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: OZYMANDIAS: A BIODIVERSITY KNOWLEDGE GRAPH
Review round: 1
Reviewer: 1

Output:
Basic reporting: This paper describes a project of the author’s to reconcile data from a number of online data sources, link the data together using RDF triples, and build a web interface to browse the data. The data are: taxonomic names of the fauna in the Atlas of Living Australia, and articles and their authors that published these names. The article also presents some pertinent analyses of the taxon/article/author data once they had been assembled.  The project itself is a valuable contribution in the field of biodiversity informatics and taxonomy, and the paper describes the project clearly and comprehensively.

My criticisms are relatively minor. Some relate to the presentation (here), some to engineering choices (section 2, below), some to conclusions (section 3, below).

1. The Abstract does not reflect the full substance of the paper. Some mention of each of the elements of “reconciliation, augmentation and meta-analysis” should be added. The analyses that the author performed on Ozymandias data are themselves small but significant contributions to taxonomic science and should be mentioned in the abstract.

2. The paper shows signs of having been submitted perhaps before one final editorial check through! There are more typos than there should be, and several missing citations.

3. The formatting of in-text citation is often incorrect. I see that PeerJ does not enforce a particular citation style, but I imagine the in-text citations should be performed by the author. E.g., “published in (Riedel & Tänzler, 2016)” should be “published in Riedel & Tänzler (2016)”, etc.

4. The paper seems just a touch too long (10-15%). Some detail could be omitted: for example, some of the the details of DOI dereferencing (LL 145-152), some of the details of the web interface (LL 296-326), and some of the text from LL 163-170 and LL 225-237. On the other hand, the details of reconciling author names (LL 204-216) is valuable and probably represents a lot of the time that went in the project.

5. A trivial question, but one most readers will have: Why the name Ozymandias? After the poem by Shelley? A short answer to that question would be appropriate in the paper.

6. There are few mentions of other biodiversity knowledge graphs, suggesting that Ozymandias is one of the very first. If we include any sharing of biodiversity data as LOD, then there are many (e.g., OpenBioDiv, Minami et al (2011, “Towards a Data Hub for Biodiversity with LOD”), Mai et al. (2011, “Linked Open Data of Ecology (LODE): A New Approach for Ecological Data Sharing”), http://bioimages.vanderbilt.edu/, taxonconcept.org, etc).

Line by line:

L 38. “representation of a knowledge graph” -> “implementation of the basic unit of a knowledge graph”

L 40. maybe list a few other ways to model a knowledge graph (KG)?

L 49. Typo: “identify” -> “identity”

L 79-80. It seems to me that reconciliation is not “built on top” of a KG, but is a key prior step in creating that KG.

L 117. Typo: “adopted to because” -> “adopted because”

L 131. “(Vicki Tardif Holland & Jason Johnson, 2014)” -> “(Holland & Johnson, 2014)” and in references list.

L 250 Typo: “write” -> “wrote”

L 378. Check commas, parentheses around the citation.

L 445. Missing citation.

L 453. Missing citation.

L 479. Missing citation.

LL 481-490, and LL 492-497. Good points!

Figures:

Fig 1. Would be helpful to add the namespace to the classes and properties. Also the layout of the figure could be improved to save space and make it a bit more “artistic”.

Fig 3. Could be omitted or made supplemental information.

Fig 4. Could be omitted or made supplemental information. Well described in text.

Figs 5, 6, 7, 8. Could be omitted or made supplemental information. While a great deal of work went into the design of the web interface, these views are available on the web.

Fig 9. Not sure about the PeerJ color figure policy, but this figure could easily be made into a black-and-white figure.

Fig 10. Legend typo: “publications for each.” -> “publications.”?  While “sparklines” (I had to look this term up) don’t have axes, this figure would better served by adding axes: “decade”, vs. “number of publications per decade”.

Fig. 11. Good figure!

Figs 12 and 13. It would be nice to also see the number of (current) synonyms as well (using both a cumulative line and as bars). This would require a new query and new figure and is not vital (for me), but would enhance the graphs. Some comment/discussion of this would also be needed in the text.

In summary, the article:

 * Is clearly written.
 * Gives sufficient context with citations (but see comments above).
 * Is acceptably structured (it is hard sometimes to fit description of software into a standard scientific article format, but the author has done this well).
 * Contains only figures relevant to its content, though some may not be needed (see above).
 * Is self-contained and an appropriate unit.
Experimental design: Since this is a software product rather than experiment, I’ll read “engineering design” for “experimental design”. Overall, Ozymandias appears to me to be very well-designed, and the choices made are nicely laid out in the text of this paper (if only all software/data products had an associated paper with this level of documentation of choices!).

I’m not a big fan of the way Schema.org models roles (http://blog.schema.org/2014/06/introducing-role.html), with it’s duplication of the `creator` property, and so would probably have tried to find another ontology/schema to model article authorship (Bibo?). However, I accept that modeling ordered lists in graphs is never easy, and also accept the value of choosing widely-used schemas (like Schema.org) where possible. One compromise if one needs to use a more domain-specific vocab is to specify the Schema.org classes in addition to the classes of the other vocabulary, which aids in discovery if not data integration.

The associated analyses (Figs. 9-13) were valuable and well described. I especially enjoyed the discussion of taxonomy as “long data”. It would have been nice to see a few more comparisons of the results of these analyses with those done by other authors (the comparison with Pullen et al. 2014 excepted).

The comparison of weevils vs. snails (Fig 12 vs. 13) was interesting and well described (LL 404-409).
Validity of the findings: The conclusions of the paper are valid, and all the data necessary to fully reproduce the software product, and the analyses, are provided (via Github, the included SPARQL queries and outputs, and via the ability to run _any query_ via the SPARQL endpoint).

A couple of comments that the author may chose to further reflect on in the text:

1. In a couple places the author states that Ozymandias was made to a deadline and so the work of matching data from different sources had to be halted. This reconciliation step is what makes the construction of the KG possible, and is also probably the most time consuming. Even though it was not needed for this product, the author alludes to the process (and problem) of matching taxonomic names (LL 182-184), and this is a critical and time-consuming step in the construction of most biodiversity knowledge graphs.  Can this reconciliation phase, which requires many “bespoke” solutions to be engineered, really be scaled up to, e.g., GBIF-scale?

2. Without calling into question the value of Ozymandias and this paper, I wonder if the additive value of Ozymandias is really a result of it being based on a graph data model (cf. LL 422-423), or of the data reconciliation steps that it required. The author’s other major data/software contributions in biodiversity informatics (e.g. iSpecies, BioNames, BioStor) are not built on graph databases (as far as I know) and have similar user interfaces. To truly demonstrate the value of the KG nature of Ozymandias, I think one would have to show that a) users were accessing the end point to ask queries not asked by the web interface, and b) that these queries feed directly into graph-like analyses and products (i.e., they need the RDF output of Ozymandias, and could not have been run as SQL queries, should there have been a SQL Query interface to the underlying Ozymandias data). Another way to demonstrate the “graph power” of Ozymandias would have been to provide some original metadata served as RDF, via “live” content negotiation with triples having an Ozymandias URI.  Finally, perhaps the graph data could be contributed to a 3rd party triplestore (Wikidata, https://datahub.ckan.io/) which would at least provide the opportunity for users to integrate Ozymandias’ new data with other graph data.

The author alludes to the question of how valuable graph data really is for biodiversity in LL 468-474, and the jury is out on where biodiversity LOD will go. But perhaps it might be more explicitly recognized in the paper that _currently_ it is really the data integration across data silo that liberates data rather than the KG nature of the output.
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: OZYMANDIAS: A BIODIVERSITY KNOWLEDGE GRAPH
Review round: 1
Reviewer: 2

Output:
Basic reporting: The author meets all basic reporting requirements.
Experimental design: The author meets all experimental design requirements.
Validity of the findings: The author meets all validity requirements.
Additional comments: I enjoyed this paper and I think it can be published with some VERY minor changes.
1. Sometimes it is important to find the last author of a paper. How would one do that using the numerical roleName?
2. This probably isn't a problem, but it seems weird to me that a TaxonName would be a synonym of a TaxonConcept. Wouldn't another TaxonConcept be the synonym and the synonymous TaxonName attached to that?

Small typos:
Line 16: should be "and examples of its application"
Lines 55-57 and 71: I don't understand why CrossRef, Altmetric, ORCID, and Schema.org are in parentheses and quotes
Line 90: I suppose its the author's preference, but I think of "data" as plural
Lines 156-157: should be "themselves have DOIs. Hence, if we"
Line 174: should be "cases the sources of data"
Lines 187-188: subordinate clauses at the beginning of a sentence should be followed by a comma. Example "For the works in AFD, I searched". There are many of these throughout the paper.
Line 241: should be "Egloff et al. (2017) have"
Line 257: Can you mentioned how many named graphs you had? Their sizes? Just a point of curiosity
Line 391: should be "Figure 11 would"
Line 398: should be "Pullen, Jennings & Oberprieler (2014) recently"
Line 408: should this be "species in leaf-litter and other habitats"?
Line 445: Is the reference to Shorthouse a pers. comm.?
Line 479: Did you forget to replace "cite citation papers" with actual citations?
Line 525: I didn't see anything but a readme in the oz-wikispecies repo. Is this correct?
Line 677: should be "in this case it displays"
Line 694; should this be "data using the knowledge"?
Line 726: should be "Fig. 12"


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: GENOTYPIC VARIATION IN DISEASE SUSCEPTIBILITY AMONG CULTURED STOCKS OF ELKHORN AND STAGHORN CORALS
Review round: 1
Reviewer: 1

Output:
Basic reporting: This manuscript describes the results of direct-graft challenge assays with multiple genotypes of Caribbean acroporid corals in Florida, to determine whether genetic resistance to disease can be identified. This manuscript is very well written in professional English, is clear, concise, and thorough. It provides clear justification for each experimental decision, and with a few exceptions outlined below, provides ample methodological information to be repeated in the future. It is self-contained and follows a professional structure, including providing sufficient background, context and references to the literature.

