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: SEARCHING FOR PHYLOGENETIC PATTERNS OF SYMBIODINIACEAE COMMUNITY STRUCTURE AMONG INDO-PACIFIC MERULINIDAE CORALS
Review round: 1
Reviewer: 2

Output:
Basic reporting: The paper surveyed endosymbiont diversity and community structure in merulinid corals and report large intraspecific variation, as well as the apparent influence of geography on community structure.  The paper is written in clear language, and is well-organised. The introduction and background is sufficient and concise, providing context to the paper’s objectives.  The research question is well-defined.  Figures and tables are well-presented.
Experimental design: The research question is well defined, relevant and meaningful in the context of biological diversity studies and to some extent global change biology.  Ethical standards for field collection and sampling, and technical standards for methodology and data analysis conform to high standards.  The methods (including data analysis) are described with sufficient detail and are appropriate.
Validity of the findings: The data is robust and self-contained.  Overall, the conclusions are well-stated, with cautionary statements on certain aspects where the data provide limited support, i.e. lines 254-258.The spatial scale of the paper is commendable, but therein also lies its limitations which the authors rightly acknowledge, i.e. the lack of finer scale sampling which may provide better insight into patterns driving community structure at smaller spatial scales.
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: SEARCHING FOR PHYLOGENETIC PATTERNS OF SYMBIODINIACEAE COMMUNITY STRUCTURE AMONG INDO-PACIFIC MERULINIDAE CORALS
Review round: 1
Reviewer: 3

Output:
Basic reporting: Searching for phylogenetic patterns of Symbiodiniaceae community structure among Indo Pacific corals written by Leveque et al examined Symniodiniaceae communities of 8 Merulinid species collected from 4 different locations in the Indo-Pacific by sequencing ITS2 using NGS. Based on the non-metric multidimensional scaling with the Bray-Curtis dissimilarity and Jaccard distance measures, authors found Symbiodiniaceae communities are not clustered according to coral species or genera and/or their host phylogenetic patterns, though intra-species patterns dominate the variation. The wide-range of Indo-Pacific sampling of phylogenetically well-defined coral spices enable the hypothesis testing proposed in the introduction. The result presented here is contrasting with the previous study of Pocillopora and worth publishing.
Overall the manuscript is well-structured and well-written. I just would like to suggest several points to meet PeerJ’s criteria.

1.	Raw data for MiSeq run should be deposited and opened before acceptance.
2.	There is no materials and methods about Fig. 1 (even if the tree is from Huang et al 2014b, it will be better to open the FASTA data and briefly describe material and methods). It will be helpful to have material and method information on them at least in the supplementary material.
3.	Regarding the spatial structure of Symbiondiniaceae, citing contrasting results by Thornhill D, (Xiang Y, Fitt W, Santos S (2009) Reef endemism, host specificity and temporal stability in populations of symbiotic dinoflagellates from two ecologically dominant Caribbean corals. PLoS ONE, 4, e6262.) would be also useful.

The title is very attractive but it will be better include “Merulinid or Merulinidae” because not all Indo Pacific coral species have been examined.
Experimental design: Overall there is no significant problem
But authors should be careful about using Go-taq because the sequences obtained using this taq contain a lot of artificial errors (1 bp over 300 bp) and caution is required especially when using NGS.
Please briefly describe how these errors can be ignored in this study (I think this does not affect the large classification used in this study. But not sure).
L188-L200 Please briefly describe the rationale for using these criteria (97% identity etc)
L204-209 The analysis conducted in this study is good enough to deduce the conclusions, but it might be also useful to use “taxonomic distinctness index”. Warwick RM, Clarke KR 2001. Practical measures of marine biodiversity based on relatedness of species. Ocean.Mar.Biol.Ann.Rev. 39, 207-231
Validity of the findings: L304 Overall, our results show that coral communities
→coral Symbiodiniaceae communities?
Fig.2 How do Arthurs interpret the relatively huge error bars in Cladocopium for G. favulus and G. retiformis)?
Additional comments: nothing


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: SEARCHING FOR PHYLOGENETIC PATTERNS OF SYMBIODINIACEAE COMMUNITY STRUCTURE AMONG INDO-PACIFIC MERULINIDAE CORALS
Review round: 2
Reviewer: 1

Output:
Basic reporting: The paper has been greatly improved especially following the recommendation by the reviewer1's comment to use "proportional abundance".
I'm happy with the revised version and I think it worth publishing.
Experimental design: Improved and met the standards
Validity of the findings: No comment
Additional comments: Please take care of ABS when you use genetic materials from abroad in the future.


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: RHOPALOCNEMIS PHALLOIDES HAS ONE OF THE MOST REDUCED AND MUTATED PLASTID GENOMES KNOWN
Review round: 1
Reviewer: 1

Output:
Basic reporting: This paper is well-written overall.  I have noted a few places on the manuscript where small wording changes might be made.  I would suggest citing the recent review paper on plastome genome evolution which appears to be missing:

Graham SW, Lam VK, Merckx VS. 2017. Plastomes on the edge: the evolutionary breakdown of mycoheterotroph plastid genomes. New Phytologist 214: 48-55.

Also, in the Literature Cited, the book shown to have been written by V. Merckx was really edited by him, so individual chapters should be cited.
Experimental design: The methods are explained in good detail.  Graphics are useful and informative.  The report of a genome that is this small is of interest and pushes our knowledge of extreme plastomes forward.
Validity of the findings: The analyses are appropriate and the conclusions follow in straightforward manner from them.  Speculative parts of the paper, such as why the genomes of these plants are so AT-rich, are identified as such.
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: RHOPALOCNEMIS PHALLOIDES HAS ONE OF THE MOST REDUCED AND MUTATED PLASTID GENOMES KNOWN
Review round: 1
Reviewer: 2

Output:
Basic reporting: This is a very interesting, well written and overall well executed study of a highly modified plastid genome in the nonphotosynthetic plant Rhopalocnemis phalloides (Balanophoraceae).
Experimental design: -- The results have not been reported on before, and this work fills in a gap in our knowledge about a poorly known group of holoparasitic plants. Like other recent reports of nonphotosynthetic plastid genomes (plastomes) in other lineages, the one described here is highly reduced through loss of photosynthesis and other genes. The plastome is unusual in other ways, including its very high AT content, as also described here. The research question is well stated.

