sequelae. You can retrive information from: Simmen D, et al. Anterior maxillary wall and lacrimal duct relationship - CT analysis for prelacrimal access to the maxillary sinus. Rhinology. 2017 Jun 1;55(2):170-174
2) The surgical technique description may be improved in view of a recently-published technical note where the relation between the mucosal incision and the maxillary osteotomy has been described (Preti A, et al. A Surgical Variant of the Pre-Lacrimal Approach to the Maxillary Sinus. Iran J Otorhinolaryngol. 2019 Sep;31(106):327-328). Following this advice, the risk of post-operative residual inferior meatotomy can be minimized.
Validity of the findings: The paper is well written and the conclusions reasonable. The analysis of post-operative outcomes of endoscopic prelacrimal approach is innovative.
Additional comments: The manuscript is really interesting but should be improved following the suggestions raised in the review process.


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: CLINICAL OUTCOME OF ENDONASAL ENDOSCOPIC PRELACRIMAL APPROACH IN MANAGING DIFFERENT MAXILLARY PATHOLOGIES
Review round: 1
Reviewer: 3

Output:
Basic reporting: Many spelling and grammatical errors strewn throughout the document
Experimental design: From initial reading - it is unclear what the study design is. Only after further reading, can one ascertain that it is a prospective study. It would be good if the authors could clearly define this at the beginning of the article.

Measuring pre-lacrimal complex parameters has no benefit in this study, as the results were not used in a meaningful manner in the article.
Validity of the findings: The study is flawed for the lack of a control group. One might argue that any patient undergoing surgery for the pathologies listed would have improved VAS and SNOT-22 scores, regardless of whether the endoscopic pre-lacrimal approach was used. In fact, if there was a control group, it would very likely show that the control group would have similar VAS and SNOT-22 scores.

It was stated that other surgeries such as surgery for a deviated nasal septum was also performed, if necessary, on patients in the study group. This could counfound the improved VAS and SNOT-22 scores. It would be useful if these patients were identified in the cohort. Furthermore, patients may have had other sinuses, including the maxillary sinus operated on, which may also have affected VAS and SNOT-22 scores. This was not clearly stated in the article.

Follow-up care is vague. There was mention of nasal saline irrigation, but it is unclear if any other medical interventions were utilized. Patients with concurrent allergic rhinitis, for example, may have been started on intranasal corticosteroids which by itself, may lead to improved VAS and SNOT-22 scores
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: CLINICAL OUTCOME OF ENDONASAL ENDOSCOPIC PRELACRIMAL APPROACH IN MANAGING DIFFERENT MAXILLARY PATHOLOGIES
Review round: 2
Reviewer: 1

Output:
Basic reporting: good
Experimental design: good
Validity of the findings: good
Additional comments: The article is much improved and the authors well answered the question raised by the reviews.
Only minor issues should be corrected as below:

p.11, line 114-115:　＂1.0-cm slices in the axial view＂should be 1.0-mm slices

p.16, line 238-239: “This study found that EPLA-treated patients reported improved VAS ratings SNOT-22 scores over time, with mean improvements of 4.2 and 17.6 points, respectively.” This sentence is unclear, respectively what?

p.17, line 263:  for full-filed surveillance should be "full-filled"


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: CLINICAL OUTCOME OF ENDONASAL ENDOSCOPIC PRELACRIMAL APPROACH IN MANAGING DIFFERENT MAXILLARY PATHOLOGIES
Review round: 2
Reviewer: 2

Output:
Basic reporting: The Authors have fully addressed all the issues raised in the revision process and the paper is now suitable for publication.
Experimental design: The study design is correct.
Validity of the findings: The results are reasonable.
Additional comments: The paper has been considerably improved and it is now suitable 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: IDENTIFICATION OF A GENOME-SPECIFIC REPETITIVE ELEMENT IN THE GOSSYPIUM D GENOME
Review round: 1
Reviewer: 1

Output:
Basic reporting: No comments.
Experimental design: 1 The Authors of the paper in the section «Material and methods» mention «in-house» Perl scripts, but do not provide links to the web resource where they can be downloaded. It is also necessary to provide a list of scripts and for each of them a brief description of what they are for.
2. Versions of genome sequences used in the work should be specified.
3. Versions of software used in the work should be specified.
4. It is not clear what the authors meant by the sentence «The insertions of repetitive sequences in genomes were illustrated by R language» in the 2.2 section of the paper. Did they mean that they wrote a script for this in R or something else?
Validity of the findings: No comments.
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: IDENTIFICATION OF A GENOME-SPECIFIC REPETITIVE ELEMENT IN THE GOSSYPIUM D GENOME
Review round: 1
Reviewer: 2

Output:
Basic reporting: The manuscript is clear and concise, I have no questions on its context, aim and conclusions.
There are few minor points to change
e.g. lines 47-48 - "The genome-specific TE is an efficient approach to study species formation and genome evolution in genome comparative research" - looks like something ("investigation of" or "analysis of") is missing after "The".
line 97 - please correct reference to Ugene, this not Edgar 2004
line 120 - this should probably be "does not exist"
Regarding data sharing, the authors use custom scripts and accoding to PeerJ guidelines they should be made available, either as supplement or submitted to a public repository.
Experimental design: no comment
Validity of the findings: My major concern is that the authors use old version of genome assemblies of G. barbadense and G. hirsutum. In the recently published versions: https://www.nature.com/articles/s41588-019-0371-5 the assembly and annotation of repetitive regions is highly improved. I recommend to re-run the analysis concerning tetraploid cottons using these new assemblies.
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: IDENTIFICATION OF A GENOME-SPECIFIC REPETITIVE ELEMENT IN THE GOSSYPIUM D GENOME
Review round: 2
Reviewer: 1

Output:
Basic reporting: I don't have any additional comments.
Experimental design: All issues mentioned in my previous review were corrected.
Validity of the findings: I don't have any additional comments.
Additional comments: I don't have any 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: IDENTIFICATION OF A GENOME-SPECIFIC REPETITIVE ELEMENT IN THE GOSSYPIUM D GENOME
Review round: 2
Reviewer: 2

Output:
Basic reporting: I found one typo in the species name: lane 90 - change G. arboretum to G. arboreum
Experimental design: no comments
Validity of the findings: no comments
Additional comments: All required revisions are made.


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: OSMOTROPHIC GLUCOSE AND LEUCINE ASSIMILATION AND ITS IMPACT ON EPA AND DHA CONTENT IN ALGAE
Review round: 1
Reviewer: 1

Output:
Basic reporting: •	This is an interesting manuscript on how osmotrophic growth affects fatty acid composition of microalgae. This is of interest to scientific readers from various backgrounds, including ecology and biotechnology and as such, I recommend it for publication following amendments and clarification.
•	The text is generally well written, however some improvement on the language and sentence structure will help to ensure the points are delivered in a more concise and structured manner.
•	The introduction was very good; however, a mention towards the role of algal lipids in the food webs was introduced, however not re-visited in the discussion. I believe a paragraph in the discussion on what the possible implications of this research are on aquatic food webs will enhance your impact. For example, if there is enhanced run-off (either due to climate change, or seasonality) leading to higher carbon / amino acids in the water, this might lead to a reduction in total fatty acids of certain algae species (e.g. in this study) and therefore reduced transfer along the food web.
•	The figures are relevant to the manuscript and illustrate the points made. For figure 1 I would like it if the authors highlighted what the isotopic difference is relative to (I am guessing the internal standards) to make this clear.
•	In Table 2 I am unsure why the authors have used different letters to denote significant differences, and then do not explain what each means. For instance a, ab, b, c, bc, abc (as in table 1, I would stick with using an asterisk to denote significance, and you could use ** or *** to denote higher significance), but I would also add to the figure legend what the letters or number of asterisks means.
Experimental design: The research questions within this manuscript have been well defined, and I believe it fills an important knowledge gap.
Specific comments regarding the methods section are included below.
•	Line 100; authors used Peridinium sp. from their own collection, but it would be useful to have information on where this organism was collected from, when etc. This allows comparison with other literature
•	Line 108; the authors used centrifugation to collect the algae, do they know if this had any impact on algal physiology? With some strains, we sometimes find that centrifugation can lead to low recovery.
•	Line 115; I would just specify here that for the short-term experiments it was; “under a constant light intensity of…”
•	Line 154; it is specified that the algal strains were grown for 15-16 days, however no mention is made to what phase of the growth cycle they were in when the cells were harvested. If it is exponential or stationary this can vastly affect the fatty acid profiles (see for isntance Boelen et al., 2017, Growth phase significantly decreases the DHA-to-EPA ratio in marine microalgae, Aquaculture International, 25 (2)). It would be nice to see (even in supplementary) the growth curves for the algae used in the long-term osmotrophy experiment. This could also be one reason why we see such different profiles for M. kalinae as algae tend to accumulate FAs in stationary phase. Similarly, what time of the light: dark cycle were the algae harvested at, as this can also affect FA profiles.
•	Line 158; The equation is given but not what the terms mean. I would just add a line saying “..using the equation 1, where Tx is the number of elapsed days during the experiment and cellsTx the number of cells, whereas T0 is at the start of the experiment etc..” just to ensure clarity to the reader.
•	Line 160; it is mentioned see the method in chapter 2.4, and in this paper chapters aren’t used, so I would change this to say “see section above” or something similar.
Validity of the findings: The data provides an interesting look at how different algal strains react to changes in glucose and leucine concentrations. This is of interest both for ecologists, as it can affect fatty acids and thus the transfer of energy rich material through the food chain, and also biotechnologists who wish to optimise yield in industrial processes. This paper makes an interesting point that the response of algae is both species and compound-specific, and I think this is an important finding for researchers’ and industry. I have made some suggestions below on some extra discussion points and questions below.
•	Line 192; it is mentioned that fatty acid profiling was conducted on all algae strains during the short-term experiment, however I cannot see this data is displayed anywhere (including the supplementary information).
•	Were duplicate measurements of freeze-dried autotrophic controls conducted? If so I cannot see this data presented.
•	Given some of the variability seen between duplicates, I would have recommended triplicate measurements of the samples (e.g. total FAs for Cryptomonas sp. at 0.5 glucose was 64.4 and 41.2 µg in mg DW). If it is possible to run triplicates based on preserved material this would be great.
•	If any of the algae were in stationary phase when harvesting, then this should be mentioned in a discussion section as a reason for any differences seen in lipid profiles.
•	Line 268; can the authors provide any hypothetical reasons for why some growth enhancement was seen during some experiments for M. kalinae and C. ozolinii? For instance possible enhanced bacterial growth at concentrations used leading to either enhanced mutualistic interactions, or possible phagotrophy of the bacteria themselves (which has been observed for cryptophytes – see Yoo et al., 2017. Mixotrophy in the marine red-tide cryptophyte Teleaulax amphioxeia and ingestion and grazing impact of cryptophytes on natural populations of bacteria in Korean coastal waters, Harmful Algae, 68, 105-117).
•	Line 293; The first line of the conclusion says that osmotrophic nutrition was wide-spread and found in different types of algae, however in this study only 3 of the 7 strains tested displayed this mode of nutrition. This disagrees with line 234-235 of the discussion and hypothesis 1.
•	The glucose concentrations used within this experiment seem very high for typical DOC concentrations, however I am not sure if this is true for freshwater systems. It would be useful if the authors in the discussion could try to contextualise the concentrations used to natural systems (e.g. is 5 mg L-1 what would be observed during highly eutrophic conditions with high runoff, or is it an extreme situation?).
•	Are any of the algae strains used within this study of interest for biotechnological applications? For instance Cryptomonas sp, and can any recommendations for these processes be made (even if it is just to try other organic compounds or repeat with different concentrations etc..).
•	It can be interesting to conduct some multivariate statistics which allow visualisation of the differences between algal strains and treatments. For instance, a principle component analysis using the lipid groups analysed within this study. I had a quick attempt at this using the data provided within Table 2 of the manuscript, and attach a file with the PCA plot output with my review as a pdf (I am happy to share the code if required also). I think this nicely shows that the Cryptomonas sp. is very different to the other 2 algae (although all are dissimilar in some way), but they also show the widest response with glucose and leucine treatment (as shown in the PERMANOVA also). It is possible to then take the principle components PC1 and PC2 and correlate them against the fatty acids to try and determine which direction the relationship is displaying in the PCA.
Additional comments: I would like to congratulate the authors on an interesting piece of work. I believe that it provides interesting knowledge on how algae respond to carbon and amino acid enrichment and on osmotrophic growth. There are a few methodological clarifications which I would like to see before publication (especially concerning the growth stage the algae were in during the long-term experiment), but I believe this paper contributes to this field. As mentioned in the review, I think this is of interest to ecologists and biotechnology researchers’ and will therefore be well received.