This work adds to the growing body of literature around identifying ideal candidates for coral restoration programs and adds to the body of basic literature around disease ecology. It also uses standardized field protocols, which advances the field by using comparable methods across studies.
Experimental design: Overall, the experimental design is sound, sufficiently replicated and appropriately analyzed. The major decisions in the design process are adequately justified, and the paper reports original primary research with clear aims and scope. The only significant concern I have regarding the design is in the use of inoculants. While the authors address the use of inoculants directly in the text (i.e. lines 144, 148 and others), I still think there is the potential for a significant bias from differentially infective inoculants.

I understand why inoculants were collected without regard to genotype, and that it is extremely difficult to know the potential infectivity of an inoculant in the field, but was there any attempt to use inoculants from ‘donor’ colonies evenly across receiving genotypes? How many different inoculant ‘donor’ colonies were used, and was there any pattern in the receiving corals related to the inoculant ‘donor’ colonies? At the very least, I think it would be useful to provide the data for which inoculant ‘donor’ colonies were used in challenge assays for each genotype, and whether there appeared to be a donor effect. Perhaps the results could be standardized in some way (maybe by rate of tissue loss on the inoculant for 2017)? In line 203, the authors write “despite variable inoculant virulence, significant variation was observed among genotypes of each species in risk of transmission’. However, if the results were not standardized to inoculant virulence, I’m not convinced that the variation in infectivity can be attributed to the genotypes of the infected corals. If they could be standardized in 2017 by rate of tissue loss on the inoculant, and the results are the same as the pre-standardization, it would add justification for this method.

This is particularly concerning given that some fragments that did not experience tissue loss in the initial trial in 2016 were re-exposed to a second inoculant. This would suggest that perhaps the authors were not convinced that the first inoculant was infective. Furthermore, only 81% of inoculants appeared to lose tissue during the 7-day trial, although I do acknowledge that some inactive inoculants were still associated with transmission (very interesting!), and appreciate the authors providing the range of transmission risk including and excluding these inactive inoculants. Thirdly, the histology of the samples suggested potentially two different diseases were impacting the inoculants (WBD and RTL), which could also result in differential transmission or level of infectivity. Therefore, I think a further justification of why these results are reliable despite potentially differentially infective inoculants is necessary.
Validity of the findings: Overall, I think the sample sizes are robust, the statistical methods are sound and there were proper controls. However, I do question whether the results are confounded by differentially infective inoculants, as discussed in the section above. I think a further justification for the use of these inoculants, or additional information about how many and which inoculant donor colonies were used for each genotype, is necessary.
Additional comments: Introduction
•	Line 62, “et al” missing appropriate punctuation
•	Line 56, it may be worth mentioning that restored populations also often involve the asexual fragmentation of corals that can potentially result in populations with lower genetic diversity than natural ones, and consequently higher risk of epidemics (Altizer et al. 2003 TREE 18(11), pp.589-596), although I do recognize that natural Acroporid populations, especially in Florida, currently have very low abundance and genetic diversity.
•	Line 64 and Line 70 – in one instance “susceptibility/resistance” is used, and in the other instance “susceptibility or resistance” is used. I suggest keeping text consistent and prefer the latter.

Results
•	Line 177, acronym WBD was used here but I don’t think it was previously defined in the text. Please define white band disease.
•	Line 179, I suggest changing ‘contributed by’ to ‘caused by’
•	Line 183-185, I think this may be an incomplete sentence. The first phrase seems unfinished.
•	Line 187, acronym RTL was used here but I don’t think it was previously defined in the text. Please define rapid tissue loss.
•	Line 196, is ‘intensities of infections’ an epidemiological term? I think it may be useful to define infection intensity in this context.

Figures & Tables
•	Figure 1, it was not clear to me what the p-values in panel C are referring to.


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: GENOTYPIC VARIATION IN DISEASE SUSCEPTIBILITY AMONG CULTURED STOCKS OF ELKHORN AND STAGHORN CORALS
Review round: 1
Reviewer: 2

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: This is a very nice study examining general tissue loss disease resistance in A. cervicornis and A. palmata using established techniques.  Overall, the paper is well written, methods are appropriate and conclusions well supported.  I commend the authors for including histology to examine the inoculate fragments but would like to see representative pictures showing the different host responses as interpreted from the slides e.g. RLOs, lysed cells, apoptosis, degraded cell spherules, etc.  It could be a supplemental Figure but would really help in the evaluation of the findings and more importantly be a tool to help train others in histo slide interpretation.

I would also like them to include the genotypes of the inoculant fragments to Table 2.  There may be some interactions between different genotypes that are affecting susceptibility in this experimental design and that added information would allow folks to evaluate that.

They state that the higher transmission rates in 2017 may be due to more infective inoculants which may be but it could also just be more susceptible genotypes were tested in 2017….or both.

I also appreciate them bringing the concepts of genotype diversity into restoration activities as many studies to date have only focused on a few traits e.g. bleaching resistance without considering disease susceptibility.  Hopefully, this paper will bring this problem out in the open so others can learn from it.


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: PROPERTIES OF A CRYPTIC LYSYL OXIDASE FROM HALOARCHAEON HALOTERRIGENA TURKMENICA
Review round: 1
Reviewer: 1

Output:
Basic reporting: . Although this is the first time to study the properties of lysyl oxidase from this archaeon as the authors claimed, the theoretical reasons for many experiment is not provided enough. Also, the manuscript is not prepared properly.  Please check the attached file for detail.
Experimental design: The rationale or methods is not provided enough. For example, the authors used two different polymerase but did not explained the reason. The information for the primers are not provided. Also, the rationale of making truncated LOX is not provided. Please check the attached file for detail.
Validity of the findings: Ther are some interesting findings but too many guesses which are not based on the results are inclued such as lines 241, 243, 244 etc.Please check the attached file for detail.
Additional comments: In this articles, the authors reported the expression and functional analysis of LOX protein of  haloarchaeon Haloterrigena turkmenica. Although this is the first time to study the properties of lysyl oxidase from this archaeon as the authors claimed, the theoretical reasons for many experiment is not provided enough. Also, the manuscript is not prepared properly. All suggestions and questions are marked on the attached file to save the space and the major concerns or problems are listed below. The authors could submit again after all this issues are resolved.

1.	Line 12: the corresponding author is not marked on the list of the authors
2.	Line 58-58: the presence of LOX gene some species cannot be the evidence of HGT.
3.	The authors used two different polymerase but did not explained the reason. The information for the primers are not provided. Also, the rationale of making truncated LOX is not provided.
4.	There are many places that are not proper in description such as Lines 88-89, 91, 93.
5.	There are many places at which the location of marked sentence in the attached file is not proper: for example, descriptions that need to be in the discussion section are in the Results section such as lines 125-128, 175-176, 178-180, 200-203, and results are in the discussion section such as lines, 230-232..
6.	There are too many guesses which are not based on the results including lines 241, 243, 244 etc.
7.	Some explanation is not theoretical: For examples the authors explained the low level expression of HTU-LOX is its moderate functional role in increasing availability of nitrogen from non-typical amines (lines 249-250), which does not accords to its function described on ln lines 53-53.
8.	Table 1. The authors said that data for bovine aorta is from other paper at which the reaction condition ist significantly different. In that case the data should not be used for comparison.
9.	There are too much unnecessary explanations in the Figure Legends which are marked in the attached file.
10.	Figure 5. Is this alignment necessary?
11.	Figure 6. The identity of HaloterrigenalimiCola is not provided.
12.	Figure 7:. The methods for selecting the sequence as the representative of each domain is not provided.
13.	Figure 8. If there is not enough space for full Species name, the GenBank accession number of each need to be provided. Figure 8 shows the relationship among different LOX gene based on the sequence of catalytic domain and it does not show the wat of gene transfer as the authors claimed on lines 232-235


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: PROPERTIES OF A CRYPTIC LYSYL OXIDASE FROM HALOARCHAEON HALOTERRIGENA TURKMENICA
Review round: 1
Reviewer: 2