-- The rrn16 analysis (not a huge part of the paper) is a bit odd because the taxon sampling used is really tiny. With the number of taxa included here, and the substantial distance among them, I think that most plastid genes, even of photosynthetic taxa, might behave oddly. For this reason I am not fully convinced that it fully demonstrates convergence or is the only reason for taxon attraction in the tree. The analysis might be improved with a better representation of other major land-plant lineages, for example.

-- Explain why rrn16 was chosen for phylogenetic analysis at the relevant point of the M&M (i.e. around l. 292: this was not really clear to me until later). This particular gene seems like an odd choice, as it is unusually slowly evolving in photosynthetic plants (because it is usually found in the slowly evolving IR) and so will comprise a more extreme example than other genes. This may add to the unusual results here. I am guessing the gene was used because it is one of the few genes retained in common between this organism and non-land-plants.

-- What steps were taken to make sure the plastid contigs did not include plastid inserts in the mitochondrial genome (I assume rejecting contigs that included mt genes).

-- I appreciated the extended explanation of the strategy used to find genes here (l. 171-214), which can be a difficult task in these highly modified heterotroph plastomes.

-- I think the contamination detection procedure (l. 216-235) is most relevant to the discussion of the nuclear genome (e.g. 536, Table S5), but this was unclear to me until I had read the whole paper. A brief explanation of the purpose of this procedure should be included in the M&M section.
Validity of the findings: -- Su et al. (PNAS doi: 1816822116) recently reported retention of trnE genes in other Balanophoraceae species that are likely non-functional in protein synthesis, but could still be functional for their alternative role in haem-biosynthesis. Could that be the case here? The tRNA analysis and/or discussion (particularly for this gene) could be re-done in light of this.

-- I also wondered if the ‘false-positive’ trnE reported here is located close to one or more genes it is found in for photosynthetic organisms (or other Balanophoraceae). More generally, how does plastid genome structure here compare to other photosynthetic or non-photosynthetic relatives (e.g., Su et al. 2018)? How about including a brief Mauve analysis to summarize this?

-- I appreciated that caveats to estimating dN/dS ratios were laid out in the Results/Discussion (l. 479-503). The logic of why genes with open reading frames in a lineage with high AT content are likely to still be under strong negative selection (estimation of no. of stop codons if random) is a simple but effective way of thinking about this.
Additional comments: Overall I thought this paper was well written and said thought-provoking things.

I’d like to see the findings reported here put into context with recently published findings for two other Balanophoraceae plastomes (Su et al. PNAS doi: 1816822116) in terms of things like AT content, gene content, unusual trnE presence/possible function, novel genetic code, genome structure, lack of inverted repeat, and other findings reported there.

I think it could be stated more directly what the authors think was the main reason for success in eventually obtaining the plastid genome after initial failure to do so. Was the main breakthrough just the substantial increase in depth of coverage (l. 94), the complex strategy used to recover plastid genes (l. 271-214), a combination of these… or something else? This could be relevant for other purported losses (e.g., Molina et al. 2014, reported for Rafflesia), or for other uncooperative taxa.

I am not sure I buy the rationale for why the AT content is so high (i.e., linking this to possible loss of nuclear recombination genes, with that favoured as a mechanism to rapidly degrade plastid genes that have lost function but are still translated and so potentially interfere with other processes, presumably). A high mutation rate (in plastid or other genomes) could have many very undesirable pleiotropic effects – would these really outweigh the benefits from getting rid of a few photosynthesis genes? However, this speculation is still worth including, and I like the suggestion to look for loss of DNA-RRR genes in other heterotroph nuclear genomes.

I understand that the authors may not want to release the raw sequencing reads yet if working on other aspects of it. The plastid genome seems to have an NCBI number (MK036331, noted in Table S1), although this could usefully be stated in the main text too. Perhaps the mitochondrial contigs could also be released here, instead of by request, as the authors state they won’t work further on these.

Minor points

Para starting l. 46 – gene loss and retention (e.g., of the five genes mentioned here) was also reviewed in Graham et al. (2017 New Phytol 214:48-55), which could be cited here.

l.23. ‘outstanding’ used in the Abstract is unusual English usage. Perhaps use something like ‘extraordinarily high’ instead.

l. 34. it is not clear that full mycoheterotrophs are fully parasitic. Workers in the field are usually cautious about claiming so; I believe the thinking is that perhaps the fungi get something other than photosynthate from the symbiosis. It is hard to disprove that they don’t of course, but mycoheterotrophs clearly started off as mutualistic, while holoparasites did not. So there may be something to this objection.

l. 70. Make clearer here that there must be relaxation of selection in these genomes (explaining photosynthetic gene loss), although that doesn’t enter into this argument much.

l. 83. It would be safer to call this a *probable* loss of a plastome in Rafflesia, as perhaps one could be recovered with additional effort (as was done here, and later suggested for Rafflesia, l. 393).

l. 153-154. Briefly state/explain the problem noted by Shah et al. (2018).

l. 297. ‘SAR’ should be explained on first usage (not just spelled out later in the same paragraph).

l. 330-331. The speculation that RNA components of the plastid ribosome (rDNA gene products) may be imported was raised in Graham et al. (2017 New Phytol 214:48-55), and has been speculated on for quite a while for tRNA genes (e.g., Wolfe et al. 1992).

Fig. 4 (and S2). I find these trees hard to read because of the very dark colours used behind taxon names. I am also red-green colour blind, so Fig. 2 is the worst possible colour scheme for me and a large fraction of the population.

l. 406 and elsewhere. Avoid ‘interestingly’ (let the reader decide what is interesting)

l.467. Remind reader than only two genes of R. phalloides were included here. The total number of genes used for Fig. S5 could also be noted in the figure legend there.

l. 503. You could usefully note that (much) better taxon sampling could help with dN/dS estimation here.

l. 581. ‘semi-crumbled’ is evocative, but ‘semi-degraded’ might sound more scientific in English?


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: RHOPALOCNEMIS PHALLOIDES HAS ONE OF THE MOST REDUCED AND MUTATED PLASTID GENOMES KNOWN
Review round: 1
Reviewer: 3

Output:
Basic reporting: The introduction and background are well done, with relevant issues presented and appropriately referenced.