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: OSMOTROPHIC GLUCOSE AND LEUCINE ASSIMILATION AND ITS IMPACT ON EPA AND DHA CONTENT IN ALGAE
Review round: 1
Reviewer: 2

Output:
Basic reporting: Generally this is a well written manuscript, but English expression requires some attention. Some points are mentioned below but this list is not exhaustive.

line 107: stable not stabile
line 108: resuspended not dissolved
line 119: 'supernatants were' not 'was'
line 127: weighed not weighted
line 160: presumably this manuscript is derived from a thesis but this reference to a chapter should be removed
References: a number of references have genus/species names that are not italicised
Experimental design: With any work on osmotrophy it is important to ensure that the  organic substrates are truly taken up by the algae themselves and rule out the possibility that bacterial breakdown of organic matter is driving the changes observed. Were the cultures axenic and how was this checked?

line 113 and line 149: It should be specified that these were independent replicate cultures, not technical replicates from a single culture (pseudoreplication)
Validity of the findings: Lines 69 -72: I am not especially convinced that buildup of triacyl glycerols (storage lipid) necessarily leads to higher levels of PUFAs. Most LC-PUFAs are found in membranes (Roessler PG (1990) Environmental control of glycerolipid metabolism in microalgae: commercial implications and future research directions. J Phycol 26:393–399) though this can change in stationary phase when LC-PUFAs can be mobilized from membranes into TAGs - see Boelen et al 2017 (Boelen, P., van Mastrigt, A., van de Bovenkamp, H.H. et al. Aquacult Int (2017) 25: 577. https://doi.org/10.1007/s10499-016-0053-6). The latter point is important in view of the long term experiment results and needs to be incorporated into the discussion
Additional comments: Overall this is an interesting study but the manuscript requires further attention before it could be considered suitable 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: A TRANSCRIPTOMIC STUDY OF PROBENECID ON INJURED SPINAL CORDS IN MICE
Review round: 1
Reviewer: 1

Output:
Basic reporting: In this work Yu-Xin Zhang and co-workers investigate the effect of probenecid in local gene expression, upon spinal cord injury in mice. Probenecid has been shown to have a neuroprotective effect in some brain disorders; however, its role in modulating the transcriptional response upon spinal cord injury (SCI) had not been addressed yet. To answer this question the authors performed an RNA-seq analysis in spinal cord samples derived from un-lesioned and lesioned mice, treated or not with 0.5 mg/kg of probenecid immediately after injury. Samples were harvested  8 hours after the lesion,  to investigate primary changes in gene expression.

The paper is self-contained and addresses a well-defined question with the appropriate methodology.  Relevant literature is cited and the results obtained are properly discussed within the state-of-the-art in the field. Maybe some more information could be extracted (and discussed) from the data (see point 4 below).

Raw data are available in a public repository as requested
Experimental design: The authors address a relevant, well-defined question that fills an important gap of knowledge regarding the transcriptional response observed during central nervous system injuries. Overall, the work is technically well done, however, several experimental and reporting caveats should be addressed before publication:

1)  The number of biological replicates for RNA-seq experiments on animals is very low (n=3). This is also reflected in the PCA analysis, where probenecid samples do not cluster together and they do not separate well from control-treated samples. The authors should consider increasing the number of treated animals to improve differential gene expression analysis and data consistency.

2) Statistical analysis for the qRT-PCR experiments in figure 3 is missing. Was the analysis performed in independent samples or in the same samples used for the RNA-seq analysis? Are the differences observed statistically significant? This info needs to be provided prior to publication. Figure legend should be also re-written to improve clarity.

3) GO and KEGG analysis on figures 5, 6 and 7 should include only enriched categories that are statistically significant. Many of the pathways represented in the figures are not significative and therefore should be eliminated to avoid confusion and to strengthen the manuscript conclusions.

4) In figure 4 the authors describe the hierarchical clusterization of the RNA-seq data. However, they do not give any indication on the genes (or gene categories) within each of these clusters. For instance, there is a cluster that is strongly downregulated in SCI_control versus SCI_sham, and upregulated again upon probenecid treatment. Are those genes related to neuroprotection? Such information is likely to provide important clues into the mechanism of action of probenecid and should be addressed in the manuscript. In addition, it is not clear the criteria used for the clusterization. Does the analysis include all DEG between the three conditions? Or is it based in the DEGs with  the lowest p  value? The authors should clarify these aspects.

5) In general, the description of the experimental approach (in particular regarding the bioinformatics analysis) and figures labelling and legends do not give sufficient detail, making it difficult to properly interpret the results at several points.
Validity of the findings: This manuscript provides a relevant dataset that could be used in future work to elucidate the mechanism of action of probenecid in SCI models. In addition they are properly discussed, with relevant references to available literature and no “over-stating” conclusions.
Additional comments: No additional comments


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

Input:
Title: A TRANSCRIPTOMIC STUDY OF PROBENECID ON INJURED SPINAL CORDS IN MICE
Review round: 1
Reviewer: 2

Output:
Basic reporting: Some typos in verbs and syntax improvements possible and the quality of the figures could be improved. Some of them are illegible (for instance Fig. 5, Fig. 6 and Fig. 7). Raw data are supplied.
Experimental design: Although the research question is well defined and it is of potential interest, the conclusions remain elusive. The effect of the drug used by the authors (probenecid) on the general gene expression is convincing, but if the drug exerts neuroprotective effects on the injured spinal cord and through which genes and/or signaling pathway/s are not investigated at all.
Anyway, methods are well-described and present sufficient details.
Validity of the findings: The conclusions are not supported by the results, but may only represent speculations. Although the research question of the authors is interesting, the manuscript in its current form does not have sufficient novelty to be published.
Additional comments: In the present manuscript Zhang and colleagues evaluated the effect of the probenecid on general gene expression in spinal cords following acute spinal cord injury (SCI). Briefly, the authors performed an RNA Seq analysis in control and SCI mice with or not the probenecid treatment.
They identified 641 differentially expressed genes in probenecid-treated SCI mice compared to vehicle-treated SCI mice, of which 286 were up-regulated and 355 were down-regulated. To validate the genome wide analysis, the authors evaluated the relative expression level of only nine genes, which represent only less than 2%. Furthermore the analysis lacks of a statistical analysis.
Finally the authors performed a Go and KEGG enrichment analysis to highlight the functional categories and signaling pathway/s, respectively, to which differentially expressed genes belong. The down-regulated genes in probenecid-treated SCI mice clustered in three GO terms (protein binding, binding and sequence-specific DNA binding), although none of the up-regulated genes was significantly enriched in Go terms. Some of the genes, resulting differentially regulated by probenecid treatment in SCI mice, belonged to some SCI-related signaling pathways (CAMs, ECM-receptor interaction, PI3K-Akt signaling pathway and Focal adhesion), as evidenced by KEGG enrichment analysis.
Although these data are of potential interest, Go and KEGG enrichment analysis can represent only the initial step of a work to better address the experiments that must be done to support and validate a scientific hypothesis, but certainly not the conclusion of a scientific report.
Furthermore, the experiments performed in this study are very preliminary and the most of conclusions of the authors represent only possible hypotheses. Moreover, an important issue that the authors did not consider is whether the treatment of probenecid in their system had “neuroprotective and repairing effects” in injured spinal cord. This point, in my opinion, represents the major weakness of the manuscript.
The manuscript is really poorly written and makes it difficult to understand the text in some points. For all these reason I believe that this manuscript should not be committed for the publication in Peer Journal.