Output:
Basic reporting: The manuscript by Pestov et al investigates the properties of the lysyl oxidase form present in the archaeon Haloterrigena turkmenica. While the validity of the findings and experimental design is discussed in the following sections, different aspects referring to the reporting of the manuscript are evaluated here. First, in general, the language used throughout the text is acceptable and structure of the manuscript conforms the journal standard. With respect to whether introduction properly shows the background of the field, there is a particular sentence that does not actually reflect what it is already known on phylogeny of LOX enzymes. Thus, in line 56 it is said that “most fungi are unknown to possess (LOX)”, whereas Grau-Bove et al, 2015 in a very comprehensive analysis of LOX isoforms in a wide variety of organisms already described the presence of homologues of this enzyme in some fungi. Regarding the figures, it is counter-intuitive to see plots composed by a group of dispersed points. My suggestion is to make bargraphs where a non-numerical x axis is displayed (Figure 2C), and line graphs in those cases representing a numerical axis (Figure 2A, B, D and Figure 3). Additionally, in the alignment shown in Figure 5, it is also difficult to see how the different deletion LOX constructs of H. turkmenica were designed and whether this makes sense in the context of the mouse sequence. Finally, information about the sequences used in the alignment shown in Figure 8 is not provided, nor the details of the method employed to generate it.
Experimental design: Original research in the manuscript is within the scope of the journal. Research question is in fact not clear: whether the authors want to analyze the properties of the H. turkmenica form or they want to have a look to the phylogeny of the LOX homologues. The former can be in fact interesting in the context of the mammalian forms, the latter has been already done in detail in the article by Grau-Bove et al, 2015.
With respect of the technical standard of the investigation, as discussed in the next section, protein characterization methodologies are of poor quality. Methods do not include any information about how alignments were done (insufficient description in the figure legend).
Validity of the findings: With respect to the findings, the authors claim that they were able to express, purify and refold a couple of deletion mutants of LOX from H. turkmenica, whereas Fig. 1 only shows the expression of one of them. To be more illustrative with the differences between these proteins and the mammalian homologues, optimization of refolding of these protein forms (Figure 2 only for one construct) should be compared with the mammalian homologue. This will answer the question of whether mouse or human LOXs present the same difficulties for purification, refolding and if they are equivalent in terms of activity. In any case, there is no information about the robustness of the data, whether they are replicates from different preparations, no statistical approach seems to be used.
The authors claim that they are generated a polyclonal antibody against the H. turkmenica protein and used it to characterize the expression of this form in these cells. In fact, quality of the blot is poor, and it is difficult to believe that the faint band observed at about 50 kDa can actually correspond to the endogenous LOX homologue, and therefore, it is very speculative to say that this can be a precursor form. Even more unrealistic is the statement that the growth of the cells without yeast extract resulted in the formation of a band of 3 kDa less mobility.
Finally, to my opinion, multiple alignment and phylogenetic analysis is also very poor, particularly considering the complete study performed in Grau-Bove et al, 2015.
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: PROPERTIES OF A CRYPTIC LYSYL OXIDASE FROM HALOARCHAEON HALOTERRIGENA TURKMENICA
Review round: 1
Reviewer: 3

Output:
Basic reporting: Please, add more details to Materials and Methods section. And all tests should be repeated in triplicate, and the authors should use proper statistical methods for the data in the paper.
Experimental design: no comment.
Validity of the findings: no comment.
Additional comments: Nikolay B Pestov et al., reported the heterologous expression and characterization of a novel lysyl oxidase from haloarchaeon Haloterrigena turkmenica. The author performed some results about the LOX, but the quality of the manuscript is far from publication. Here are some suggestions:

Major points:
1.	The “Method” part did not introduce the methods of tests used in this study such as Line 171-174 how to prepare the test of properties with different buffers; Line 190-208 activity measurement of substrates with this enzyme; Fig. 5-7, the aliment of LOX protein sequences; Fig. 8, the reconstruction method of LOX phylogenetic tree.
2.	The “Method” part should be more detailed on: 1) “activity assays”, how to define the activity unit should be added, because the activity should have the Unit and should be compared using Unit. 2) the primers used in this study should be summarized in a Table, and the sequences of genes used in this study should be submitted to Genbank or shown in supplementary files. And it is not clear what HTU-AA, HTU-QV and HTU-LOX represents.
3.	All tests should be repeated in triplicate, and the authors should use proper statistical methods for the data in the paper.

Minor points:
Line 31, “usual” to “regular”.
Line 38, why use “may be”?
Line 50, “LOX catalytic domain” to “Catalytic domain of LOX”.
Line 55-56, please provide a reference for the conclusion.
Lien 57- 61, please provide a reference for this conclusion.
Line 87-101, please just provide clear description of medium used in this study, but not results and discuss.
Line 94, what dose “modified from” mean?
Line 113, “Purified polynucleotide fragments HTU-AA and HTU-QV”. What does they refer to? Please give a specific explanation or reference.
Line 152 and 154, “TBST” to “TBST buffer”?
Line 179-180, “refolding efficiency is not significantly affected by NaCl concentration (Fig. 2B)”. In Fig. 2B, the activity of the enzyme change from 100% to <70%, which is obviously changed. Please revise this conclusion carefully. In addition, two peaks were showed in Fig. 2B, please reperform and check the results carefully.
Line 186, “in the cold”? please give a clear temperature or temperature range.
Line 187-189, the data should be shown in the manuscript, because this is important results. Or please delete this conclusion.
Line 213, “this band was reliably detected only at a high sensitivity”, how is the sensitivity? And the images in figure 4 are not in good qualities. Please reperform the experiment and get a better picture.
Line 226-227, please give the method and data about this result.
Line 230, more discussions are needed on sequence alignments of Fig. 5-7.
Line 232, “hyperconserved amino acid residues like Cu-binding His and TPQ formation.” Please provide the references for it.
Line 233-235 and Fig 8, please give a clear note of each genes (from which species and their classification in kingdom or phylum).


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: PROPERTIES OF A CRYPTIC LYSYL OXIDASE FROM HALOARCHAEON HALOTERRIGENA TURKMENICA
Review round: 2
Reviewer: 1

Output:
Basic reporting: Effort has been made in proper citing previous works on the field and in the presentation of experimental results. No more comments about this particular section.
Experimental design: I still feel that manuscript is a complicated mixture of phylogenetic and experimental results that hardly fit together.

Protein methodologies are still no acceptable. As explained below, I do not yet understand why authors do not validate their antibody with the recombinant proteins they have generated. Conclusions raised on these results are fully speculative.
Validity of the findings: With respect to the findings, the authors still claim that they were able to express, purify and refold a couple of deletion mutants of LOX from H. turkmenica, whereas Fig. 1 still shows only the expression of one of them. It is true that supplemental figures shows the two bands in a gel, but there is no information about how bacterial expression of the HTU-AA is induced.
The authors still claim that they are generated a polyclonal antibody against the H. turkmenica protein and used it to characterize the expression of this form in these cells. In fact, quality of the blot is still poor, and it is difficult to believe that the bands now appearing are in fact the proteins they expect. Validation of the antibody using the recombinant proteins is missing and this would be an important control. I feel it is too premature to say that high molecular weight precursors of LOX are expressed in these cells just by looking those heavy bands in the gel. Even more unrealistic is the statement that the proteolytic events are taking place to explain the different bands.
Additional comments: In general, I feel that the manuscript still presents serious concerns regarding the detection of the lox forms expressed in H. turkmenica cells as well as requires further characterization of the generated recombinant proteins.


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: PROPERTIES OF A CRYPTIC LYSYL OXIDASE FROM HALOARCHAEON HALOTERRIGENA TURKMENICA
Review round: 3
Reviewer: 1

Output:
Basic reporting: 'no comment'
Experimental design: 'no comment'
Validity of the findings: 'no comment'
Additional comments: The authors tried to show the presence of HTU-LOX in cultured cell. However, there is discrepancy in the molecular weight of proteins. The calculated molecular weight of the recombinant with the His-tag is 24.3kDa but the protein expressed in E. coli was 34 kDa. However, they detected a protein with 50kDa in cultured cell in addition to 210 kDa protein. So, the relationship among these proteins can not be explained. Furthermore, the 50kDa protein was detected with the non-specific antibody as shown Fig. 4.
If the authors expect that the 210kDa protein is a multimer, I wonder why they did withelectrophoresis with but that do not contain mercaptoethanol.
Also, the authors need to show the enzyme activity from host cell extract where they detected the protein by western blot.
Therefore, despite of revision, the questions raised from the first review have not been solved yet


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: PROPERTIES OF A CRYPTIC LYSYL OXIDASE FROM HALOARCHAEON HALOTERRIGENA TURKMENICA
Review round: 3
Reviewer: 2

Output:
Basic reporting: The article is clearly written and its experimental conclusions are well-supported, with some reservations regarding the validity and relevance of their phylogenetic analyses (see below).

Comments on the introduction:

Line 56-58 – The authors claim that “all animal genomes sequenced to date” have LOX genes (with exceptions). The cited study (Grau-Bové et al. Sci Rep 2015) does not support this strong claim. I suggest it is amended to “most animal genomes” or a similar phrase. Incidentally, the placozoan animal Trichoplax adhaerens should also be included among the exceptions (alongside Nematoda and Ctenophora).

Line 62 – The fact that archaeal LOX genes derive from a bacteria-to-archaea HGT is not an established fact, but rather remains a hypothesis. Confirmation of this hypothesis should include at least the identification of a donor bacterial lineage (currently impossible, given the lack of statistical supports in the phylogeny (Grau-Bové et al. Sci Rep 2015)). Thus, the authors should mention HGT as hypothetical (here and elsewhere in the paper), and then state that it is indeed the most parsimonious explanation given current phylogenetic and taxonomic knowledge.

Line 77 – I would suggest that the authors introduced a new paragraph here (“Haloterrigena turkmenica was isolated from Turkmenistani sulfate saline [...]”), for clarity.
Experimental design: See specfic comments about methods for phylogenetic analysis in "Validity of the findings". No further comments
Validity of the findings: Line 303-306 – The authors claim: “Although the second option is less likely due to splitting of animal ORFs into exons, it allows a more parsimonious explanation of the evolution of the catalytic LOX domain both in animals and bacteria”. This is a very strong claim that does not fit neither their results (Fig7 – see problems listed below) nor previous work (Grau-Bové et al 2015 Sci Rep). Animal LOX genes are monophyletic (bacterial sequences do not appear within the animal part of the tree) and actually descend from a LOX homolog that was already present in the last common ancestor of animals, ichthyosporeans and fungi.