The structure of the paper conforms to disciplinary and Peer J standards.

As far as the raw data being supplied, if what is meant here is that, in the case of this particular paper, appropriate GenBank and SRA accession numbers were provided, then yes, the raw data were supplied.

The writing uses reasonably professional English and is certainly better than most papers I see that are written by non-native English speakers.  However, there are a number of places where the writing could be improved (e.g., rewrite the phrase “many times discussed phenomena” on page 19) or where an inappropriate word is used (e.g., also on p. 19, change “reparation” to “repair”, specifically “DNA repair”).  If possible, the authors should enlist a native-English-speaking scientist who is reasonably close in field to this paper to give it a quick read for grammar and word choice.

Three of the main figures should be improved as follows:

Fig. 1.  Font sizes should be increased significantly throughout the figure.  The colored arrows indicating genes should heavier/thicker so that the color differentiation is more apparent.  The internal (boxed) legend should either be deleted (and this information provided in the regular legend) or moved below the actual figure so that the figure can be larger and more readable.  In this figure, in other figures, and in the text, numbers larger than 999 should include commas (as in “2,000”) rather than a space (as in “2 000”).

Fig. 4.  Please reduce the intensity/darkness of the three colors used so that the black lettering is more easily read.

Fig. 5.  The current format makes it very hard to readily compare the various values on the branches.  I think this figure would be much improved if it were recrafted so that the cladogram is shrunken greatly in width, with the only numbers on it being 1-8 (one number on each of the eight branches), and with the d, dn, etc. values placed in a table underneath the figure, with the 1-8 numbers being the first column in the table.
Experimental design: The experimental design is fine as is, with the Materials and Methods section being appropriately thorough, detailed, and informative.  This study represents a rigorous investigation.
Validity of the findings: The data in this paper are robust, and the conclusions are generally well stated and drawn – in the context of the relevant literature available to the authors at the time of manuscript submission – and are relevant to the questions and results of this study.

There is, however, a big issue here.  On Dec. 31, 2018, subsequent to submission of the current manuscript, a very highly relevant paper was published online in PNAS with the title “Novel genetic code and record-setting AT-richness in the highly reduced plastid genome of the holoparasitic plant Balanophora”.  (Note that this I was a coauthor on this PNAS paper.)  It is imperative that the current MS be thoroughly revised to include all relevant comparisons to and discussion about the Balanophora findings, this being a member of the same family of parasitic plants as Rhopalocnemis, and with the two sets of plastid genomes sharing a number of interesting, derived features – most notably with respect to high AT content – yet also differing in one or two important ways.  Among these revisions are:

A)	Please point out wherever appropriate, starting with the abstract, that the two sequenced Balanophora plastid genomes are slightly more AT rich than the R.p. genome, and, naturally enough, have very similar codon and protein usage biases.

B)	The authors should consider comparing the AT richness and lengths of those genes that are held in common by the R.p. and Balanophora genomes, as it evident from both papers that there is considerable variation in AT-richness on a gene by gene basis, while the Balanophora paper also reports a significant reduction in size for a number of its protein genes, with ycf2 being exceptionally reduced in size (but apparently less reduced in R.p.).

C)	Both Balanophora genomes should be included in Fig. 2.

D)	It would be best to include at least Balanophora genome in Fig. 3.

E)	One, and possibly both, Balanophora genomes should be included in Fig. 5.

F)	The authors should carefully consider whether the Balanophora genomes should be included in any of the supplementary figures and tables.

G)	Please compare the inferred gene content between the two genomes.  As annotated, the R.p. genome contains two genes (rpl16 and rpl36) that appear to be absent from the Balanophora genomes.  Vice-versa, the Balanophora genomes contain four protein genes (rps2, rps4, rps11, rpl14), one tRNA gene (trnE), and one rRNA gene (rrn4.5S) that appear to be absent from the R.p. genome.  I think it is incumbent on the authors to investigate even more closely than they already have done the possibility that they have failed to detect one or more of these six genes (as part of this they may of course choose to see whether, conversely, the Balanophora study missed rpl16 or rpl36).

H)	Somewhat related to the last point, the authors should calculate how much of R.p. genome is non-genic (i.e., not exons or introns, including, as done in the Balanophora paper, excluding a guesstimate for the length of the trans-spliced intron of rps12) and compare this value to those of the Balanophora genomes.  The Balanophora paper makes a big point about how compact the the two Balanophora genomes are, while Fig. 1 suggests that the R.p. genome is a good bit less compact.  On the other hand, the larger spacer regions in R.p. do raise the possibility that the authors, despite the care and thoroughness with which they’ve already addressed this issue, somehow failed to detect one or more plastid genes.

I)	Assuming, as is probably the case, that R.p. does in fact lack the trnE gene, then this is a particularly important gene content different relative to Balanophora that deserves a bit of discussion, especially given that the Balanophora trnE gene is proposed to function only in heme synthesis, but not in protein synthesis.

J)	The authors should consider pointing out one notable difference between these genomes, which is that the Balanophora genomes have lost all cis-spliced introns, whereas the R.p. genome contains 4 cis-spliced introns, all of them located in genes that are present in the Balanophora genomes.

K)	The authors MUST discuss the most important apparent difference between these genomes, which is that the Balanophora genomes use a novel genetic code, in which TAG has been reassigned from stop to tryptophan, whereas implicit in the fact that there is no mention of the genetic code in the current manuscript is that R.p. presumably uses the standard genetic code.  As part of this, the authors should carefully examined their alignments and annotations to see if there’s any possibility that R.p. might share with Balanophora the TAG code deviation.  Is it possible that there is only a few, even just one, amino-acid position in an R.p. protein gene in which TAG appears in-frame at a site that is normally TGG/trptophan in other plants?  Related, could some of the current annotations be extended with respect to homology to other plants by reassigning an in-frame TAG from stop to Trp?  One possibility that should be considered in assessing a possible code change in R.p. is that in contrast to Balanophora, where the genetic-code change is “complete” (i.e., TAG is used only for Trp, i.e., never as a stop codon), it could be “incomplete” in R.p.  That is, is it possible that R.p. uses TAG for BOTH Trp and Stop.  Relevant here are references 56 and 57 in the Balanophora paper, which report cases where two unrelated green algal plastid genomes use TGA in this manner, i.e., as both stop and sense codons.