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

Input:
Title: A TRANSCRIPTOMIC STUDY OF PROBENECID ON INJURED SPINAL CORDS IN MICE
Review round: 1
Reviewer: 3

Output:
Basic reporting: I suggest a small improvement in background citations (see comments section)
Experimental design: Spinal Cord Injury (SCI) is a devastating clinical condition resulting mainly from traumatic damage that
leads to neuronal cell death and concomitantly, different degrees of motor function loss and sensitivity.
Neuroprotective treatment is one of the promising strategies to alleviate SCI symptom and increase the successful of others pharmacological or rehabilitating strategies. Probenecid is a urate transporter-1 (URAT-1) inhibitor, that has been recently demonstrate to exert a neuroprotective and repairing effects in some brain disorder.
In this manuscript Zhang and colleagues analyze and make available an RNA-seq dataset of spinal cord tissue derived from control, SCI and Probenecid treated SCI mice.
The manuscript is clear and well written and the raw sequence data have been deposited into Sequence Read Archive. Zhang and colleagues analyzed 3 replicates for each experimental point (CTR, SCI and SCI+ Probenecid), using a classical RNA-seq approach.  The experimental methods are clear and well explained with sufficient amount of information.
Validity of the findings: A basic but well performed analysis of SCI and Probenecid treated SCI compare to control tissue was presented. The conclusion of the manuscript is supported by the performed analyses. Although the authors have not entered in deep into the merits of the mechanism of action of the drug, I believe that the published dataset is an excellent starting point for other studies and a valid tool for designing new pharmacological strategies in the treatment of spinal contusion. Indeed, authors identify macrophages and more in general the inflammatory response as a primary target of Probenecid treatment, speculating that this drug can exert some beneficial effects in SCI therapy.
Additional comments: From my point of view, I think that the publication in this journal is appropriate.
Nevertheless, I would suggest the following minor changes to the authors in order to improve the manuscript:
-Line43: correct “rats” with “mice”
-Line81-90: background can be expanded to other studies involving Probenecid and nerve injury. Some example are listed bilow:
a) Probenecid and N-Acetylcysteine Prevent Loss of Intracellular Glutathione and Inhibit Neuronal Death after Mechanical Stretch Injury In Vitro. Du L, Empey PE, Ji J, Chao H, Kochanek PM, Bayır H, Clark RS. J Neurotrauma. 2016 Oct 15;33(20):1913-1917.

b) Pannexin 1: a novel participant in neuropathic pain signaling in the rat spinal cord. Bravo D, Ibarra P, Retamal J, Pelissier T, Laurido C, Hernandez A, Constandil L. Pain. 2014 Oct;155(10):2108-15.

c) The L-kynurenine-probenecid combination reduces neuropathic pain in rats. Pineda-Farias JB, Pérez-Severiano F, González-Esquivel DF, Barragán-Iglesias P, Bravo-Hernández M, Cervantes-Durán C, Aguilera P, Ríos C, Granados-Soto V. Eur J Pain. 2013 Oct;17(9):1365-73.

-Line: 126-127: specify if a cut-off was used or not for fold change.
-Line146: a comment on the result depicted in the PCA would be useful (distance between CTR and SCI etc ..)
-Line 148: I would change the title of the paragraph with: “The effect of SCI and Probenecid treatment on gene expression”

-Line162/Fig3: I would suggest to shows readcounts replicates in the graph.
-Table 1: there is an error (probably derived from a cut and paste) in the sequence of Itga1 primers (not match)


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

Input:
Title: A TRANSCRIPTOMIC STUDY OF PROBENECID ON INJURED SPINAL CORDS IN MICE
Review round: 2
Reviewer: 1

Output:
Basic reporting: The authors have now address all my concerns. I consider the article suitable for publication, but I suggest it undergoes PeerJ English language editing services to improve the clarity of the message.
Experimental design: See above
Validity of the findings: See above
Additional comments: See 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: A TRANSCRIPTOMIC STUDY OF PROBENECID ON INJURED SPINAL CORDS IN MICE
Review round: 2
Reviewer: 2

Output:
Basic reporting: see comments for the author below
Experimental design: see comments for the author below
Validity of the findings: see comments for the author below
Additional comments: In the revised version of the manuscript the authors have sufficiently improved the quality of the figures and the English language. Anyway, we strongly recommend using the language editing services provided by PeerJ journal.


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: QUESTIONING THE PROVERB ‘MORE HASTE, LESS SPEED’: CLASSIC VERSUS METABARCODING APPROACHES FOR THE DIET STUDY OF A REMOTE ISLAND ENDEMIC GECKO
Review round: 1
Reviewer: 1

Output:
Basic reporting: Your study is important as it provides the first results on the diet of an endemic species. The main focus seems to be on the comparison of the two techniques so there should be a more clear-cut conclusion as why the question in your manuscript title is posed and what are your views on it. The discussion could include a rational on when to use one technique or the other or on how to use them complementary. I would also like to see some more discussion of the ecological significance of the results.
Other studies have identified plant material in lizard faecal pellets using a morphological technique (e.g. Pietruzka et al., 1986; Rodríguez et al., 2008; Ibrahim, 2004). What do you think is the reason for you not having found plant material in the faecal samples using the classical approach, despite their being an important component of Selvagens gecko diet?
Sadek (1981) conducted a diet study on a different lizard species in Selvagens islands and found considerable amounts of plant material in the stomachs he analysed. Maybe you could quote this study and discuss ecological interactions and potential resource competition between the two species?
Another study that could be worthwhile quoting is Matias et al., 2009, who also found that the Madeiran wall lizard in Selvagens is able to prey on seabirds.
The English language could be improved throughout the manuscript. Some sentences are too long and a bit confusing. Some examples where the language could be improved include lines 7 & 8, 51 & 52, 95 to 101, 286 to 288.
When quoting references throughout the manuscript please follow PeerJ instructions and add a “,” between the author and the year. For example, in line 3 write (Whittaker et al., 2017), instead of (Whittaker et al. 2017).

ABSTRACT
“Reptiles are often neglected in islands systems” -  Should be “island” (singular not plural)
“remote and integral reserves of Selvagens Archipelago “ – Should be “reserve” (singular not plural)
“revealed that this species is a generalist eater” – consider replacing “generalist eater” by “generalist feeder”.
“The traditional method of morphological identification of prey remains in faecal pellets collected over a long period was compared with” – Instead of saying “over a long period” I would say “over a longer period”. That is a longer period than the one day sampling for NGS, but not really an extended period covering the entire year/seasons spectrum.
“feeding on invertebrate, plant and even vertebrate items” – please, remove the word “even”. It is not unusual that lizards feed on vertebrates.
 “Results of this study are useful to highlight the applicability of rapid surveys on remote islands of difficult access around the world.“ – not really new, the same could be concluded from the results of Pinho et al., 2018.
The abstract is too general; you should include some more of your results, namely the dietary shift between late Spring and end of Summer. Please also discuss the ecological significance of your results and provide some feedback on the question in the title and what you consider the best approach for a future dietary study.
INTRODUCTION
Lines 5 to 10 – All the three sentences begin with the word “These”. Please try to make the writing more fluid.
Lines 11 to 13 – Diet studies are not only essential as a one-off experiment for unstudied species. Diet studies are also especially important as a line of research in the long run and over several years to allow for comparisons and detect environmental changes.
Line 14 – Please follow PEERJ instructions for quoting an article with three authors. It should be (Brock, Donihue & Pafilis, 2014), instead of (Brock et al. 2014).
Line 22 - Please follow PEERJ instructions for quoting an article with three authors.
Lines 31 and 32 - Few studies were performed using next-generation sequencing (NGS) techniques to assess the diet of reptiles, but none (to our best knowledge) on Tarentola geckos (Seguro 2017).

This statement is incorrect. Not only have Tarentola species diets been studied by NGS, but you include the study in your references list – Pinho et al., 2018. Please remove the sentence and make sure to quote Pinho et al. 2018 in the Introduction.
Lines 37 and 38 – Metabarcoding can be advantageous for diet studies, mainly for insular species from remote areas, as with less effort and time it is possible to obtain a large amount of data.
I don´t agree, metabarcoding it is useful for a broad-spectrum of study sites, not mainly in remote islands. Additionally, if you are comparing effort and time of different methods, you should also include cost in the equation.