Line 306 – LOX HGTs are not “obvious facts”. While they are a likely explanation and this is a valid background for the interest of LOX in Haloterrigena turkmenica, they remain hypothetical (see discussion in the introduction).

Line 262 and Fig. 7 – I do not agree with the authors’ interpretation of their phylogenetic analysis. They claim that LOX genes appeared in actinomycetes and then ‘spread’ to other prokaryotes. This assertion is ambiguous: do they refer to HGT or vertical inheritance? If they were talking about to HGT, how many HGT events do they claim did occur? My interpretation of their tree would not confidently support any (see below). If they meant vertical inheritance, they provide no rationale for that assertion of an actinomycete origin (e.g. some independent information that allows them to root the tree). Most importantly, the tree does not address the specific HGT event that is hypothesized to have transferred LOX to the ancestor of Haloterrigena turkmenica.

In addition to these questions of interpretation, there were major methodological issues which I will list below.

However, the phylogenetic analyses here performed are not essential to interpret the valuable functional analyses detailed in the results section. Thus, given that it does not affect the validity of the rest of the work, I would recommend that this analysis and discussion be removed from the present manuscript before publication. Whenever needed, the authors can refer to the results from previously published analyses (Figure S1 and S2 from (Grau-Bové et al. Sci Rep 2015)).

As an alternative, the authors could address all the methodological concerns (but this would require exhaustive reanalysis and rewriting – I  leave this choice for the consideration of the editor and authors).

These are the methodological issues:

(i) The aim of the phylogenetic analysis is not clear. As an example, it would have been interesting to identify the possible donor for the bacteria-to-archaea HGT. However, this issue is not addressed in neither the discussion nor the results. As a specific example, they could include a wider taxon sampling to identify close bacterial relatives of archaeal LOX that could be candidate donors (it would require some improvement in the taxon sampling compared to previous analysis, which is not the case – cf Figure S1 and S2 from (Grau-Bové et al. Sci Rep 2015).

(ii) The authors claim to have used approximate likelihood ratio test as statistical supports for their nodes, but this information is not displayed in Fig. 7. The authors should always display the statistical supports in all nodes of the tree. In addition, the interpretation of the aLRT metric in isolation can be unintuitive (see for example Minh et al MBE 2013, PMID: 23418397), and it is good practice to report more than one method of statistical support for the ML phylogeny (e.g. standard bootstrap replicates or UFBS). For example, Fig7 shows Sorangium cellulosum as sister group to Haloterrigena turkmenica, but without statistical support it cannot be determined whether the relationship is significant or not.

(iii), the authors should have used a phylogram instead of a cladogram (i.e. where branch lengths are proportional to phylogenetic distance). Following the previous example, this would allow the readers to examine whether Haloterrigena turkmenica is sufficiently close to Sorangium cellulosum to be deemed a good candidate for HGT donor.

(iv) Finally, the authors should include a more detailed description of their phylogenetic methodologies in the main text (Methods section). These details are important and should not be confined to a figure’s caption text. These improved methods must include: 1) how were the sequences retrieved from GenBank (blast? HMMER? From a previous publication? Etc.); 2) how did the authors define the catalytic region they used for the analysis; 3) how were the sequence alignments constructed and how many positions were included; 4) how were the alignments trimmed and how many positions were included; 5) which substitution model did the authors use (LG, WAG, did they include gamma categories, etc.) and how/why did they choose it (model selection can be assessed with dedicated software e.g. ProtTest; or with IQ-TREE, RaxML, etc.); 6) finally, as mentioned above, the authors should include, at least, statistical supports from standard bootstrap replicates (implemented in all major phylogeny inference tools e.g. Phyml, IQ-TREE, RAXML).
Additional comments: I fear that the use of ‘cryptic’ in in the title can be misleading. It suggests a ‘hidden’ role for the enzyme (e.g. in comparison to something else), but in actuality it is simply ‘unknown’ (hence the study). Elsewhere in the article, the correct meaning is clearer.


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: PROPERTIES OF A CRYPTIC LYSYL OXIDASE FROM HALOARCHAEON HALOTERRIGENA TURKMENICA
Review round: 5
Reviewer: 1

Output:
Basic reporting: No more comment
Experimental design: No more comment
Validity of the findings: No more comment
Additional comments: Line 187: Is this true mutants? It is a shorten form of LOX protein and not a real mutated form of an organism.
Line 194: the authors clam that the slow mobility of this protein is because it is acidic protein. Does the removal of N-terminal signal peptide the acidity of this protein? How was the mobility of full length HTU-LOX?
Line 126. Please indicate the location of AA and QV in LOX.
Lines 258-261 can be discussed in the Discussion section
Lines 296-297. What result supports this discussion (Refer the Figure).
The authors reported the assay for enzyme activity, but did not indicated how much enzyme they used for enzyme activity. On line 28 of the abstract, the authors reported that cultured H. turmenica did not have any enzyme activity but it is not described in the material and Methods, and Result section. How this experiment conducted? How much cell extract was used. Althoug they did not detected enzyme activity from the cultured cell, they detected a protein of 210kDa. If there is that much enzyme that present in the cell, why there is no enzyme activity? By running a known amount of purified protein with the cell extract, the authors can tell whether the absence of the activity is because the amount of enzyme in the cultured cell. Is the absence of enzyme activity because of the presence of antifuntional signal peptide on the 210kDa form? This could be discussed..


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: DIET AND MITOCHONDRIAL DNA HAPLOTYPE OF A SPERM WHALE (PHYSETER MACROCEPHALUS) FOUND DEAD OFF JURONG ISLAND, SINGAPORE
Review round: 1
Reviewer: 1

Output:
Basic reporting: Overall the basic report is quite good. However, I have some specific suggestions below.

1. Abstract - third sentence is not a complete sentence. Could change to something like “Here we report on the identification of stomach contents and mitochondrial haplotype of this individual, as well as hydrodynamic modelling used to determine the possible geographic origin of the individual.”

2. Lines 45-48: This sentences is a bit awkward. Perhaps something like “Genetic analyses have found that sperm whales are subdivided into a large number of groups based on mitochondrial DNA, whereas there is little-to-no structuring based on nuclear markers (refs). This fits with what is known about their social structure, where females show strong fidelity to specific areas, whereas males may wander around the globe.”

3. Line 93: I would change “consumed” to “present” because the animal could have consumed items without hard parts, which therefore would not be detected. I would therefore argue that it is impossible to tell everything that the animal consumed, all you can do is report on what was present in the stomach; there is a difference.

4. Line 115: Change “larger” to “large”

5. Line 126: Should be “tropical conditions”

6. Line128: Should be “ended up close to”

7. Line 214: Should be “known to forage”
Experimental design: Not so much of an issue with this paper, at least at a “big picture” scale, as it is just describing what was found in a dead sperm whale that washed ashore. However, the protocols used, and analyses conducted, are appropriate for the goals of the study and the data available.
Validity of the findings: Good. The authors summarize their findings well, and interpret them appropriately in the context of other available information on this species.
Additional comments: Overall it is a good paper. However, there is one fairly big topic that the authors should elaborate on. In lines 286-288 they mention that it had a large spinal injury, possibly caused by a ship strike. This seems extremely important, but is only given one sentence. If the authors are planning on reporting on the necropsy and its findings in a separate paper, then they should say so here (as readers will be expecting more information). Otherwise, they should greatly expand on this finding. For example, is there evidence of haemorrhaging that could indicate if the damage occurred pre- or post-mortem? Again, this is extremely important, because mortalities from ship strikes are an increasing threat to marine mammals around the world, but there are few data from this region, so this would be an important data point.


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: DIET AND MITOCHONDRIAL DNA HAPLOTYPE OF A SPERM WHALE (PHYSETER MACROCEPHALUS) FOUND DEAD OFF JURONG ISLAND, SINGAPORE
Review round: 1
Reviewer: 2

Output:
Basic reporting: No comment
Experimental design: No comment
Validity of the findings: No comment
Additional comments: I have previously seen this manuscript when it was submitted to PLOS ONE (don’t worry, I was the supportive Reviewer #1!). I have checked to see whether previous comments on the manuscript were addressed (most of which were), and if not, have re-mentioned them here (so I can see in your reply letter why you chose not to include those suggestions – perfectly fine if there is a valid reason...which might just be hoping not to get the same reviewer again!), as well as doing a fresh read through of the manuscript. I stand by my statement on the last version of the manuscript – this paper does a great job gleaning as much as possible from a stranding event in order to surmise what is going on in the nearby sperm whale population, and I’m very glad the authors have submitted it to a different journal. I have just minor editorial comments below.

Abstract
Line 24: Might be worth pointing out that one of the reasons that mtDNA haplotypes are worth looking at in this species are the strongly defined patterns of female philopatry.
Line 28: The comma following ‘Mesopelagic squids’, makes it seem like mesopelagic squids and then these specific species were found in the sperm whale. If these specific species are mesopelagic squids then it would be better to say ‘The mesopelagic squids X, Y, Z’ and then list the species names.
Lines 33-34: This is a pretty audacious claim - there have been other studies on sperm whales in south-eastern Asia so I think you need to modify the language to make it sound less like nothing is known of sperm whales from this area.
Line 34-36: Suggest modifying the language of this sentence to “The combined analyses of stomach contents, DNA, and hydrodynamic modelling could provide a context for future studies of sperm whale strandings, and have broader applicability for other marine mammals in the region.”