Here are a few suggestions, mostly minor, for improving the text as currently written:

The last two sentence of the brief Conclusions section are not conclusions but constitute a statement about an intended follow-up study on the nuclear genome/transcriptome of R. phalloides.  One solution here is to change the title of the section to “Conclusions and future studies”.  On the other hand, the nuclear study is just one of many possible future/follow-up studies and it is already mentioned in lines 537-539.  So it might be better to exclude the last two sentences of the Conclusion section and beef up the actual conclusions part.

Abstract:  Please change “Their genomes, especially plastid genomes, markedly differ…” to “Their plastid genomes markedly differ…” as I don’t think one can safely generalize int that the mitochondrial and nuclear genomes of heterotropic plants “differ” (much less “markedly") from those of photosynthetic plants.

Lines 57-58:  I suggest qualifying “ycf1 (encodes a component of the translocon…) to something like “ycf1 (though to encode a component of the translocon).

Line 83:  Please point out that in the case of Rafflesia, it’s possible that the authors of the Rafflesia study failed to detect a highly divergent plastid genome, a possibility raised in both the Hydnora and Pilostyles papers, as well as by the authors of the current studies themselves, later on in the manuscript.

Line 322 and in the legend to Fig. 1, it’s important to qualify/change “circular” to “circular-mapping”.
Additional comments: Overall this is a well done, thoughtful, and interesting study.


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: RHOPALOCNEMIS PHALLOIDES HAS ONE OF THE MOST REDUCED AND MUTATED PLASTID GENOMES KNOWN
Review round: 2
Reviewer: 1

Output:
Basic reporting: See below
Experimental design: See below
Validity of the findings: See below
Additional comments: The revised MS is nicely improved, with appropriate inclusion of (in text, figures and tables) and comparision to the recently published Balanophora plastome sequences, as requested by myself and reviewer.

My only substantial concern is with Fig. 4, where I agree with reviewer 2’s point about the tiny taxon sampling (I feel quite amiss that I didn’t raise this point myself in my review) and disagree with the author’s rebutta.  To do a meaningful phylogenetic analysis of this issue requires far more comprehensive taxon sampling, not only of other land-plant lineages but also a thorough sampling of the SAR group.  To include only the best BLAST hits to Rhopalocnemis seems almost preordained to lead to the kind of result shown in Fig. 4B.  Keeping the figure as is will only detract from an otherwise fine study.  If the authors are not prepared to do a proper sampling of taxa than I think it would be in their own best interests to delete this figure and analysis, and to just let the BLAST results stand on their own as evidence of potential convergence due to high AT-content.  In this respect, the authors might consider stating in the text the AT content of the 16S rRNA genes of Nitschiza, Plasmodium, etc.

Line 449:  “is supposed to be” unnecessarily (and I’m sure unintentionally) casts suspicion, doubt on the conclusion of Su et al. that Balanophora uses a non-canonical code.  Please reword, preferably to “is” as in “…the genetic code in the plastid genomes of Balanophora is non-canonical….”


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 AND EXPRESSION ANALYSIS OF AQUAPORINS IN SALT CRESS (EUTREMA SALSUGINEUM)
Review round: 1
Reviewer: 1

Output:
Basic reporting: The paper is interesting and merits publication in PeerJ, but manuscript needs major improvements before being suitable for publication.

The use of english must be improved and need a style revision. In the present form many sentences are confusing or difficult to understand.

Literature references and introduction is Ok.

Article structure, figures and table presentation is Ok.

The hypothesis and the way to adress it is Ok.
Experimental design: Table1 has a major problem. All the localizations are in the plasma membrane or the tonoplast, and only 1 in the chloroplast. It is difficult to believe that Eutrema has no mithocondrial located aquaporins (TIP5;1 is an aquaporin specifically targeted to pollen mitochondria and is probably involved in nitrogen remobilization in Arabidopsis thaliana
Gabriela Soto  et al) or in the ER (Plant Cell Environ. 2018 Dec;41(12):2844-2857. doi: 10.1111/pce.13416. Epub 2018 Sep 12.BvCOLD1: A novel aquaporin from sugar beet (Beta vulgaris L.) involved in boron homeostasis and abiotic stress.Porcel R et al) Clearly the algorithm is not working properly. Authors must complete the table with the published localization of the conserved aquaporins in order to give more accuarte information.
Validity of the findings: Findings are interesting, but the presentation of them is very poor and must be improved.

The problem with the present paper are not the experiments itself, but the way they are commented or the conclusions drawn need major improvement.

The discussion is too long and the conclusion is merely descriptive. it is clear that aquaporins are very similar to the ones found in arabidopsis, as expected, but which are the diferences? Do these diferences justify the differential salt tolerance of both species? Authors should redran the discusion and the conclusion and focus in which are the major findings of this papers, specifically, in the differences from arabidopsis, rather than just a lengthy description of the conservation with arabidopsis, very redundant with the results section. Which aquaporins are differentially expressed? Which of them are not conserved, if any? These questions should be clearly adressed and the reader should not look for them among a mess of irrelevant descriptions.

Data is ok.
Additional comments: Please, mind the previous suggestions and the paper will be greatly improved.


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 AND EXPRESSION ANALYSIS OF AQUAPORINS IN SALT CRESS (EUTREMA SALSUGINEUM)
Review round: 1
Reviewer: 2

Output:
Basic reporting: The study presents the genome-wide analysis of aquaporin (AQP) genes in salt cress (Eutrema salsugineum). However, the English language of the manuscript should be improved to ensure that your international audience can clearly understand.
Experimental design: In this manuscript, two genome assemblies at scaffold or chromosome level were used in this study. In my opinion, the chromosome-scale assembly is more suitable, however, the authors first identified AQP genes from the scaffold-level assembly and then aligned to the chromosome-scale assembly. Please explain why? Moreover, comprehensive comparison of AQP genes from two assemblies is highly recommended.
Validity of the findings: Several analyses (e.g. gene structure and motif analysis) presented in this study were purely based on the automatic genome annotation. I am afraid it would be misleading. More evidence should be provided.
Additional comments: Other suggestions are as follows:
1.	Change “functioned” to “functioning” in line 17.
2.	Rewrite lines 24-33.
3.	What is the latin name for moss in line 59.
4.	Rewrite lines 60-61.
5.	Change “stress” to “stresses” in line 79 and 92.
6.	Rewrite lines 83-85.
7.	Add for after “search” in line 101.
8.	Change “Characters” to “Characterization” in line 148.
9.	Synteny analysis between salt cress and arabidopsis is highly recommended.