Line 41 – You have referred to the advantages of metbarcoding for studying diet, but please also add some references to the limitations of the method.
Line 43 – Please add the word “located” before “about 250 km”
Line 44 and 45 – You could add a reference to the figure, where the name of the three islets that hold Selvagens gecko populations can be seen, e.g. add (see Fig 1)

Line 55 – As suggested for the abstract I would rather read “over a longer period of time” than “over a long period of time”.
Line 51 to 57 – Confusing at the moment, the research question and aim need to be more clearly defined. The most important aspect of the study is not to assess the applicability of NGS methods in remote areas and this is not a good punch line for the introduction. This has been done extensively for several taxa, such as seagrass, birds, mammals and reptiles. In my view the most important aspect is that you are characterising for the first time the diet of an endemic species. You should also describe that you prepared a reference collection (DNA and morphological), as this is an important part of the study and has no mention in the aims. Did you have any intention of using the data collected with the pitfall traps to infer (or at least speculate on) food availability in the two seasons studied (Spring and Late Summer)? If so you should state in the in aims.

Maybe you could list your funding sources after the acknowledgments?
Table 1 - Please format the alignment of the values in column 2 (under 9/10).
Table 2 – Please include the English name - Selvagens guecko - in the legend of the table.
Figure 2 – Title is very basic, please expand.
Supplemental material
Please include the titles for Tables S1, S2, S3 and S4.
Table S1 – Taberlet et al. 2007 is missing from the References list. The sequences of primer 12sv5 are reversed. “TTAGATACCCCACTATGC” is for primer R and not F. Similarly, the sequence given for primer F is in fact for primer R.
Table S2 – Please indicate the units for the reagents in the table.
Table S4 - Supplemental excel file – Please correct line 6. Brassicales is an order, not a classe. Brassicaceae is a family, not an order. Lobularia is a genus, not a family.
Thank you for providing the raw data. You don´t include a table with the sequences of the arthropods, plants and vertebrates sampled for the DNA collection. I supposed you have made them available at an online reference collection / GenBank? If so please include the accession numbers in the manuscript. If not, maybe you could include it as Table S5?
Experimental design: The focus of the study is well within the aims and scope of PeerJ.

The research question is relevant and meaningful but could be better defined.

The methods are sufficiently detailed but should be organised in a more rational/coherent form.

MATERIALS and METHODS
Lines 62 & 63 – No need to repeat the years when sampling was conducted. You mention it below in lines 83 and 84, under the “Data collection and analysis”, so please do not have it there too.
Line 67 – Classified as threaten by whom?
Lines 72 and 73 – quote appropriate reference
Line 74 -  remove “for which these islands embody one of the last sanctuaries in the world“

Line 77 – please provide the English name of Teira dugesii
Lines 77 and 78 – You refer to the occurrence of reptiles in a very casual manner. You could state here that one of the species will be the object of your study. Further along in the discussion it would be interesting to see some comparison of the two species.
Lines 81 to 137 – This section needs improvement and a more coherent description. At the moment, it is all mixed, including the description of the two techniques. Lines 110 to 114, describing how and when the samples were collected, should came at the beginning. Only after should you refer to the reference collection justifying why it was needed. And finally, you should describe the methods, one after the other. It would even be better if you split it in different sub-sections, e.g. sampling, morphological analysis, NGS or Metabarcoding and Statistical analysis.
Line 83 and 84 – Simplify, maybe you could say “and late spring of 2011, from 9 to 30 of May.”, instead of “and late spring of just one year, from 9 to 30 of May 2011.”
Line 96 – Should be “tables” instead of “table”
Lines 97 and 124 – Please add the word “gene” after 16S rRNA.
Line 95 to 101 – Sentence too long, please divide in two or three.
Lines 94 and 106 – Please indicate the name of the experts that identified the OTU’s. Or at least list them in the acknowledgements.
Line 105 – should be tables in plural
Line 108 – Please add the word “intron” after (UAA).
Line 111 - Please add the words “the abdomen” after “gently pressing”.
Line 122 – Taberlet et al., 2007 is not in the references list.
Line 135 – Here you should also quote Borges et al., 2008a
Line 139 – Please indicate which software you used for the statistic calculations
Line 152 -  Is there no permit reference # for 2010 and 2011?
Validity of the findings: RESULTS
It would be clearer to have two sub-section on the results, one on the reference collection and one on the gecko diet.
Lines 162 and 163 – Please simplify…For example, say “ In 2017, we collected nine species of plants and two species of vertebrates for the DNA library (Table 1).”, instead of “For plants and vertebrates, we collected in 2017 a total of nine and two species, respectively (Table 1).”

Line 164 – “two sampling seasons”. If you are referring to Spring and Late Summer than just say “the two seasons”. If you are referring to the three years of sampling then say “three sampling periods/years”. The way it is now - “two sampling seasons” - adds unnecessary confusion.
Line 169 – The relative importance in terms of biomass is not so different between September 2010 and May 2011.
Line 193 to 196 –If you only need five or more pellets to characterize the diet using a morphological approach than you can probably do it in one day, while if 27 pellets are not enough to characterize the diet using DNA techniques, you probably need more time. So how does this affect the idea you put forward before that you need a long time period for morphological studies and a short time period for DNA studies?
DISCUSSION
Line 200 – “diet of the endemic” instead of “diet of endemic”
Line 200 to 206 – This would be better in the introduction or in the methods to justify your sampling periods.
Lines 213 to 216 – This sentence is unclear and too long. Please rephrase it.
Line 232 – species should not be in italic
Line 234 and 234, Line 264 – please use the English name instead of the Latin one.
Line 240 – “In fact” implies connection with the previous statement so it shouldn´t be the start of a new paragraph.
Line 243 – The sampling period was one and a half week in 2010, not three weeks.
Line 246 - Please follow PEERJ instructions for quoting an article with three authors.
Line 259 – should be “DNA pooling and sequencing” instead of “DNA pooling, sequencing”
Line 261 – Please follow PEERJ instructions for quoting an article with three authors.
Line 270 – 6 pellets out of the total of 27 pellets analysed is not so much. Please give some more details into why you think secondary consumption is not the main cause for the large proportion of plants you found in the Selvagens gecko diet.
Line 280 – please add English name or sp. in front of Podarcis if you are referring to a group of species.
Line 291 – it should be “their remains” instead of “there remains”
Line 313 - Please follow PEERJ instructions for quoting an article with three authors.
Line 320 – What do you mean by “natural diet”?
Line 323 - Please follow PEERJ instructions for quoting an article with three authors.
Line 347 – By allying do you mean that they should be conducted simultaneously? Explain better what you mean by “allying” in practical terms.
Line 349 – Please replace the word “allied” by connected/linked or coupled to avoid using allying and allied so close together in the text.
Lines 351 and 352 – Are you confident of the importance of plants in the diet?
Line 359 – You suggest DNA sampling in different seasons; are you confident that this will enable you to detect changes in the diet given the limitation in the DNA method you discussed before?
Lines 361 to 364 – Several studies have shown that ants can feed extensively on seabirds. See, for example Boieiro et al., 2018 and Plentovich et al., 2018. How do you know that the seabird items you found in the diet of the Selvagens gecko are not due to secondary consumption? You should at least discuss the possibility.
Boieiro, M., Fagundes, A.I, Gouveia, C., Ramos, J.A. & Menezes, D. (2018) Small but fierce: invasive ants kill Barolo Shearwater (Puffinus lherminieri baroli) nestling in Cima islet (Porto Santo, Madeira Archipelago). AIRO - Portuguese Society for the Study of Birds, 25:44-50.
Plentovich, S., Russell, T. & Fejeran, C. C. 2018. Yellow crazy ants (Anoplolepis gracilipes) reduce numbers and impede development of a burrow-nesting seabird. Biological Invasions 20: 77-86.
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: QUESTIONING THE PROVERB ‘MORE HASTE, LESS SPEED’: CLASSIC VERSUS METABARCODING APPROACHES FOR THE DIET STUDY OF A REMOTE ISLAND ENDEMIC GECKO
Review round: 1
Reviewer: 2

Output:
Basic reporting: The grammar needs to be improved substantially for clarity and readability of the manuscript. I have offered suggestions on this for the Introduction, but this was too time-consuming to do for the entire manuscript.

References are sufficient but more references to recent DNA metabarcoding studies would be welcome. Sufficient background/context was provided.

The article structure could be improved with clearer paragraph breaks and further subdivision of the 'Data collection and analysis' section. Table 2 is very long and difficult to absorb the information. I feel these data could be more easily interpreted as a figure(s).

The authors need to clarify various aspects of their DNA metabarcoding workflow to ensure reliability and reproducibility of their results, namely sample collection, reference database curation, sequencing, bioinformatics, and data accessibility. In particular, I am quite concerned about the lack of detail provided on sequencing (e.g. reagent kit used, number of sequence reads passing each stage of bioinformatic filtering, percentage of sequence reads that were taxonomically assigned), bioinformatic processing, and data accessibility. The raw sequence reads should have been deposited on the NCBI Sequence Read Archive, and bioinformatics/R scripts along with associated data should have been deposited in a data repository (e.g. DRYAD, GitHub with archival on Zenodo). These issues must be addressed to allow the reader to fully assess the authors’ workflow, results and discussion.
Experimental design: I cannot confirm whether the experimental design has been performed to a high technical standard due to details on DNA metabarcoding that were not provided in the manuscript or supplementary information. I do not feel that the methods have been described with sufficient detail that would allow them to be replicated.
Validity of the findings: I cannot confirm whether the underlying data are robust, statistically sound and controlled as these do not appear to have been archived in a data repository. I would like more discussion on how the findings could be incorporated into current/future conservation and management strategies for this gecko species.
Additional comments: General comments

This is an interesting manuscript that uses DNA metabarcoding in conjunction with morphological identification of prey items to assess diet of a remote island endemic gecko. The study nicely shows that DNA metabarcoding can complement and enhance traditional dietary assessments to improve understanding of resource exploitation and trophic adaptation in threatened species.