Introduction:
Line 40-41: “They are typically found in regions with deep seas” seems a little stilted. Potentially, “They are typically found in deep, off-shore waters”.
Lines 45-48: This sentence seems a bit overcomplicated. What about something along the lines of: “Sperm whale population structure and studies of the maternally-inherited mitochondrial DNA have shown differentiation between populations and suggest female philopatry, which has also been of interest to biologists [13–17].”
Line 51: “western” should not be capitalised – description, not location.
Line 65: Suggest clarifying why the fully erupted teeth point is important (e.g. ageing etc)
Lines 72-73: Suggest focusing on the broader take-homes I mentioned in response to comments on the abstract rather than on this narrower take.

Methods:
Line 77: Was her age-class as an adult established because of her size? This would be a good point to add in if so.
Line 85: Think this reference should be pointing to [28] not [27]
Lines 91: Could these references just read [27-31]?
Line 93: By number, or by estimated biomass?
Line 99: “sequences” rather than “sequencings”
Line 98-99: How many replicates? Two replicates for each tissue type? Need to be more specific.
Line 114: Think you can ditch the “the” in front of “larger tropical storm”
Line 116: Suggest removing ‘model called the”,  adding “was” before built, and “and” before was used

Results:
Line 137: Again, need to clarify whether percentage total is by number or biomass. Also suggest removing the word “represented”.
Line 138: Need to point out that this estimated mass is per individual squid, not in aggregate across all Taonius pavo.
Line 143: “was” not “is”
Line 165: “included” not “include”
Line 171: “Mitochondrial DNA haplotype identification” instead of “haplotype identification”

Discussion:
Although the authors do a good job summarising the broader implications of the diet analysis to lead off the discussion, I think you might need a broader opening highlighting the other main findings of your study
Line 192: But were fish were underrepresented due to digestion pressure?
Line 197: Need a space between typically and have.
Line 198: “different the different” – remove the first different.
Line 210: You can remove “a finding” from this sentence.
Line 211: Add an “a” before majority.
Line 214: Add a “to” before forage.
Line 223: Suggest changing “their” to “the male’s” because there is some confusion about what the subject of this sentence is e.g. the pyrosomes or the male sperm whales.
Line 241-242: Interesting point!
Line 258: Think this should be “common haplotypes” not “common haplotype”
Line 286-288: Or could the spinal injury have caused her immediate death?

Conclusion:
Line 297-298: Again, same comment about broadening this sentence out to make it seem less like it is the first research on sperm whales in southeastern Asia.
Line 300: Add “mitochondrial DNA” before “control”

Tables and Figures:
Table 1: I feel like this table might be better presented as a series of histograms/bar graphs/scatterplots representing this information graphically rather than in a long-form table. Would make it easier to compare between species at least. Either way, will need to clarify for the figure legend whether this percentage total is calculated by number or estimated biomass.

Figure 1 legend: suggest adding “simulated” in front of drogues

Figure 4: Add location of whale when it was located. Suggest adding “simulated” in front of first mention of “the drogues”. Line 187: suggest changing wording “that will end up” to “to have ended up”. Also suggest adding “on 10 July 2015” (because of the point you made in the manuscript that there are different strong seasonal effects).

Supplementary Materials
The video is a cool resource – I look forward to the day when such materials can actually be embedded in articles. One modification if it is not too difficult to make (and not necessary if it is), would be to mark the location of where the whale stranded on Jurong Island on this animation, so folks can easily see which of the drift simulations could have resulted in the whale being deposited there (similar to recommendations for Fig 4, and what the authors have already done for Fig 1).

The table with the squid lengths in it needs a little more context (potentially a readme tab?) so that it can be interpreted if someone just opens the file e.g. define LRL, what are the units, what is “-“ given for some records (e.g. beak too degraded to get measurements off?)?


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: HERVS, IMMUNITY, AND AUTOIMMUNITY: UNDERSTANDING THE CONNECTION
Review round: 1
Reviewer: 1

Output:
Basic reporting: In this manuscript, Greenig reviews and synthesizes recent findings relating to endogenous retroviruses in immunity and autoimmunity. Overall the review is very well written and was a pleasure to read, being both informative and insightful. The figures are also original and extremely well done. While there have been a few reviews relating to HERVs and immunity (in an evolutionary sense) or autoimmunity (from a disease-causing sense), no other review has directly bridged these largely distinct subfields. The unique cross-disciplinary perspective is properly introduced. Overall this is an excellent review which should be of interest to both specialists and newcomers to the topic of endogenous retroviruses and immunity.
Experimental design: The author first introduces the concept of ERVs. Then, he summarizes key findings of ERVs co-opted for immune responses for both protein-derived and regulatory functions. Key cases include mouse Fv1 and Fv4, and human MER41. In the next section, he summarizes our current understanding of HERVs in autoimmune diseases, focusing on multiple sclerosis and lupus as examples. The author next proposes several mechanisms linking co-opted ERVs to disease-causing ERVs.  First, he notes that AIM2 is regulated by an ERV, and aberrant AIM2 expression is linked to autoimmune diseases including lupus. Therefore, epigenetic dysregulation of a co-opted ERV promoter may contribute to disease. In a second example, he discusses HERV mRNA as a potential source of TLR7 overstimulation in lupus. In a final third example, the author speculates that polymorphism of HERV-K alleles may have modifier effects on Trim5a activity, which may contribute to multiple sclerosis.

Major comments:
-While several key studies are reviewed as illustrative examples, it would be useful to add a comprehensive table (or two) summarizing all published/experimentally validated examples where ERVs are contributing to immune function or disease with references, species, level of evidence, etc.
-Another example of co-option could be listed, ERV-derived dsRNA can also contribute to immune “priming” (Canadas et al, Nature Medicine 2018) which may connect with the proposed mechanism with TLR7.
-More disease-related examples could be drawn from neurodegenerative diseases, including ALS and schizophrena, which have some mechanistic overlap with autoimmune diseases.
-The sections (“HERVs and autoimmune disease” and “HERVs and autoimmune pathogenesis”) could be distinguished more as separate sections (or simply combined). The current organization is currently a bit confusing jumping back and forth across autoimmune diseases. Perhaps the first could specify “Current evidence associating HERVs and autoimmunity” and the second may be “Potential mechanisms linking HERVs to autoimmunity”

Minor comments

Line 196: Not necessary to report the P value from another study
Line 330: This paragraph (HERVs and SLE) was difficult to follow and contained many details that were not relevant from a clear argument. It should be rewritten for clarity.
Validity of the findings: The conclusions of the review are well-stated and provides insightful outlook relating to the challenges of linking HERV activity to autoimmune disease. I would suggest adding some additional discussion on how our understanding of HERV contributions to disease may impact the use/interpretation of animal models in autoimmune research.
Additional comments: This is a very nice and thoughtful single-author review, and very impressive considering the career stage of the author (undergraduate).


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: HERVS, IMMUNITY, AND AUTOIMMUNITY: UNDERSTANDING THE CONNECTION
Review round: 1
Reviewer: 2

Output:
Basic reporting: In this manuscript, the author reviewed literature about endogenous retroviruses with an emphasis on their impact on the host immune system, which fits in the general scope of PeerJ.  However, some fundamental issues needs to be addressed to make it suitable for publication, so we recommend a major revision.

The scope of the review needs to be better defined.  The manuscript is a update of recent advances in immunity-related topics of endogenous retrovirus.  This pertains to two major areas: ERV co-option into the host immune system, and ERV-related autoimmune disease, both of which have been reviewed recently.  Although some recent reviews have been cited, the author needs to layout a clear scope of what have been covered in those recent reviews and what have not been, then clearly define the scope of this review and focus on it.

The structure of the writing borders on superficial treatment of an extremely broad range of topics and points. The author should define the important points to address the above designed scope and build a clear organization of the text body. One useful revision would be to add a number of subheading to orient the reader and structure the logic.

The body of the manuscript equally addresses ERV co-option and ERV-related autoimmune disease, whereas the introduction almost exclusively talks about the former.  We recommend condensing the ERV co-option section of the introduction, and extracting some of the points of the ERV-related autoimmune disease section and move it to the introduction, if not re-writing the whole introduction.
Experimental design: When citing previous literature, the author tends to talk about the finding/conclusions in them.  Instead, the author should talk about the evidence found in the former studies, and then synthesize the evidence to support/reject certain points.  Some of these cases are listed in the "general comments" section of the review.

Key points need to be emphasized to improve the manuscript.  The author listed findings of previous works in the field, but these findings are disconnected given how they are written.  As suggested above, a clear definition of the scope of the review will help the author finding these key points, and other parts of the manuscript should revolve around the key points.  Such key points could be but not limited to: Why is ERV co-option a unique way to gain immunity, and what are the possible consequences (beneficial or deleterious) to the ERV and the host? Why are ERV-related autoimmune disease interesting compared to those triggered by other pathogens?

Some highly related topics have only been superficially mentioned without further discussion.  One of them is the ERV-host arms-race: the impact of ERV on host genome, particularly the evolution of the host innate immune system, is very relevant given the title of the manuscript.  The other case is rheumatoid arthritis in ERV-related autoimmune disease. There are rich literature about ERVs in rheumatoid arthritis, but it was only mentioned once in the manuscript without further discussion. This is very surprising given the detailed review of ERVs in MS and SLE in the manuscript.