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 AND EXPRESSION ANALYSIS OF AQUAPORINS IN SALT CRESS (EUTREMA SALSUGINEUM)
Review round: 1
Reviewer: 3

Output:
Basic reporting: The mauscript is clear and unambiguous.
Experimental design: The original primary research and the research question are fine.
Validity of the findings: The findings in the manuscript seem fine. Its quality  wil be much better if the predictions could be tested by experiments
Additional comments: Qian et al have characterizes the AQPs in E.salsugineum, which will favor further studies on AQPs roles played in abiotic stresses. However, I think the manuscript could been  improved from the following points:
1.	The authors should combine function analysis (AQP function or plant physiological function), but not only the expression data on mRNA level.
2.	The primers used should be listed. The PCR efficiency for each primers should be checked in qPCR. The authors did not show the reference gene were stable in stress conditions. Multiple reference gene should be tested and the qPCR should be done according to Vandesompele et al.2002.
3.	The authors clarified that all the 35 AQPs were expressed in all organs. How to explain the TIP3s usually specific expression in seeds in other species, for instance in Arabidopsis?
4.	Do the authors believe the prediction of the subcellular localization by these two kinds of soft used? The prediction should be tested with AQPs, whose localization has been investigated in other species. And the prediction should be confirmed by experiments.
5.	The English should be improved. For instance, the sentence in Line 83 - 85 is not complete.

Minor points:.	The authors should explain what is MEME motif.


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 AND EXPRESSION ANALYSIS OF AQUAPORINS IN SALT CRESS (EUTREMA SALSUGINEUM)
Review round: 2
Reviewer: 1

Output:
Basic reporting: Authors have substantially improved the manuscript, also the english is much better, but please change "conversed" by "conserved"
Experimental design: Meets with the criteria
Validity of the findings: Meets with the criteria
Additional comments: Authors have made a great effort to meet the criteria of the journal


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

Input:
Title: GENOME-WIDE CHARACTERIZATION AND EXPRESSION ANALYSIS OF AQUAPORINS IN SALT CRESS (EUTREMA SALSUGINEUM)
Review round: 2
Reviewer: 2

Output:
Basic reporting: Most concerns have been adressed. However, the English language of the manuscript should be improved before publication.
Experimental design: No comment.
Validity of the findings: No comment.
Additional comments: Most concerns have been adressed. However, the English language of the manuscript should be improved before publication.


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

Input:
Title: MENTAL PRACTICE IN ISOLATION IMPROVES CERVICAL JOINT POSITION SENSE IN PATIENTS WITH CHRONIC NECK PAIN: A RANDOMIZED SINGLE-BLIND PLACEBO TRIAL
Review round: 1
Reviewer: 1

Output:
Basic reporting: ok
Experimental design: ok
Validity of the findings: ok
Additional comments: TITLE
1.	ok.
ABSTRACT
1.	ok

INTRODUCTION
2.	ok
METHODS.
3.	okhas been used.
RESULT
4.	ok
DISCUSSION
5.	could be re-written to describe in brief.
Tables
6.	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: MENTAL PRACTICE IN ISOLATION IMPROVES CERVICAL JOINT POSITION SENSE IN PATIENTS WITH CHRONIC NECK PAIN: A RANDOMIZED SINGLE-BLIND PLACEBO TRIAL
Review round: 1
Reviewer: 2

Output:
Basic reporting: The English language is appropiate, the background and literature references are correct, figures and tables are very useful, raw data is provided and relecant results are presented.
Experimental design: The aims are with the scope of the journal, the research question is well defined. Please, see comments to authors in order to improve this manuscript. Ehics information is added. Methods are well described.
Validity of the findings: The validity of findings is well described. Conclusion are supported by the study findings.
Additional comments: Thanks for the opportunity to review this interesting manuscript. This is a well designed clinical trial that provided importants considerations for the biomedical field. Please, consider the following issues/comments according to the presented manuscript:

ABSTRACT
-Please, consider add subheadings to the abstract.

INTRODUCTION
-Paragraph from line 40 to 55: Please, a phrase linking sentral sentization to motor control alterations would be useful at the end of tis paragraph (please, reference this paargraph appropiately).

METHODS
-The study was adequately designed and registered prospectively at clinicaltrials.gov.
-References to support inclusion and exclusion criteria would be useful. In addition, the Declaration of helsinki should be cited.
-Randomization, blinding and interventions were well-detailed.
-Reliability coefficients and minimum detectable changes were adequately provided for the primary outcome.
-Reliability coefficients need to be provided for some secondary outcomes tools such as Tampa scale, NDI and level of physical activity.
-A sample size calculation is lacking. Please, provide this calculation or if it is not possible, please, include this issue in limitations and detail this manuscript as a pilot study.

RESULTS
-Tables and figures were well presented and provided useful information.

DISCUSSION
-Please, consider to dicuss about some future studies comparing AO and MI with other interventions such as dry needling or ischemic compression of myofascial trigger points (Acupunct Med. 2019 Jun;37(3):151-163. doi: 10.1177/0964528419843913) and mobilization of the upper cervical region (J Oral Rehabil. 2019 Feb;46(2):109-119. doi: 10.1111/joor.12733) to improve cervical motor control in subjects with non-specific neck pain.


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: MENTAL PRACTICE IN ISOLATION IMPROVES CERVICAL JOINT POSITION SENSE IN PATIENTS WITH CHRONIC NECK PAIN: A RANDOMIZED SINGLE-BLIND PLACEBO TRIAL
Review round: 1
Reviewer: 3

Output:
Basic reporting: well written paper covering an interesting area of physiotherapy.
Experimental design: No comment
Validity of the findings: No comments
Additional comments: Please see attached 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: MENTAL PRACTICE IN ISOLATION IMPROVES CERVICAL JOINT POSITION SENSE IN PATIENTS WITH CHRONIC NECK PAIN: A RANDOMIZED SINGLE-BLIND PLACEBO TRIAL
Review round: 2
Reviewer: 1

Output:
Basic reporting: Ok
Experimental design: Ok
Validity of the findings: Ok
Additional comments: I congratulate to the authors due to the manuscript has widely improved and the authors have responded all my prior comments. Thanks for your contribution to the physical therapy field.