Metabarcoding revealed the gecko was a generalist, with a broader diet than found previously, and identified more diversity at higher taxonomic resolution than morphological identification of prey remains. The findings should improve conservation and management of this gecko species, although I would like more discussion on how the findings could be incorporated into current/future conservation and management strategies. Nonetheless, the study demonstrates that DNA metabarcoding could be applied to diet of other lizards in continental or island settings. The experimental design, sampling strategy and statistical analyses are largely sound.

However, I have two primary concerns which when resolved would substantially improve the quality of the manuscript. Firstly, the authors need to clarify various aspects of their DNA metabarcoding workflow to ensure reliability and reproducibility of their results, namely sample collection, reference database curation, sequencing, bioinformatics, and data accessibility. In particular, I am quite concerned about the lack of detail provided on sequencing (e.g. reagent kit used, number of sequence reads passing each stage of bioinformatic filtering, percentage of sequence reads that were taxonomically assigned), bioinformatic processing (e.g. OBItools programs and parameters used), and data accessibility. The raw sequence reads should have been deposited on the NCBI Sequence Read Archive, and bioinformatics/R scripts along with associated data should have been deposited in a data repository (e.g. DRYAD, GitHub with archival on Zenodo). These issues must be addressed to allow the reader to fully assess the authors’ workflow, results and discussion.

Furthermore, grammar needs to be improved substantially for clarity and readability of the manuscript. I have offered suggestions on this for the Introduction, but this was too time-consuming to do for the entire manuscript. I have a number of minor comments to help improve the clarity of the manuscript, which I have detailed in the specific comments to the authors below.

Specific comments

Abstract

Line 3: Change ‘islands’ to ‘island’.

Lines 4 & 6: Change ‘on’ to ‘about’.

Line 7: Change ‘supposed to be exclusively insectivorous’ to ‘but they are assumed to be exclusive insectivores’.

Lines 8-10: Change ‘However, using next-generation sequencing (NGS) techniques only one study was performed thus far for this genus and very few for reptiles in general’ to ‘only one study has used next-generation sequencing (NGS) techniques for this genus thus far, and very few NGS studies have been employed for reptiles in general’.

Line 14: Change ‘with metabarcoding methods associated with rapid sampling surveys’ to ‘with metabarcoding of samples collected during rapid surveys’.

Line 15: Remove ‘eater’.

Line 16: Change ‘even though’ to ‘whereas’.

Line 17-20: Change ‘This method identified a higher diversity of dietary items and with high taxonomic resolution. On the other hand, with the traditional method it was possible to calculate relative abundances and biomasses of the ingested arthropods, a parameter that was not possible to measure with metabarcoding’ to ‘Metabarcoding identified a greater diversity of dietary items at high taxonomic resolution, but morphological identification enabled calculation of relative abundances and biomasses of ingested arthropods’.

Line 20-22: Change ‘Results of this study are useful to highlight the applicability of rapid surveys on remote islands of difficult access around the world’ to ‘Results of this study highlight the global applicability of rapid metabarcoding surveys for understudied taxa on remote islands that are difficult to access’.

Main text

Line 3: Change ‘present’ to ‘host’.

Line 8: Change ‘being’ to ‘that are’.

Line 9: The authors should clarify what “These studies” are. Do they mean studies of endemic taxa on remote islands?

Line 12: Change ‘continuing to be’ to ‘and are’, and ‘for which there is’ to ‘with’.

Line 15: Remove ‘the’.

Lines 17-18: Paragraph breaks should be indicated with an indentation or new line here and throughout the rest of the manuscript.

Lines 18-19: Change ‘worldwide, inhabiting mainly warm climate regions,’ to ‘worldwide (mainly in warm climate regions),’. Change ‘including’ to ‘include’.

Lines 22-23: Remove ‘classical’ and commas before ‘based’ and after ‘prey’.

Line 25: Change ‘The former’s diet, in northern Egypt’ to ‘In northern Egypt, the former’s diet’.

Line 26: Remove ‘also being reported’.

Line 28: Change ‘ ground dwelling’ to ‘ground-dwelling’.

Lines 29-30: Change ‘while in the historical centre of Rome, Italy, it is composed mainly of flying arthropods, such as Diptera and adult Lepidoptera’ to ‘but is composed mainly of flying arthropods, such as Diptera and Lepidoptera, in historical Rome, Italy’.

Line 31: Change ‘were’ to ‘have been’.

Line 32: Change ‘but none (to our best knowledge)’ to ‘and none to the best of our knowledge’.

Line 33: Change ‘Metabarcoding’ to ‘DNA metabarcoding’. It is important to distinguish DNA from environmental DNA in the context of metabarcoding.

Line 34: Insert ‘of’ before ‘standardized’.

Line 35: Remove ‘the’ and change ‘general’ to ‘universal’.

Lines 35-36: This is too repetitive of the previous sentence. Change ‘This method is based on the mass-amplification of DNA using general or group-specific primers, followed by the cloning and sequencing of amplicons for individual taxa identification’ to ‘Highly variable DNA regions that enable species-level identification are amplified using universal or group-specific primers which bind to conserved sites across multiple taxa’.

Line 37: Change ‘mainly for insular species from remote areas, as with less effort and time it is possible to obtain a large amount of data’ to ‘especially for insular species from remote areas as less effort and time is required to obtain large datasets’.

Line 38: Change ‘Additionally, in relation to classic methods’ to ‘ Compared with traditional methods,’.

Line 39: Change ‘resolution, and’ to ‘resolution as well as’.

Line 40: Change ‘; ultimately’ to ‘. Ultimately,’.

Lines 43-44: Change ‘, occurring’ to ‘. It occurs’.

Line 45: Change ‘. It is considered’ to ‘, and is considered’.

Line 48: Change ‘with’ to ‘ to’.

Line 49: Insert ‘it’ before ‘separated’.

Line 53: Move ‘in this study’ to come after ‘approaches’.

Lines 55-56: Change ‘with metabarcoding methods associated with rapid sampling surveys’ to ‘with metabarcoding of samples collected during rapid surveys’.

Lines 56-57: Change ‘Results of this study are useful to highlight the applicability of rapid surveys on remote islands of difficult access around the world’ to ‘Results of this study highlight the global applicability of rapid metabarcoding surveys for understudied taxa on remote islands that are difficult to access’.

Line 79: Why is taxa italicised?

Line 86: Why did the number of occasions differ?

Line 87: Please state the percentage of alchohol used, and whether absolute ethanol was used or some other variation.

Line 88: Use of “scattered” seems vague. It would be better to say “randomly selected”, but even so I wonder why the authors did not conduct more systematic trapping transects?

Lines 102-103: The authors should really provide a summary of what species present on the island were represented on GenBank/BOLD, what species were missing, and what species were barcoded in their study.

Line 103: Sanger sequenced where? How were the Sanger sequences validated to ensure they was no sequencing error/misidentification of source DNA?

Lines 104-105: I’m curious as to why the authors chose a saline method instead of a commercial kit, such as the Qiagen DNeasy Blood and Tissue Kit for vertebrate DNA extractions?

Line 116: Stored at what temperature?

Line 118: How long were samples left in the incubator? The standard method of removing ethanol is generally to pour the majority out and leave the pellet to air dry in a fume hood at room temperature.

Lines 123-124: I wonder why the authors did not use COI metabarcoding primer set as well as or instead of 16S rRNA given the amount of invertebrate reference sequences available for the COI region in addition to COI sequences the authors generated themselves.

Line 127: 8ul of blocking primer (10uM) seems excessive – did the authors optimise PCR conditions?

Lines 130-132: What MiSeq reagent kit was used for sequencing? Were COI, 12S and 16S amplicons prepared as one library on a single sequencing run, or were separate sequencing runs performed for each marker? This information should be reported to allow readers to assess whether results were influenced by sequencing depth and coverage.

Line 133: Although OBItools is a standard bioinformatics pipeline to process metabarcoding data, the programs employed across studies vary drastically. I would like the authors to supply more detail on which OBItools programs they used, either in the main text or supplementary information.

Line 134: GenBank is a public reference database – not a method of taxonomic assignment. I suspect that the authors used BLAST for taxonomic assignment. In this case, the BLAST identity (%) and query alignment to reference sequences (%) should be reported.

Line 351: I think the authors need to be careful about inferring ecological networks from their results as they did not actually construct species or trophic networks.

Table 2: It is difficult to compare the morphological and metabarcoding results in table format. A figure (e.g. venn diagram, upset plot, barplot) would be much more intuitive.


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: QUESTIONING THE PROVERB ‘MORE HASTE, LESS SPEED’: CLASSIC VERSUS METABARCODING APPROACHES FOR THE DIET STUDY OF A REMOTE ISLAND ENDEMIC GECKO
Review round: 2
Reviewer: 1

Output:
Basic reporting: Grammar in the Abstract and Introduction is much improved, but some text can be more concise. The use of third person when referring to methods or species should also be minimised and names used for clarity throughout the manuscript. I have highlighted these instances in my specific comments to the authors. There are some outstanding grammar issues within the Methods and Discussion that need to be addressed as detailed in my specific comments.

The additional references are appropriate and the article structure is improved. The inclusion of Figure 2 makes it easier to interpret the dietary data and I accept the retention of Table 2. I am satisfied with the authors’ efforts to clarify various aspects of their DNA metabarcoding workflow to ensure reliability and reproducibility of their results, namely sample collection, reference database curation, sequencing, bioinformatics, and data accessibility.
Experimental design: The reorganisation of the methods section and the extra detail that has been added is sufficient. However, I think justification of primer choice should be provided if in silico analysis was not undertaken. If in silico analysis was performed, these results should be reported.
Validity of the findings: I accept that the authors will deposit their data and code on appropriate data repositories upon acceptance.  The added text to the discussion on future management strategies for this gecko species is adequate. I accept the validity of the findings bar the issue of in silico analysis.
Additional comments: Abstract

Line 3: Change ‘they’ to ‘these species’.