The figures are essentially adaptations of textbook or other article figures without synthesis to support the focus of this review (when defined). A summary figure that shows how ERVs can affect the immune system will be very helpful to introduce the readers to the topic.  Figure 1 can potentially be adapted to such a figure with elements added. Indeed, although figure 1 shows the life cycle of retrovirus, it probably isn't worth a figure on its own.
Validity of the findings: no comment / see above and below
Additional comments: Title: the author needs to be careful on where to use "ERV" and "HERV".  Although the manuscript is titled to talk about "HERVs", some of the points discussed in the paper are not specific to humans. In fact, the co-option stories are all based on rodent genomes. Also, in the introduction, only use "HERV" when a certain property of an ERV is specific to human or only human-based evidence is available.

Figure 2: there is significant evidence that ERV transcripts are also important regulators of immunity (Wysocka, McFarland, Coyne, others).

Multiple lines: "et. al" should be "et al." and italic.

Line 71-72: retroviral RT happens in the cytoplasm (fig1 is correct).

fig1 do all retroviruses enter via endocytosis, not membrane fusion?

78-80: confusing presentation. Are you talking about new ERVs? Integration could be in gamete, germline, or early embryo.

80: "now" should be changed to "then".

82-85: humans are exceptional, as there are tons of species with active ERVs

89: "most significant" not appropriate

90: syncytins, also expressed in placenta not just precursor

94: syncytins are not HERV domestications

98: some intact ERV ORFs are not old enough to expect them to decay, decay is probabilistic

100: tissue breadth, not proportion of transcriptome

103: 'biological role' unclear meaning

110: intuition suggests (Delete)

115-126: "propositions are novel…fair and accurate…" This whole section is inappropriate; these are judgments of reviewers.  We suggest rewriting or removing it.

147: ERV not HERV, also see comments on the title

165 and fig3: there is no mention of 'rough ER', etc in text. Citation needed.

212-213: the connection here needs to be clarified.

215: need a definition of an autoimmune disease.

219: “MZ” only appeared once in the text and do not need to be abbreviated.  We suggest going through the manuscript and see if there are additional cases like this.

233-234: EBV-HERVs? This connection appears abruptly and only later discussed.

236-243:  there is a lot of statements without the important part which is the data. This section jumps around a lot - MS, EBV, others, MS, EBV…

252, 253: why is nexo underlined?

291: need a reference

303-305: this could probably be said for many immune genes.

402: "gag" should be italic. We suggest doing a global search in the manuscript for latin-derived nomenclatures. Also, "Env" should be "env" and italicized in multiple locations of the manuscript.


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

Input:
Title: HERVS, IMMUNITY, AND AUTOIMMUNITY: UNDERSTANDING THE CONNECTION
Review round: 1
Reviewer: 3

Output:
Basic reporting: This is a review on a very interesting topic, human endogenous retroviruses and their role in immunity and autoimmune disease. My overall feeling is that the review is not sufficiently broad to cover the title. The review focuses on very specific aspects of HERVs in relationship to immunity and autoimmunity and fails to cover all relevant aspects. Having said that i feel that if the review title was more specific, then it could make a sensible contribution, although I think the authors need to find how exactly this is going to happen. Some examples of missing important aspects (not an exhaustive list) relevant to the topic are info on the immunosuppressive properties of retroviral envelopes, the role of HERVs in relevant diseases (Aicardi Goutieres, ALS) etc.
Experimental design: See comments above
Validity of the findings: See comments above
Additional comments: -This is a review focusing on human ERVs, thus I am not sure what exactly is the purpose of the fV-4 in mice and why exactly the fV-4 example and not another ERV?
-ERVs are not only transmitted vertically
-"This hypothesis align with the observation that nearly all human ERVs....": I think this is not exactly true. There are active ERVs in animals which are also hosts thus if the above statement was the explanation of ERV inactivation this should be true for all hosts.
-"...pertaining to present day HERV activity...": I am not sure that "present day HERV activity" is a good choice of words for the specific situation.
-"Epigenetics are indisputably": are we so sure about that?
-Please check if the use of the term "homology" is correct where it is used. I think similarity would be more accurate. Homology means common ancestry, thus it is a "black-white" term. Two genes are either homologous or not.
-The acknowledgement of the manuscript suggests that Dr Mike Tristem has made a significant contribution, however he is not involved in the authorship? According to authorship criteria he could probably be a co-author. Has he been offered the opportunity to be a co-author (i.e. read the manuscript and comment etc) according to the authorship criteria recommended by ICMJE?


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: HERVS, IMMUNITY, AND AUTOIMMUNITY: UNDERSTANDING THE CONNECTION
Review round: 2
Reviewer: 1

Output:
Basic reporting: I have read the revised manuscript which has been significantly improved in its clarity and organization. I feel it is now suitable for publication in PeerJ.
Experimental design: NA
Validity of the findings: NA
Additional comments: Great job!


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: HERVS, IMMUNITY, AND AUTOIMMUNITY: UNDERSTANDING THE CONNECTION
Review round: 2
Reviewer: 2

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: In this revision, the author nicely addresses our previous concerns. The manuscript is now clearly defined in scope, focusing on the interaction between ERVs and the host immune system, with particular focus on HERVs and examples of human autoimmune diseases.  Compared to the last version, citations are now more concept-oriented instead of data-specific.  The introduction of EVEs, then gradually moving to ERVs and HERVs made the introduction more logically sound.  The added section pertaining to rheumatoid arthritis not only adds an example, but also highlights the potential role of HERV gag in autoimmune disease.

Overall, we believe that the revised manuscript is suitable for publication but would like to point out the following minor issues:

1) It would be worthwhile to revise the titles and/or figure legends of figures 2-4 to highlight the general principle addressed with the specific example drawn out in the figure. For example, Figure 2 is co-option against the receptor of an exogenous retrovirus.

2) The “host cell” in Figure 1 should be “host germline cell” for the ERV colonization and then co-option to make sense.

We also add that we very much appreciate the constructive tone and approach of the rebuttal letter.

Best,

Rick McLaughlin and Lei Yang


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: NONINVASIVE AND SAFE CELL VIABILITY ASSAY FOR EUGLENA GRACILIS USING NATURAL FOOD PIGMENT
Review round: 1
Reviewer: 1

Output:
Basic reporting: This is an interesting manuscript. However:
-	The language would benefit from revision to improve clarity. Sentences are often complex and difficult to understand. E.g. lines 73-75 state that E. gracilis can grow in heterotrophic cultures by photosynthesis, which is probably not what the authors meant. There are too many examples of unclear text to define them all.
-	The authors should use past tense to describe results and also results reported in the literature. Past and present tense should be used consistently throughout the manuscript. E.g. line 137 - "8 natural food pigments WERE tested in this study..." not currently being tested ("are") as indicated in the text.
-	The authors should avoid use of "etc." and "and the like". If there are more relevant points which they wish to raise, please state them. If not, please don't leave the reader guessing at what is being left out.

-	The introduction shows appropriate context and the literature is relevant, although the authors rely heavily on citing their own work, which may raise questions concerning the thoroughness of the background review. They could consider including methylene blue in their description of popular vital stains. Methylene blue is considered less toxic than trypan blue and in my experience is much more popular as well.
	- The statemen in lines 107-109 "The mechanism for TB to stain cells ..." should be supported by citing relevant literature.

-	The structure conforms PeerJ standards.

-	The figures are relevant and of reasonable quality. However, both spectra and photographs would benefit from having clear labels. i.e. the peaks in the spectra should be clearly labelled and arrows used to indicate stained and unstained cells in the photos in figures 4, 5 and 6.
-	Figures should be referred to where relevant. In general, Figures are not relevant to the section headings in the methods and should only be referred to in the methods if the figure illustrates the method - which I did not find to be the case in this manuscript.
-	It was not possible to access the supplementary (raw) data files, which were not in a format accessible to me. The file extension did not appear to be one accepted by PeerJ either.

-	The results are self-contained and relevant to the hypothesis, but quantitative data would be desirable.
Experimental design: -	The research presented was original and with the journal scope.

-	The research question (can non-toxic food dyes be used to stain algae?) was defined, but the description of why they considered it relevant and meaningful was not convincing. The manuscript would benefit if data would be provided which would demonstrate how the authors envision the stains being used in practice, along with control data obtained using currently available dyes. The authors claim that non-toxic stains are needed so that cells can be monitored in situ, but then always remove cells from the system in order to study them. They claim that cells should be monitored in situ so that the same cell can be monitored over time, but provide no suggestions as to how that same cell would be found within the larger population, if it were not removed for staining and was allowed to grow freely with its neighbours.

-	The investigation does not appear to have been rigorously performed, although the ethical standard is okay. There is no quantitative data presented, and wherever numerical data is mentioned, it is only as an estimate. This diminishes the usefulness of the research and reduces the probability of the dyes being adopted for use outside the lab which has generated the results.

-	The methods are difficult to understand and would be difficult to replicate. Improving the English will help improve method clarity. In addition, the authors should consider describing methods, rather than experiments. E.g. How were the the cells cultivated? What was the medium (-a) used? If C. reinhardtii was cultivated differently from E. gracilis, this should be in the cultivation section, not in a separate section where someone looking for methods might not find it. Did bright field microscopy differ depending on which samples were viewed? If so, this could be described in a microscopy section, not each variation being given its own section.
Validity of the findings: The paper presents spectra and photos obtained from staining the photosynthetic protest Euglena gracilis with 2 food-grade dyes. The authors demonstrate that the dyes penetrate and stain dead or dying cells, and speculate that this could be useful in monitoring the population over time. The work is interesting because good vitality stains are useful in many microbiological application. However, the paper is disappointing in that it does not include any quantitative data, nor any evidence of how they propose the stain(s) could be used in practice that would differ from the use of any currently used vital / viable stain.