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: MENTAL PRACTICE IN ISOLATION IMPROVES CERVICAL JOINT POSITION SENSE IN PATIENTS WITH CHRONIC NECK PAIN: A RANDOMIZED SINGLE-BLIND PLACEBO TRIAL
Review round: 2
Reviewer: 2

Output:
Basic reporting: The basic reporting is clear and professional English is used throughout.
Experimental design: Good experimental design.
Validity of the findings: Conclusions and supported by research findings.
Additional comments: The authors have revised the manuscript very well and have attended to all comments made.
Well done on this very interesting piece of work. Well done on providing very clear clinical implications.


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: RELATIONSHIP BETWEEN ROS PRODUCTION, MNSOD ACTIVATION AND PERIODS OF FASTING AND RE-FEEDING IN FRESHWATER SHRIMP NEOCARIDINA DAVIDI (CRUSTACEA, MALACOSTRACA)
Review round: 1
Reviewer: 1

Output:
Basic reporting: 1. Line 53, I suggest inserting the preposition "in" before the word adverse.

2. Line 55, I suggest inserting the article “the” before the word processes.

3. Line 62, It is suggested to improve the description of the function and response of mitochondria in the oxidative stress.

4. Line 68, It is  suggest mentioning some components of the superoxide dismutase family and their importance in oxidative stress.
Experimental design: Line 163. It is suggested that the authors describe the number of fields analyzed per experiment.
Validity of the findings: There is no description of figure 3 in the Results section.
Additional comments: The experiments are well performed and the findings are are in agreement with previous studies. I suggest discuss the importance of N. davidi in other types of studies and as a model to understand oxidative stress at the cellular level due to long-term starvation.


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: RELATIONSHIP BETWEEN ROS PRODUCTION, MNSOD ACTIVATION AND PERIODS OF FASTING AND RE-FEEDING IN FRESHWATER SHRIMP NEOCARIDINA DAVIDI (CRUSTACEA, MALACOSTRACA)
Review round: 1
Reviewer: 2

Output:
Basic reporting: The study is interesting. The authors analyzed the production of ROS and the presence of the MnSOD in freshwater shrimp after starvation and refeeding in hepatopancreas and intestine. They report that long term starvation provokes an increase in ROS and in MnSOD and after refeeding, the values decrease.  The association of starvation with oxidative stress is a very interesting topic.
In general the ms is clear and well presented in all the sections.
Experimental design: The experimental design is appropriate and clearly presented. In materials and methods, it is important to inform that the antibody used for the inmunolocalization of MnSOD does not cross react with other proteins and it was validated in Western blots.  Additionally, several crustaceans have a citosolic MnSOD in addition to the classical mitocondrial enzyme and the antibody used might be detecting the 2 MnSODs. . Check paper published by Dr. Brown group and also data for  l. vannamei and  chinese crab, here are the DOIs
DOI 10.1042/BJ20030272, DOI: 10.1016/j.cbpb.2012.03.003, DOI:10.4238/2014.November.11.8
Validity of the findings: The resultasaare well presented and clear. I am not sure that the format used for the tables is the one required by Peer J., since the title should be at the top and not at the bottom and this should be concise, while the additonal information presented shoul be a footnote .
In figure 2, in the legend, a sentence is duplicated.
Additional comments: The resultss are well presented and clear. I am not sure that the format used for the tables is the one required by Peer J., since the title should be at the top and not at the bottom and this should be concise, while the additonal information presented shoul be a footnote .
In figure 2, in the legend, a sentence is duplicated.
General comments for the authors
It is an interesting study that adds information about responses of aquatic animals to oxidative stress derived from starvation.  Check spelling and gramar in the whole paper, for instance the inset in figure 3, “ intestine” is not spelled correctly


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: RELATIONSHIP BETWEEN ROS PRODUCTION, MNSOD ACTIVATION AND PERIODS OF FASTING AND RE-FEEDING IN FRESHWATER SHRIMP NEOCARIDINA DAVIDI (CRUSTACEA, MALACOSTRACA)
Review round: 1
Reviewer: 3

Output:
Basic reporting: 1. English: correct, not professional enough
2. Literature references: week
3. Structure of the article: Abstract and Introduction have unnecessary details, but relevant information are omitted. Figure and table legends are not appropriately described
4. Results are not relevant to hypotesis and discussion is rather speculative
Experimental design: Nothing original.
Not well described with some unexplained issues.
Tecnical and ethical standards not given.
Validity of the findings: Not satisfied.
Additional comments: The manuscript entitled “Relationship between ROS production, MnSOD activation and periods of fasting and re-feeding in freshwater shrimp Neocaridina davidi (Crustacea, Malacostraca)" by Włodarczyk et al. represents the study of the digestive system in Crustacea where the authors use transmission electron microscopy and histochemical methods to detect and localize the ROS positive cells and MnSOD enzyme after periods of fasting and refeeding in shrimps.

There are to many speculative conclusions based on the scarce data.
The major flaw of this manuscript is that the authors insist on oxidative stress and elevated MnSOD activity after different periods of refeeding in shrimps.
Oxidative stress is much more than simple increase of ROS positive cells and localization of MnSOD.
Although mitochondria are the main intracellular source of ROS generation, these ROS also have physiological function, likeH2O2 which is used as a secondary messenger to coordinate oxidative metabolism with changes in cell physiology.
 At least, authors could measure the activity of MnSOD, not just concentration, because elevated concentration doesn't seem its elevated activation. Some proteins can be synthesized in large quantities, but inactivated (some protein modifications)at some stage of their activation.
In addition, mitochondria are enriched with the antioxidant defenses required to degrade ROS, beside MnSOD, glutathione (GSH), thioredoxin (TRX) as well as CuZnSOD settled in its intermembrane space.
One should analyze a battery of antioxidants to be relevant for discussion on oxidative stress.
MnSOD and CuZnSOD catalyze the dismutation of O2•− to H2O2. Although mitochondria do produce O2•−, the dominant ROS in the matrix environment is H2O2 (Ryan J. Mailloux, “Mitochondrial Antioxidants and the Maintenance of Cellular Hydrogen Peroxide Levels,” Oxidative Medicine and Cellular Longevity, vol. 2018, Article ID 7857251, 10 pages, 2018.)