Line 7: Insert ‘by the Portugese Red Data Book’ after ‘Vulnerable’. Change ‘their’ to ‘this gecko’s’ and ‘they are’ to ‘it is’.

Line 9: Insert ‘have’ before ‘used’.

Line 16: Insert ‘up’ after ‘opened’.

Line 19: Provide binomial name for the Madeira lizard and change ‘high’ to ‘higher’.

Line 21: Change ‘detection of’ to ‘detected’.

Line 23: Remove ‘do’.

Line 24: Insert ‘,’ after ‘approach’ and move ‘only’ after ‘sampling’.

Introduction

Line 28: Change ‘unique’ to ‘uniquely’, ‘it’ to ‘islands’ and ‘presents’ to ‘present’.

Line 30: Change ‘in’ to ‘on’.

Line 33: Change ‘allow sampling more thoroughly’ to ‘allows more thorough sampling’.

Line 38: Remove ‘both’ and change ‘to’ to ‘for’.

Line 39: Change ‘to contribute’ to ‘contributing to’ and remove ‘already’.

Line 42: Change ‘islands’ to ‘island species’ or ‘island reptiles’ and ‘the’ to ‘their’.

Line 47: Change ‘in’ to ‘across’.

Line 54: Insert ‘,’ after ‘however’.

Line 56: Change ‘specifically’ to ‘specific’. ‘Dietary habits’ of which Tarentola species?

Line 58: Change ‘contrastingly’ to ‘contrasting with’.

Line 68: Change ‘habits,’ to ‘habits as’.

Line 69: Change ‘that would have to be invested without the existence of this tool’ to ‘needed by conventional tools’.

Line 70: Change ‘this technique’ to ‘metabarcoding’

Line 71: Insert ‘,’ after ‘ultimately’.

Line 73-74: Change ‘. One of them would be that it only provides the species presences, and not their proportions in the samples’ to ‘, one of these being that it only provides species presences and not their proportions in samples’.

Line 79: Change ‘metabarcoding techniques evolve in accelerated rates’ to ‘metabarcoding continues to evolve at an accelerated rate’.

Line 83: Change ‘South’ to ‘south’. Direction is only capitalised when referring to the name a place, e.g. South America.

Line 89: Change ‘17,5’ to ’17.5’.

Line 92: Change ‘referred’ to ‘inferred’ and ‘avoid’ to ‘avoids’.

Line 94: Change ‘small’ to ‘poor’ and ‘its diet’ to ‘Selvagens gecko diet’.

Line 95: Move ‘for the first time’ to the end of the sentence.

Line 99:  Change ‘to infer on’ to ‘deduce’.

Methods

Line 107: Change ‘in’ to ‘on.

Line 113: ‘considered threatened’ by who? The Portugese Red Data Book? IUCN? Government?

Line 114: Provide binomial names for house mouse and rabbit.

Line 119: Insert ‘an’ after ‘as’.

Lines 121-123: Change ‘and specifically for the protection of Cory’s shearwater Calonectris borealis (Cory, 1881) the archipelago plays a key role, sheltering one of the largest breeding colonies in the world’ to ‘and the archipelago plays a key role in the protection of Cory’s shearwater Calonectris borealis (Cory, 1881) by sheltering one of the largest breeding colonies in the world’.

Lines 125 -126: Change ‘object’ to ‘species’. Change ‘Despite the segregation in the period of activity of the two,’ to ‘Despite having segregated activity periods,’.

Line 128: Change ‘in the Island’ to ‘on the island’.

Line 135: Remove ‘our’.

Line 136-137: Change ‘in’ to ‘with’ and ‘of some groups regarding seasonal variation’ to ‘in Selvagens gecko diet due to season’

Lines 137-139: This is very convoluted. I suggest changing to ‘Sampling took place in intermittent years at the end of summer (6th – 15th September 2010 and 10th – 11th September 2017) and in late spring (9th – 30th May 2011)’.

Line 141: Change ‘,’ to ‘;’ or vice versa, but separation of list items should be consistent.

Line 144: Remove ‘,’ after ‘ethanol’.

Lines 148-151: Reword as ‘To obtain a reference collection of the island’s arthropods for morphological identification, five traps containing water, 70% alcohol and detergent were placed in each of four 1 ha squares at evenly spaced intervals along the island plateau, and left open overnight in 2010 and 2011’.

Line 159 - 160: Replace ‘the most common ones’ with ‘those’ and change ‘in GenBank’ to ‘on GenBank’. I appreciate that the authors have provided a table of sequences generated by DNA metabarcoding for pellets and Sanger sequencing for voucher specimens. However, I stand by my previous comment where I said that the authors should really have performed in silico analysis and reported the results.

Results should include a summary of which species present on the island were represented on GenBank/BOLD, which species were missing reference sequences, which species were barcoded in their study, and which species amplified with each primer set using a program that simulates PCR, e.g. ecoPCR. A species list for their study area, Selvagem Grande, could be constructed using public databases (e.g. GBIF, https://www.gbif.org/), based on previous studies or based on their own trapping efforts. In fact, the authors mention such a species list in Lines 224-225. With this species list, they can perform a GenBank/BOLD search to identify which species are represented and which species have no sequence records. They can highlight which gaps they filled through their own DNA reference database construction and which still remain for future studies to procure. They can download sequence data from GenBank/BOLD for species in their list and combine it with their own barcoding efforts to create a single fasta file to convert to an ecoPCR database. ecoPCR can then be performed for each primer set to identify which species should have amplified. Any additional species that PCR-amplified but did not amplify in silico will likely be species with reference sequences that do not include both the forward and reverse primer. Any species which amplified in silico but did not PCR-amplify would indicate amplification bias.

If the authors did not conduct an in silico analysis, they should provide justification for their primer choices. Why do they expect the selected primers to amplify taxa in their study area or more broadly, geographic region?

Lines 171-172: ‘following the criterion of the minimum number’ – I have not heard of this before. Could the authors add a sentence and briefly describe please?

Line 176-189: I suggest moving this section on sequencing of pellet samples below Lines 191-207 on reference database construction, or use subheadings to distinguish the two sections. Typically, you would construct your reference database before doing any DNA metabarcoding. Perhaps the authors did not do things in this order, but it is a more logical flow to the manuscript.

Lines 178-184: Change to ‘Three different DNA fragments were chosen to identify the distinct prey groups presumably preyed by the study species. For plants, the g/h primers that target the short P6-loop of the chloroplast trnL (UAA) intron were used (Taberlet et al., 2007). For invertebrates, modified IN16STK-1F/IN16STK-1R primers that target the mitochondrial 16S rRNA gene were used (Kartzinel & Pringle, 2015). For vertebrates, the 12sv5F and 12Ssv5R primers that target the V5-loop of the mitochondrial 12S rRNA gene (Riaz et al., 2011) were used (Tables S1, S2 and S3)’.

Line 193: Change ‘to allow the match with’ to ‘to match’ and ‘Standard’ to ‘The standard’.

Line 195: Change ‘confirming’ to ‘confirmation of’.

Line 210: Insert ‘the’ after ‘following’ and move ‘protocol’ after ‘Preparation’.

Line 213: Insert ‘,’ before ‘diluted’ and change ‘in’ to ‘on’.

Line 222: Insert ‘by’ before ‘comparing’.

Lines 225 - 227: Remove ‘of’ before ‘BLAST’. Reword as ‘Sequences with less than 90% BLAST identity were assigned to class level. Sequences with 90–95% BLAST identity were assigned to family level. Remaining sequences with 95% BLAST identity or higher were assigned to species or genus level’.

Line 227: ‘Prey items’ – I assumed the authors mean morphological prey items? Please clarify.

Line 233: Insert ‘assignments’ after ‘level’.

Lines 237-238: Reword as ‘This approach was not used for the 2017 samples as it is not possible to estimate the number of individuals belonging to each prey species using metabarcoding’.

Results

Lines 298-301: I actually interpret this as DNA metabarcoding is revealing hidden dietary diversity that the traditional sampling is not capturing, and that is why the accumulation curve does not reach a plateau. This is a main conclusion from the Discussion.

Discussion

 Line 309: Insert ‘,’ after ‘beetles’ and after ‘biomass’.

Line 318: Insert ‘,’ after ‘May’.

Lines 322-325: I believe the authors are describing results from Hódar & Pleguezuelos (1999) here but this needs to be made clearer, e.g. ‘Hódar & Pleguezuelos (1999) showed that between April and July, …’.

Line 353: Change ‘notice’ to ‘note’ and ‘does not allow to obtain’ to ‘do not provide’.

Line 354: Change ‘impossible the detection of’ to ‘it impossible to detect’.

Line 356: Insert ‘always’ before ‘happen’.

Line 366: Remove ‘with’.

Line 367: Change ‘so in’ to ‘thus’.

Line 372: Change ‘fairly’ to ‘roughly’.

Line 374: Change ‘those’ to ‘these’.

Lines 374-377: Reword as ‘The small size of this gecko (average snout-vent length circa 6 cm) may prevent ingestion of large seeds or harder plant parts, which may explain the lack of plant material detected using morphological identification of Selvagens gecko pellets’.

Line 381: Change ‘ contains’ to ‘contents’.

Line 382-383: Change ‘Applying this method to’ to ‘Assessing stomach contents of’.

Line 386: Change ‘But’ to ‘However,’.

Line 387: Remove ‘could’.

Lines 393-394: Reword as ‘Due to the low number of terrestrial predators, it is common for insular reptile species to become diet generalists as they can reach high densities and face higher competition for food’.