-	The results are of interest to the scientific community.

-	No statistical analysis is provided. No quantitative data is provided. There is no evidence provided that staining the cells provided more accurate or useful data than assessing the population state without staining (see Figure 1 which clearly shows that dead cells of Euglena could be monitored without staining by making measurements arond 510 or 690 nm. Where is the experimental data comparing the results with and without staining? Where is the experimental data comparing the result with the new stains with a selection of old stains?

-	Speculation is extensive in the conclusions. It goes well beyond the data provided in the results. That it is speculation is obvious to a knowledgeable reader, but it is not always clearly identified.
-	The conclusions go beyond the original research question and are not supported by the results. E.g. The authors claim that a non-toxic stain is needed to allow in situ staining and monitoring, provide evidence that one of their stains is toxic to the organism being stained, and then conclude that this is a good non-toxic stain and recommend its continued use.

- The statement in lines 466-467 that "... this method... can be applied to the research such as basic study on the metabolic mechanism of the cell and breeding." has no basis in the results presented, nor do the authors indicate any way in which the method could be applied for this purpose.
- Claims concerning animal cell culture are irrelevant to the research presented.
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: NONINVASIVE AND SAFE CELL VIABILITY ASSAY FOR EUGLENA GRACILIS USING NATURAL FOOD PIGMENT
Review round: 1
Reviewer: 2

Output:
Basic reporting: The manuscript deals with viability test of microalgae Euglena gracilis with edible dyes. The reviewer suggests the author get some professional English proofreading, especially in table and figure legend section. (e.g. I could not understand the meaning of the legends of table 1 and 2) In some of parts, more references are needed. I would like to show detail in "General comments to authors" section. In my reviewing, I could not open .opj file with Origin viewer because of the permission trouble. So I could not say detail about the raw data.
Experimental design: In my understanding, the purpose of the experiment of the article was whether edible dye can used  to determine the cellular viability of Euglena. Because of the shortage of the control experiment, their results could not show these facts.
Validity of the findings: The authors need to add the appropriate control experiments to improve the manuscript.
Additional comments: Line 134-136. They mentioned the possibility of natural pigment in adding antioxidant function. Synthetic food dyes also have the same possibility. The authors need to show the references.

Line 159. The author shows AP interacts with both cellulose and pectin. In my knowledge, Euglena does not contain significant amount of these polysaccharides. If you have further information about polysaccharide content of Euglena (whether they contains cellulose and pectin), please show references.

Line 166-167. The author mentioned the possibility to use the DET method with edible dye in animal cells if they succeed in the experiments using Euglena. Cell structures of Euglena and animal cells are completely different. So I think they cannot estimate the possibility whether they can use the method in the animal cells or not.

In my simple view, when I compare the spectrum in Figure 1, 2, and 3, Figure 1 spectrum shows the most significant difference between living cells and dead cells. The author need to show importance of DET process to determine cellular viability in the method.
The author did not set the positive control experiment whether the all dead cell stained with MP or AP dye. For example, when Euglena mixed with Lugol’s iodine solution (contains iodine and potassium iodide), all the cells dies immediately. (In the comment about Table 1 (e.g. line 237-238), the author suggest the states of staining of dead cells, but the reviewer could not understand how they determine these states.) They need further description especially in the method section (line 233-238).
I also think the author need to show the comparison between the DET results with edible dyes and traditionally used dye in Euglena viability assay e.g. trypane blue or propidium iodide, because the reviewer could not tell the results shown here is reasonable or not.

In the results of Figure 5, they mentioned stained cell rate were different between AP and MP. In my understanding, if the viability of Chlamydomonas can evaluate with DET method with AP and MP, the ratio with these dyes shows almost same value. How do you understand the result?

The meaning of the experiment around Figure 6 was difficult for me. Would you tell me why you did the experiment? What is the purpose of this experiment?

There are many small mistakes in English (e.g. spelling of “Monascus” in line 274). Please check whole manuscript again.


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: NONINVASIVE AND SAFE CELL VIABILITY ASSAY FOR EUGLENA GRACILIS USING NATURAL FOOD PIGMENT
Review round: 2
Reviewer: 1

Output:
Basic reporting: In previous review, I suggested some English proofreading, especially in table and figure legend section. But I think the author did not changed the legends completely. I could not understand the meaning of legend for Table 1 (I could not understand the first sentence of Table 1, especially. The expression "food of pigment" is difficult to understand the meaning what the authors want to say.), as I mentioned. In addition, Table 1 contains the author's experimental data. I recommend revision of the title. Title of Table 8 is also need to revise. The authors need to check titles of text section (especially in Materials and Methods section 4 and 5). I recommend the authors to get English proofreading by native English speaker.
Experimental design: In Abstract lines 16-18, the authors mentioned natural pigments showed growth promoting effect and synthetic dye showed inhibiting effect. There are no statistic information in Figures 6 and 7, so I could not tell difference between control and natral pigment groups (it looks almost same). If they would like to show difference between them, they need to show statistic information for Figures 6 and 7. The y-axis of Figure 6 shold be started 0 (not 4x10E5). The author would better to check the cell density range of Figure 7 (a) and (b). I could not understand the meaning of values in Table 5 (data for Figures 6 and 7). If the experiment were done as Table 5, cell suspension only added in CM, MB, and MB+G culture, and the volume of cell suspension in CM and other two were different. The growth curve experiment should be done in same cell concentration condition among all groups. And I think the data in Table 5 is something wrong. Method about Figure 6 (line 302), the author wrote "precipitated", and I could not understand the meaning of this process. The authour would like to say "centrifused to concentrate"? (In usual case, appropriate dilution is recommended. In the method, first dilute the sample, and after that concentrate it, I felt it is not very good process.)

The following is additional comment. The growth of both culture almost same (Fig.6, 2.5x10E6/ml in CM 92h vs 3.1x10E6/ml in CM+Glc 86h), but in usual case, growth of Euglena gracilis in glucose added medium showed much faster than autotrphic culture (around 10 times faster). I could not understand why CM+Glc did not increase the growth.
Validity of the findings: The fact that natural food dye could stain dead Euglena gracilis was clearly showed. This is the most strong point in the manuscript, and I agree. But, I could not agree their conclusions written in last sentence of Abstract and last sentence of Introduction. It looks overestimate, because cellular structure and component of Euglena gracilis is completely different from animal cells and bacterial cells used in the medical and industrial use, as I said in previous revision comments.
Additional comments: As comments in above section, use of natural food dye in determination of cellular viability is important finding. In contrast, I could not catch the importance of the usage of scan-free absorbance spectral imaging to find the effectiveness of natural food dye (because the staining of AP and MP were very clear in visible image with normal microscopy). I recommend to the author to emphasis the importance of scan-free absorbance spectral imaging system to find the fact in this paper.


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: NONINVASIVE AND SAFE CELL VIABILITY ASSAY FOR EUGLENA GRACILIS USING NATURAL FOOD PIGMENT
Review round: 3
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: HEMATOLOGICAL PARAMETERS IN LARGEMOUTH BASS (MICROPTERUS SALMOIDES) WITH FORMALIN-PRESERVATION: COMPARISON BETWEEN WILD TOURNAMENT-CAUGHT AND CAPTIVE-RAISED FISH
Review round: 1
Reviewer: 1

Output:
Basic reporting: In general, the author’s use of language is clear and unambiguous.  There are a couple of minor awkward phrases, which will be identified below.  The abstract is concise and follows accepted format.  Enough information is presented so that the reader can grasp the intent, methods and results.  However, I would suggest that more emphasis is placed in the abstract and elsewhere on the relatively new hematologic procedures used and the benefits that the procedure provides for field studies such as this one.  While the authors rightly point out that there is a relative paucity of data on LMB hematology, I think this study could highlight the benefits of the 10% NBF treatment method to extend specimen viability in studies where it would be difficult to perform cell counts in a timely manner.  The slightly greater value of this article might be the demonstration of the specimen preservation techniques, which encourage more research in this area.  Perhaps introducing the need for these techniques in the background section and/or discussion of the abstract would enhance the manuscript.

The sections of the manuscript including introduction, materials and methods, results and discussion is well constructed following scientific formatting, concise, and well written.  I generally found the tables and plate of microphotographs well-structured and easily interpreted.  The authors provide a thorough introduction that provides relevant background information and problems associated with hematology of fish and other poikilotherms.  However, the authors did not present their hypotheses correctly (see lines beginning on 110).  They state that their null hypothesis is that there would be a disparity between treatment groups.  A null hypothesis in experimental design is that there is NO difference between or among treatment groups.  Predicting a significant difference in leukocyte counts would be an alternative hypothesis, of which there may be several.  The authors do need to rethink and restate their hypotheses.

The authors generally provide enough information in the materials and methods, including appropriate references so that the procedures they used are understood.  I would have liked to see a bit more details on how they handled individual blood specimens during the fishing tournament.  How soon after blood smears and PCV preparations did the authors mix and dilute whole blood into formalin?  They state that pcv and total solids were performed within 8 hours, but it is more important to know how the authors handled remaining whole blood (chilled?  agitated?) and the total time until blood-formalin dilutions were prepared.  The authors need to provide some additional information on this topic.