It is known that crustaceans experience starvation periods during their growing process as a result of sequential molting. Like authors emphasized, the starved condition has pro-oxidant effects due to the reduction of the antioxidant defense levels. But what about the adaption of these organisms?
Also it is not clear, why authors used both females and males in their study (how many females and how many males). Is there any sex difference in response to starvation?
Certainly it is not enough to argue the results obtained on just four or five individuals.

Overall, I don't recommend this article to be published in Peer J.


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: RELATIONSHIP BETWEEN ROS PRODUCTION, MNSOD ACTIVATION AND PERIODS OF FASTING AND RE-FEEDING IN FRESHWATER SHRIMP NEOCARIDINA DAVIDI (CRUSTACEA, MALACOSTRACA)
Review round: 2
Reviewer: 1

Output:
Basic reporting: In the introduction section the observations that I suggested were taken into account, so now it is clearer, the description of the oxidative stress in Neocaridina davidi is better understood.

The components of the superoxide dismutase family and their importance in response to oxidative stress were included as part of the introduction.
Experimental design: They describe in the section of material and methods the number of samples analyzed in this study.
Validity of the findings: The description of the results of figure number 3 are now described in the results section.
Additional comments: In general, the manuscript describes much better the experimental work done by the authors. In this article, they describe the importance of Neocaridina davidi as a biological model to understand the enzymatic role in oxidative stress under starvation conditions.


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: PLANT HOST AND DROUGHT SHAPE THE ROOT ASSOCIATED FUNGAL MICROBIOTA IN RICE
Review round: 1
Reviewer: 1

Output:
Basic reporting: Clear language was used throughout unless otherwise noted.

Sufficient background, literature, and context provided.

Good article structure.

Specific Comments:

This is not the first study to analyze the fungal microbiome in rice when exposed to drought, as the authors state in lines 359-360 and 383 - 384. Santos et al, 2017 (which the authors have cited) conducted a greenhouse experiment analyzing the bacterial and fungal microbiome. I certainly understand the authors missing that the fungal microbiome was analyzed in the Santos study. The analysis is almost hidden as the last supplemental figure in the paper and it was not heavily discussed. I only know this because I was a co-author on the paper. There are nice similarities between the two analyses, and this certainly does not take away from the strength of the manuscript here. Andreo-Jimenez et al. conducted a field experiment (compared to a greenhouse experiment) and used 18S sequencing, which in my opinion is a better barcode than ITS. The authors include 17 genotypes (compared to 4). The authors only need to adjust the language here to reflect how their study substantially expands upon our knowledge of the rice fungal microbiome under drought and well watered conditions (which it definitely does).

I am very confused by the paragraph on lines 330-334. It could use rewriting.

Line 386: I think the word “enrichment” is misleading here. The authors intend to mean an increase in alpha diversity, but enrichment could mean other processes such as overall abundance of fungi are increasing under drought or specific OTUs are increasing under drought. The authors have not shown whether either of these scenarios are happening, therefore they should be more precise when discussing this as an increase in alpha diversity.
Experimental design: This is original primary research that falls within the Aims and Scope of PeerJ

The research question is well defined and definitely meaningful.

Rigorous investigation was performed.

Specific Comments:

An interesting aspect to this manuscript that remains under analyzed is the effect of genotype. The authors report a significant effect in the variance partitioning, but it would be nice to visualize the difference between genotypes in drought and control conditions separately. For example, a PCoA, NMDS, or CAP ordination for each treatment would be nice to see. Do the similarities between the genotypes change depending on the treatment? Is the genotype effect more evident under drought or control conditions, i.e. is there a significant interaction term between watering treatment and genotype in the PERMANOVA? Is there one genotype which is very divergent (in terms of fungal microbiome structure) compared to the others? I think these are all interesting questions that geneticists and breeders will ask when they see the experimental design.

I think it would also be possible to factorize the rice genotypes in the authors analysis, rather than using kinship values. For example, the genotype variable could be coded [G1, G2, G3...Gn] rather than having numerical values. It is unclear in the literature (at least from what I have read) the extent to which bacterial and fungal microbiomes correlate with relatedness - rather than simply varying between genotypes. For instance two closely related genotypes may differ substantially, while two distantly related genotypes may host microbiomes that are relatively similar. I think this should be an optional analysis for the authors to conduct - but still it interests me greatly.
Validity of the findings: In this manuscript, Andreo-Jimenez et al. survey the fungal portion of the root-associated microbiome of field grown rice varieties in control and drought conditions. The authors find that drought stress impacts the composition of the root fungal microbiome, with drought stressed roots hosting a fungal microbiome with greater diversity than control plants. They also find that genotype significantly affects the composition of the fungal microbiome. Because the authors collected yield data for the selected rice varieties, they were also able to correlate the abundance of specific fungal taxa with grain yield. After finding interesting correlations, the authors used isolates of Arthrinium phaeospermum to examine how root and shoot phenotypes would be impacted by the addition of putatively beneficial fungi. The authors found the A. phaeospermum isolates affected root growth negatively. I think these are very interesting results and are relevant to plant breeders and microbial ecologists alike.
Additional comments: Great job. I really like the structure of this study from going to the field and collecting microbiome and plant yield data and then trying to take what was learned from the study to follow up in the greenhouse. I think the field could use the structure of this study as an example. It is a shame that the fungal isolates did not yield conclusive results - but I like how the authors discussed the various possibilities around that and how to move forward with 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: PLANT HOST AND DROUGHT SHAPE THE ROOT ASSOCIATED FUNGAL MICROBIOTA IN RICE
Review round: 1
Reviewer: 2

Output:
Basic reporting: ARTICLE STRUCTURE.
Overall, the paper is clearly written and well organized. I only have a few comments that would hopefully improve the interpretability of some of the figures:

Fig.1.
Even though the y-axes in all panels are labeled the same, it seems the actual values displayed are only log-transformed in some of them. In particular, the values on the y-axis in the Phylum panel clearly represent the actual number of OTUs displayed on a log10 axis (the sum is ~900, which matches the reported number of OTUs found in the study), while the numbers displayed on the y-axis for the rest of the taxonomic ranks seem to be log-transformed values. For consistency, I would suggest to use a uniform format to avoid any confusion.