Line 396: Change ‘of options, causing them to’ to ‘and even’.

Line 402: Change ‘of’ to ‘for’.

Line 407: Insert ‘,’ after ‘approach’.

Line 408: Remove ‘,’ after ‘OTUs’.

Line 409: Remove ‘case of’.

Line 412: Change ‘could show’ to ‘showed’.

Line 416: Change ‘could detect’ to ‘detected’.

Line 419: Change ‘evidences’ to ‘evidence,’.

Line 425: Insert ‘Selvagens gecko’ before ‘DNA’.

Line 428: Change ‘number’ to ‘range’.

Line 431: Move ‘only’ to come after ‘sampling’.

Line 433: Change ‘integrate information’ to ‘integrate the information they generate’. Insert ‘,’ after ‘generate’.

Line 465: Change ‘it mainly consists in’ to ‘diet mainly consisted of’.

Line 466: Remove ‘,’ after ‘feathers’.

Line 474: Insert ‘,’ after ‘limitations’.

Line 475: Remove ‘the’ before ‘successful’ and change ‘the range’ to ‘a range’.

Line 476-481: Reword as ‘DNA-based diet studies have been shown to describe the diet profile of a wide range of species with higher resolution and greater efficacy than classical methodologies, e.g. birds (Trevelline et al., 2016), bats (Hope et al., 2014) and sea lions (Hardy et al., 2017). Therefore, DNA metabarcoding is a valuable and revolutionary tool for conservation research and management (Allendorf & Luikart, 2009)’.

Line 484: Change ‘studies’ to ‘methods’.

Line 485: Change ‘species diet’ to ‘species’ diet’.

Lines 486-488: Reword as ‘Metabarcoding methods, even when deployed as extremely quick surveys, can deliver holistic results on diet composition, diversity, and ecological networks at relatively low cost (Lopes et al., 2019)’.

Line 489: Change ‘in’ to ‘to’ and ‘this insular species’ to ‘the insular Selvagens gecko’.

Lines 489-491: Remove line break to read as one paragraph and change ‘high’ to ‘large’.

Line 493: Remove ‘and’.

Line 494: Change ‘as it is the case of’ to ‘as is the case for’.

Line 499: Change ‘In this particular case,’ to ‘Based on our results,’.

Line 500: Insert ‘Selvagens’ before ‘gecko’.

Line 502-503: Change ‘the diet. Enlightening the referred matters’ to ‘its diet. Shedding light on these questions’ and ‘in’ to ‘for’.

Lines 504-505: Change ‘We hope our work has contributed to establishing guiding actions of future management planning’ to ‘Our work will contribute towards establishing guidelines for future management of the Selvagens gecko and its habitat’.

Table 1: Insert ‘,’ after ‘2017’ and ‘species’ before ‘sampled’.

Table 2: Change ‘Selvagem gecko’ to ‘the Selvagens gecko’.

Figure 2: Change ‘classical and metabarcoding methods’ to ‘morphological identification and DNA metabarcoding’.

Figure 3: Change ‘studies’ to ‘identification’.


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: BIOACTIVITY OF COMPOUNDS SECRETED BY SYMBIONT BACTERIA OF NUDIBRANCHS FROM INDONESIA
Review round: 2
Reviewer: 1

Output:
Basic reporting: The title is suitable and reflects the research that has been carried out. The results were fairly discussed with sufficient support from previous findings, and with a good flow. However there are certain parts that need correction and further clarification (as commented in the draft). Moreover, the usage of English language is bad and critically needs improvement. Author should re-write many sentences in the drafts (as highlighted) due to poor English command. Minor spelling mistakes spotted in the draft as highlighted in the draft. The research approaches in this study need to be justified.
Experimental design: The article fits the aims and scope of the journal. Methodology is not clearly described, hence not repeatable by practitioners, especially the bioactive compound extraction method. Some of the method have no citation.
Validity of the findings: The amount of data is not sufficient especially in the antimicrobial analysis part. Agar overlay and disc diffusion method is qualitative analysis. Quantitative analysis of antimicrobial study is needed to support the discussion. The validity of the results is doubtful because, the author used incorrect solvent to dissolve the extract. Also the growth of indicator strains in agar plate during the agar overlay assay is very weak. Agar overlay analysis results is reported in duplicate. No statistical analysis shown.
Additional comments: The English language should be dramatically improved to make sure that the readers can understand the text easily. Certain terms and phrasings are confusing. Some of the confusing sentences need to re-phrased, as highlighted in the draft.

Need to correct the symbol of temperature throughout the draft-correct is: °C

Figure 3
-Need to re-write the caption. Need to state the picture was taken from the agar overlay method or disc diffusion method.
-From the picture, it can be seen that the growth of indicator strains is very week especially in picture A. This indicates some error in the experiment, hence the results are doubtful. In actual scenario, the indicator strains must grow well in the area that are not exposed to test strains. Author should clarify on this. Example of typical inhibition zone picture can be found in the following article doi:10.3390/biomedicines5020031

Figure 2 is not clear
The labelling of (A) and (B) on the photo is missing. Also, there is no flowchart of isolation as mentioned in the caption of Figure 2

Citation format error occurred in many places, especially when author cited more than 1 reference for 1 sentence. The correct way to cite more than 1 reference is for example (citation 1; citation 2;citation 3)

Line 325 to 326 :“The ethyl acetate extract showed that anti-MRSA activity of P. rubra was higher than V. salarius at concentrations of 500-1000 µg/ml.”
The difference is not much. Moreover, in Table 2, both isolates scores “++”. So it is not proper to state that anti-MRSA acivity of P. rubra is higher than V. salaries. Unless, quantitative analysis was done to further confirm it.

After the isolates was identified as P. rubra, the correct way to state the scientific name for the author’s strain is by mentioning the code of the isolate as well e.g. P. rubra KJB-07

What is the justification of screening for the three bioactivity (anti-MRSA, cytotoxicity, and anti-viral) and not other bioactivity like antioxidant, etc). From the results obtained, what will be the potential application of the extract obtained from P. rubra KJB-07?

Line 332 to 336: Table 2 shows that the two extracts had different effects on the MRSA. In this research, P. rubra as a gram-negative bacteria had cell walls that were thinner than V. salarius as gram-positive. Pseudoalteromonas rubra was able to prevent the loss of intracellular proteins, and reduced access of hydrolytic enzymes and some compounds produced by S. aureus

This fact is not correct. Nothing to do with the cell wall of P. rubra. The compounds produced from P. rubra will effect the cell wall of MRSA. This sentence critically needs correction.


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: BIOACTIVITY OF COMPOUNDS SECRETED BY SYMBIONT BACTERIA OF NUDIBRANCHS FROM INDONESIA
Review round: 2
Reviewer: 2

Output:
Basic reporting: Reviewed article is interesting. It is written in proper English. Structure of article is classic.
Experimental design: Authors studied anti-MRSA, anti-HSV-1 and cytotoxic activity of two extracts obtained from Pseudoalteromonas rubra and Virgibacillus salarius. Both extracts acted anti-MRSA. The extract of P. rubra was cytotoxic to Vero cells, but the extract of V. salarius presented no cytotoxicity. No anti-HSV-1 was observed. Authors isolated also three compounds from P. rubra culture supernatant identified by LC/MS. I'm surprised why Authors not studied activity of these 3 substances? Which substance is responsible for the observed effects? Where is LC/MS for V. salarius?
Validity of the findings: Studies are incomplete. It is lack of LC/MS for V. salarius and were not done studies of activity of compounds obtained from supernatant.
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: BIOACTIVITY OF COMPOUNDS SECRETED BY SYMBIONT BACTERIA OF NUDIBRANCHS FROM INDONESIA
Review round: 3
Reviewer: 1

Output:
Basic reporting: Sufficient field background
Experimental design: fit the aims of the journal.
Validity of the findings: good
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: SAGITTAL SPINAL MORPHOTYPE ASSESSMENT IN 8 TO 15 YEARS OLD INLINE HOCKEY PLAYERS
Review round: 1
Reviewer: 1

Output:
Basic reporting: The authors present a study on the assessment of sagittal spinal morphotype in young Inline-Hockey players. This study presented the assessment protocol of “Sagittal Integrative Morphotype” composing by the evaluation of sagittal spinal curvatures in a relaxed standing position, slump sitting position, and maximum flexion of the trunk. The protocol provided a diagnosis of sagittal spinal morphotype using the unilevel inclinometer. The author concluded that this method provided considerable reproducibility and validity with a good correlation with the radiographic measurement. Several issues need to be addressed before this work is suitable for publication.
Experimental design: 1. Patients with radiographic evidence deformity may present a normal spinal morphotype, therefore, the limitations and shortcomings should be recognized and reflected upon.

2. Other reports assessing spinal morphology in young athletes to assist the preemptive care of early-stage spinal deformities need to be discussed.

3. How many observers are there in this study?

4. Quantitative statistics need to be performed to acknowledge the possible significant difference between groups.
Validity of the findings: 1. The authors need to provide information/data regarding the interrater reliability of this protocol.