The results are presented concisely and clearly.  However, a little rewriting for the benefit of the reader may be in order.  I would suggest rather than state that results are “presented in tables” that the authors provide a short summary of relevant results in the text; such as “the median weights and lengths of fish in each treatment group were not significantly different (Table 1)”,  or, “the median weights and lengths of wild fish were greater than captive fish, but the differences were not significant (Table 1).”  The authors should state the most important results and let the readers then evaluate the tables, images, etc.  This is more compelling and interesting reading than simply providing a list of tables.

The discussion raises interesting points and presents the data of this study in appropriate context with previous work.  However, the authors used a relatively new technique to extend specimen viability that many fish health workers may not know about.  There needs to be a paragraph of discussion on the use of this technique including benefits/limitations and the author’s experiences thus far.  Do the authors have any comments on the usefulness or practicality of this technique during a field study in which it may not feasible to perform hemocytometer counts in a timely manner?  To me, increasing awareness of the 10%NBF technique to a broader community that may find it useful in fish health surveillance or research may be just as important, or more, then the actual results.  I’d like to read what the authors think since they are well-versed in hematological practices.

I have further comments regarding the discussion, which will appear below (general comments section).
Experimental design: I find the experimental design to be sound in general.  The results of this study provide useful information and comparison between captive fish and those that undergo the stresses of angling in a tournament setting.  The experimental design is limited in that a direct causal relationship of angling on hematologic parameters cannot be determined, and the authors were correct in not overstating conclusions.
Validity of the findings: The conclusions reached by the authors based on the methods employed and results obtained are valid and will provide useful information on LMB responses to the stresses of angling.
Additional comments: 25	Background:  why use 10%NBF?
42	I don’t necessarily agree that the results indicate superior health in wild fish.  It may be, but here are some other possibilities.  The decreased leukocyte count in wild fish may be a leukopoenia, which is a common stress reaction in animals.  Also, you detected a significantly elevated PCV in wild fish but not in RBC.  To me this suggests a shift in plasmatic water resulting in hypovolemia, or in other words, a “hemo-concentration”.  The elevated PCV in wild fish does not necessarily mean increased oxygen carrying capacity as the RBC numbers were comparable.  There may be the same number of RBC but in a smaller volume of plasma in wild fish.  Shifts in water to/from plasma as a result of osmotic changes to the gills and other tissues are a common stress reaction and should not be overlooked.  However, maintaining fish in aquaculture is also no guarantee of superior health.  There are a lot of stresses encountered in captivity based on type of containment, stocking density, DO levels, etc.  There are papers that demonstrate wide differences in hematology and plasma chemistry based on differences in conditions.  The following paper is 1 example.  There are others.

Matsche MA, Arnold J, Jenkins E, Townsend H, Rosemary K (2014) Determination of hematology and plasma chemistry reference intervals for 3 populations of captive Atlantic sturgeon (Acipenser oxyrinchus oxyrinchus). Vet Clin Pathol 43:387-396

So while this study does provide useful information, it is important to not overstate conclusions.  The authors have stated more than once that numerous factors influence hematology and there are likely marked differences in conditions and water quality between wild and captive fish.  The authors need to rethink and restate this discussion.

44	“…increased sample size…”  This is a primary benefit of specimen preservation!

110	Your null hypothesis should be N0: wild fish parameter = captive fish parameter.
	Alternatives may include: N1: wild fish parameter > captive fish parameter, and
	N2: wild fish parameter < captive fish parameter.

140	No need to make assumptions.  State that the fish sampled in this study appeared robust with no external lesions, if that was the case.  Save any discussion of cryptic health problems for the discussion.

150	Sample preparation in formalin.  I need to see more details as to how long before formalin dilution was performed and how specimens were handled prior to dilution.

174	Restructure awkward sentence.  Suggest starting paragraph with “Hotelling’s t-test was performed to compare…”, or similar.

191	Delete the word “contrarily”.
Discussion in general	As mentioned before, there needs to be discussion of the benefits/limitations of the 10%NBF technique to preserve specimens.

197 and on	The authors should restate this section based on changes that are needed regarding the statement of the null and alternate hypotheses.

Tables 1 and 2		Min-Max are presented in parentheses, which distinguishes this information from medians.  I would therefore remove parentheses from around samples sizes to avoid confusion.  There appears to be enough room to include sample size “n” as a separate column in the tables, but current layout is ok.


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: HEMATOLOGICAL PARAMETERS IN LARGEMOUTH BASS (MICROPTERUS SALMOIDES) WITH FORMALIN-PRESERVATION: COMPARISON BETWEEN WILD TOURNAMENT-CAUGHT AND CAPTIVE-RAISED FISH
Review round: 1
Reviewer: 2

Output:
Basic reporting: The work of Michelle C Whitehead et al. studies the Evaluation of A Hematological parameters in largemouth bass (Micropterus salmoides) with formalin-preservation:comparison between wild tournament-caught and captive-raised fish.

The study also included the analysis of the some Hematological indices.

Overall, the study contains good and valuable content; however, the novelty and the data of this article can be presented as a short communication finding, not as a complete article.

But there are several important issues that should be addressed before publication. The coherence of the text must be improved
Experimental design: See below
Validity of the findings: See below
Additional comments: Abstract:

It is suitable

Introduction:

- Line 86-87: “Talking about dietary lipid is excessive and useless.

 Materials and Methods:

The weighted average of the two groups is very different, and we can not generalize the results with respect to this weight difference for all categories.

Results:

It is suitable

Discussion:

In line 217: The changes in the count of white blood cells were related to age, size, and weight, but as I explained in the material and method the difference in weight and size in the tested fish is high and no countable results can be obtained.

Lin235-244: Because cortisol has not been measured in the present study, discussion of this is not feasible.


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

Input:
Title: GENOME-WIDE CHARACTERIZATION OF THE AUXIN RESPONSE FACTOR (ARF) GENE FAMILY OF LITCHI (LITCHI CHINENSIS SONN.): PHYLOGENETIC ANALYSIS, MIRNA REGULATION AND EXPRESSION CHANGES DURING FRUIT ABSCISSION
Review round: 1
Reviewer: 1

Output:
Basic reporting: My comments are included in "General comments for the author".
Experimental design: My comments are included in "General comments for the author".
Validity of the findings: My comments are included in "General comments for the author".
Additional comments: Zhang et al. reported comprehensive identification of auxin response factor (ARF) genes from lirchi, a commercially important fruit tree, especially in China. The litchi ARF proteins were named according to the phylogenetic analyses with ARFs from other model plants, Arabidopsis, rice and tomato. The introns and exons structures of the genes were compared among LcARFs, and conserved domains of ARF were identified. The authors also found target sites of miRNAs in the LcARF genes and discovered a novel miRNA pathway to regulate LcARF9s expression. The authors analyzed expression of all LcARFs in various organs/tissues by qRT-PCR and also analyzed effects of abscission-inducing treatments by RNA-seq. Throughout the manuscript, the experiments were well done and the results were satisfactorily and concisely described. However, the profiling of RNA seq data should be re-analyzed with a normalization method other than RPKM because comparison among samples with RPKM has been demonstrated to be inappropriate (Wagner GP, Kin K, Lynch VJ. Measurement of mRNA abundance using RNA-seq data: RPKM measure is inconsistent among samples. Theory Biosci. 2012, 131(4):281-5.).

Other points
1. In line 162, “Class I” should be collected as “Class II” according to Fig. 1.
2. In line 195, the authors described that “all LcARF genes were expressed in all organs/tissues studied”, although in some tissues, the expression levels are very low based on the results in Fig. 4. Generally, by qRT-PCR, it is difficult to distinguish very low accumulation of mRNAs from contaminated genomic DNAs or non-specific amplification. In the context of the manuscript, it is unlikely necessary to claim the significant expression in all tissues. The authors can mention that expression of all ARFs was detected at least in several tissues, which still means that all ARF genes found in this study are likely functional but not pseudo genes.
3. In line 197-199, the authors described that the class I and class III ARFs were expressed at higher levels in “ML”. The authors have defined that ML is “mature leaves”, thus these genes may function in mature leaves but not in “male flower development”, which is described in line 199.
4. In line 242, the authors described none of the LcARFs seems to be activator. As the authors mentioned, there may be no activators with glutamine rich AD, but the data could not exclude the possibility that some ARFs may have other types of ADs.
5. Please indicate SE or SD for the error bars in Fig. 4B.
6. In the graph for LcARF9B in Fig. 4B, the numbers of Y-axis are not located at equal interval. I don’t know the journal rule, but I have never seen the numbers in axes of graphs showing as “.5” and like that.


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

Input:
Title: GENOME-WIDE CHARACTERIZATION OF THE AUXIN RESPONSE FACTOR (ARF) GENE FAMILY OF LITCHI (LITCHI CHINENSIS SONN.): PHYLOGENETIC ANALYSIS, MIRNA REGULATION AND EXPRESSION CHANGES DURING FRUIT ABSCISSION
Review round: 1
Reviewer: 2

Output:
Basic reporting: 'no comment
Experimental design: 'no comment
Validity of the findings: Please do some RACE to confim some miRNA target ARFs
Additional comments: The article is  in good writing. But "phylogenetic analysis" and "evolution" are much different. There are some TasiRNA in “miRNA regulation” . Please modify your title of manuscript.