2. More clinical implications of the present study need to be highlighted.
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: SAGITTAL SPINAL MORPHOTYPE ASSESSMENT IN 8 TO 15 YEARS OLD INLINE HOCKEY PLAYERS
Review round: 1
Reviewer: 2

Output:
Basic reporting: The paper is well written, clear structured, and easy to follow. The introduction provides a good overview on previous works up to the state of the art. The hypotheses for the paper is clear defined. Raw data are provided with the manuscript. Only improvement I can see with the data is to provide it in English language.
Experimental design: The design of the experiment is adequate. Although it would be of interest to analyze the spinal shape changes with more recent techniques, such as wearable technologies that focus on the spinal shape and allow for long term measurement. The presented technique only takes a snapshot in lab conditions. Further, some more statistics about the distribution of the subjects into the age groups would be of interest. Similarly, it would be interesting how long the subjects are engaged in IH before the measurement was taken. This would help to see how the change in posture progresses. Regarding the statistical analysis it would be interesting to see a histogram of the data, as they are not normal distributed. Was any other defined distribution found?
Validity of the findings: Findings reported in this work are valid and sound.
Additional comments: This paper is in a good shape, I only have minor remarks that should be addressed, mentioned above.
In summary:
- convert raw data to English language.
- Add statistics about the distribution into the age groups.
- Add information about engagement time into IH, if available.
- Histogram or additional information about the data distribution.


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: SAGITTAL SPINAL MORPHOTYPE ASSESSMENT IN 8 TO 15 YEARS OLD INLINE HOCKEY PLAYERS
Review round: 2
Reviewer: 1

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


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

Input:
Title: SAGITTAL SPINAL MORPHOTYPE ASSESSMENT IN 8 TO 15 YEARS OLD INLINE HOCKEY PLAYERS
Review round: 2
Reviewer: 2

Output:
Basic reporting: The authors improved the manuscript in this revision, new tables have been added and the raw data have been translated to English.
Experimental design: Experimental design hasn't changed, but was fine in the first revision, already.
Validity of the findings: The authors have improved the article, and thus the findings are better supported, now.
Additional comments: No additional comments


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

Input:
Title: EFFECTS OF LOW PH AND FEEDING ON CALCIFICATION RATES OF THE COLD-WATER CORAL DESMOPHYLLUM DIANTHUS
Review round: 1
Reviewer: 1

Output:
Basic reporting: The manuscript is professionally structured, with sufficient literature references, figures, and tables, and contains the appropriate raw data.

Overall the manuscript is well formulated, however there were several sections in which the English used could be improved. Suggestions:

Line 30-32: Sentence is too long and unnecessarily wordy.
Line 39: There is an extra space after the words 'efforts have'
Line 51: Shoaling of the saturation state 'horizon'
Line 70: ROV should be ROVs (plural).
Line 76: Replace 'First studies' with 'Early studies'
Line 77, 92, 284, 390: Check to make sure scientific names are italicized (check through the whole manuscript).
Line 84: Consider rephrasing 'evidenced signs of stress', perhaps 'exhibited signs of stress'
Line 95: Replace 'and at two' with 'and under two'
Line 98: Replace 'especially at' with 'especially in'
Line 100: Replace 'at unusual shallow' with 'at unusually shallow'
Paragraph beginning line 170: Should be moved to immediately after (or combined with) the description of the pH regulation system (paragraph starting line 137).
Line 250: Figure 5 is referred to prior to Figure 4 in the main text.
Line 265: Replace 'being those' with 'those being'
Line 270: replace 'have undertaken' with 'performed'
Line 272: This sentence is too long, should be reworded for clarity.
Line 324: The part of this sentence beginning with 'we did not...' needs to be reworded.
Line 328-330: sentence has several grammatical errors, and may need to be reworded.
Line 337: remove 'or other stressors'
Line 341-343: sentence is unclear, needs rewording.
Paragraph starting on line 365: This seems like it would be better placed in the methods.
Line 372-375: Sentence is unclear - the current wording seems to run counter to the findings of the experiment.
Line 391: It is unclear what is meant by this sentence.
Experimental design: The experiment was well designed, and a logical expansion of previous work to explore original research. The research questions were well defined, and represent a meaningful contribution to the field.

In the methods section, the manuscript would be improved by the addition of the following information:

1) The authors report a general range of pH values for the Comau Fjord, however the range reported is quite large (7.4-8.2). They collect their specimens at two different sites, and do not report local measurements of pH or other potential differences between collection sites, nor if there was significant differences in response during the experiment of corals from one site vs. the other (at least not in the main text). Including this information would help place their study in a broader context.

2) The authors do not report the pH and feeding conditions that the specimens were subjected to during their 9 month acclimatization period, nor the initial pH of the experiment. These conditions may have had considerable effect on the differences in the onset of OA effects relative to other experiments (i.e. where the corals primed by high feeding conditions, or a lower initial pH?).
Validity of the findings: Overall, the manuscript is robust and shows clearly statistically sound results that represent a significant contribution to the field. There are however several improvements that need to be made to make the work robust:

1)  Despite the assertion of the authors on line 256 that the correlation shown in figure 4 is both significant and strong, it does not appear as such. There is also no r value presented (which appears to be very low). Perhaps the outlying specimen at initial weight of ~22.5 is driving this relationship, or perhaps its an artifact of over-massaging the data. There is also no explanation given for assigning an exponential function to the data. I recommend removing this result from the manuscript, as I have concerns about the validity of conclusions drawn from it.

2) It is unclear what conclusion is being made in the sentence beginning on line 346. The argument references a follow up experiment (which is again referenced on line 419), which is unpublished. If the authors wish to use the results of this experiment to justify their conclusions, those results should be included in this paper, or published elsewhere and then used in support. As stated, the main findings of that experiment are unclear and therefore confuse the points being made - especially if using it to justify the difference in results from previous experiments.
Additional comments: No additional comments


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

Input:
Title: EFFECTS OF LOW PH AND FEEDING ON CALCIFICATION RATES OF THE COLD-WATER CORAL DESMOPHYLLUM DIANTHUS
Review round: 1
Reviewer: 2

Output:
Basic reporting: I reviewed the paper “Effects of low pH and feeding on calcification rates of the CWC Desmophyllum dianthus”, which aimed to investigate the long-term response of this CWC to different levels of pH and aragonite saturation at different feeding regimes. Overall, Martinez-Dios et al., found that D. dianthus is vulnerable to low pH conditions and low food availability. This is a well performed study on the calcification and feeding of an important CWC, with clear and correct use of English language, informative and well-structured introduction that provides sufficient and updated background of the topic. However, some of the references are inaccurately cited and need closer look where appropriate. Specific comments will follow this review.

Some references are missing from the text as in line 61, and others do not correspond to what it is stated in the paragraph such as in lines 78-80. There is a mix-up between short-term and long-term incubation thorough the manuscript. I would suggest defining in the methods or intro section what is considered for the authors long-term because otherwise the discussion becomes confusing. Just to give a few examples, Gori et al (2016) and Carreiro-Silva et al (2014) are both long-term and should not be discussed as short-term experiments (at least from the perspective of experimental biology and CWC in particular) or Form and Riebesell (2012) is a long-term experiment and  line 81, Gomez et al (2018) is a short-term experiment. The same applies for lines 314-316: The authors citing Movilla et al. (2011) state “This previous study, although performed only at two levels of pH (8.1 and 7.8), revealed an effect of time in the appearance of potential effects, prompting for the need to undertake long-term experiments”. Again, do the authors consider 11 months incubation short-term?

Lines 85-87 the authors stated that “Regarding other stressors in addition to OA, few studies have been carried out with CWCs, and only two have examined their response to low pH and food availability (Maier et al., 2016; Gómez et al., 2018). However, Georgian et al (2016), and Busher et al. (2017), are also studies performed with CWC and included feeding variables, which should be included in here.

The authors need to double check references that are missing from the reference section but are cited in the text, as in lines 332-333, line 453 or lines 357-361, just to mention a few.
Experimental design: Well-designed experiment with a state-of-the-art set-up methodology that goes well according to the questions and main goals of the study. This study is well replicated thus the inference is very robust. All the methods were clearly stated in order to be replicated by other researchers.

Considerations: Please provide more detail about the n, such as replication units (fragments replication vs tank replication). Based on the statistical results and DF I will assume you used single fragments for your statistical inference?

Please check for typos as in lines 133, 214, 390, 423, or better use of English as in lines 25 or 48. Check redaction as in lines 372-375, which needs some rewording.
Validity of the findings: Findings are robust with the novelty of being one of the longest incubations of low pH and feeding regimes performed for CWC corals, although to my concern there is an excessive use of this novelty thorough the document. It is true that this study is longer than any other performed on this species, nevertheless, it is not the “only” long-term study and that should be stated.

There is only one concern about the environmental data from the collection site, such as pH, omega aragonite and alkalinity. I believe the authors provided information in general about the range in some environmental variables in the Fjords based on other studies and taking into account the whole bathymetric range for the species. If possible, they should provide the environmental range of the collection site at the depth of the collection, since the discussion has an important ecological component and comparison between studies with different biogeographic settings. Are the environmental values from the Fjords comparable to the ones found in the Mediterranean? Could it have an influence in the response found in the present study with other D.dianthus from elsewhere?

I would suggest moving the table for the models (i.e. ANOVA) from supplementary and include it in results.

In general, all the results are sound, meaningful and conclusions are well defined and supportive to the findings of the study.
Additional comments: Lines 361-364:
I do not think it is necessary to bound around this argument that has been mentioned all over the document. I would suggest ending the paragraph in line 361.

Lines 365-367:
Authors state: “In order to simulate as well as possible the natural field conditions and to compare with
published literature, corals in this experiment were fed taking into account both the food type and the optimal prey size observed in natural habitats of D. dianthus”. I wonder if the authors characterized this variable in the collection sites.

Line 445: double check references in here. I do not think Georgian fits in the argument.

Table 4:
Please check accuracy of incubation time for studies shown in the table. Also, I noticed there are some studies published recently that are not included in the table (specially for L pertusa). Is it supposed to be an exhaustive search? If the authors want to include a table (which I consider useful), I would suggest focus only on the studies more relevant to this study, perhaps only those related to OA and feeding with D. dianthus