Fig. 2.
Indicate which groups showed statistically significant differences between drought and control communities.

Fig.5.
Label the occurrence ratio axis.

DATA AVAILABILITY.
The raw data is available, although I would also encourage the authors to share their scripts to facilitate the reproducibility of the analyses described in the paper.
Experimental design: RESEARCH QUESTION.
The authors tackle a relevant question in the field of plant microbe interactions: how do environmental stresses affect the community structure of the plant-associated microbiota and how does the host genetic makeup shape this response. By including 15 rice varieties, the authors generated a useful dataset to delve into this problem. While they found some interesting patterns in the alpha and beta diversities of these communities, I wished they would've expanded on some other aspects. For example, even though the authors found that drought significantly shifted the overall composition of the fungal microbiota of rice roots, they didn't go into a more detailed analysis of the changes in relative abundances of  individual OTUs responding to this environmental stress. A small discussion about drought-mediated changes in frequency (Fig. 5), yet this analysis is restricted to the OTUs positively correlated to yield. In my opinion, including a section describing the differential OTU abundance patterns between drought-stress and well-watered communities could further improve the breadth of this study: how many OTUs are driving the changes in microbiome structure? are there any taxonomic patterns behind drought-enriched or drought-depleted OTUs? Are the changes in relative abundance affected by the host genotype? Similar analyses have been recently applied to drought-stressed root bacterial communities on rice (Santos-Medellin et al. mBio 2017) and other grasses (Naylor et al. ISME 2017).

METHODS.

- Many of the results discussed in this manuscript are based on the patterns of presence/absence of OTUs between the various experimental factors (e.g., richness, OTU overlap between treatments, and occurrence ratios). Yet, there is no mention as to how the authors dealt with the potential differences in sequencing depth across samples. While they filtered out low-abundance OTUs (those with less than 10 sequences in the whole data set), this approach doesn’t account for uneven total sequence counts between libraries. If there are big differences in sequencing depth between samples, this could complicate the interpretation of diversity patterns as the absence of OTUs could be a consequence of relatively shallow sequencing in some libraries. An option to deal with this issue is to rarefy the libraries to a uniform depth (see Weiss, S. et al. Microbiome 2017: 5:27)

- Lines 295 - 298. I am not totally sure why the authors didn't use raw Bray-Curtis dissimilarities to perform the PerMANOVA as this is the standard approach in microbiome studies. Also, I don't really know what they mean by rank orders. Are these the ranks of the raw BC pairwise dissimilarities? I ask the authors to elaborate more on their rationale to follow this approach or to redo the analysis with the absolute BC dissimilarities.
Validity of the findings: The authors did a good job on stating their conclusions and not overstating their findings. Furthermore, I commend them on including the experiment testing the effect of fungal isolates on the plant fitness even though it yielded results opposite to their expectations.

I only have a couple of minor comments:
Lines 265 - 269. These two sentences seem to contradict themselves. The first one indicates that taxonomic resolution was at the species level, while the second one states that most OTUs couldn’t be classified at the species level. Rephrase.

Lines 403 - 406. These two sentences contradict themselves. The first one states that drought affected Glomeromycota richness, but the the second one states the opposite.
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: PLANT HOST AND DROUGHT SHAPE THE ROOT ASSOCIATED FUNGAL MICROBIOTA IN RICE
Review round: 2
Reviewer: 1

Output:
Basic reporting: Fine.
Experimental design: Fine.
Validity of the findings: Needs a few tweaks in the Discussion. Please see attachment.
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: PLANT HOST AND DROUGHT SHAPE THE ROOT ASSOCIATED FUNGAL MICROBIOTA IN RICE
Review round: 2
Reviewer: 2

Output:
Basic reporting: Most of my previous comments were satisfactorily addressed.

Minor comments:

- While the authors added a scale at the bottom of Fig. 5, I still think the x-axis should show the values to ease interpretation.

- L293 - should be Fig S3 instead of FigS4
Experimental design: The authors addressed my previous comments
Validity of the findings: No further comments
Additional comments: No additional comments


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

Input:
Title: MONITORING PUBLIC AWARENESS ABOUT THE ENDANGERED NORTHERN BALD IBIS: A CASE STUDY INVOLVING PRIMARY SCHOOL CHILDREN AS CITIZEN SCIENTISTS
Review round: 1
Reviewer: 1

Output:
Basic reporting: The overall English is good, I have a few suggestions to improve the language. For example: Line 33 instead of local population, use local community; Line 66: since you use “as well as”, you can delete “both” in this sentence; Line 68: place “indeed” before “benefits”; Line 132: please replace “since” with “for”. Overall, this sentence should be re-structured since it reads rather awkwardly.

In my opinion, the provided results are not sufficient to properly address the stated hypothesis/research question. The abstract as well as the introduction suggest that educational activities with children as multipliers led to an increase of knowledge on the ibis population in the region. However, this has not been tested; the results are purely answers to the same knowledge questions in 2012 and 2016 and their descriptive comparison.
Experimental design: The research questions were twofold: 1) assess differences in the knowledge about the local ibis population in 2012 and 2014 and 2) evaluate the effectiveness of educational activities. Whereas the first research question was answered, albeit only descriptively and with no statistical analysis, the second question has only been addressed speculatively in the discussion. There was neither a data collection regarding educational activities (i.e. question about participation in activities in the questionnaires) nor a description of any of the activities that were on offer. I therefore think that the methods, subsequent results and their interpretation do not match the research questions of this study. Furthermore, if it was just the first research question (without speculation about the effect of educational activities), I have further suggestions for the analysis:
Line 152: I would call it evaluation instead of investigation.
Line 159: how were children taught to make sure that people were only interviewed once?

Line 162-165: This is just a descriptive analysis of the data, why not use Anova or binomial GLm to test the effect of year on the answers? This way, you can statistically assess, whether there actually is a significant difference between the years. For each question, you could run a model (answer ~ year) and assess the significance.

The results are just descriptive and I think that even with the few, rather knowledge-based questions, more statistically valid results could have been extracted. For example, people were asked if they feel well informed about the local colony – a regression between those answers and correct answers could have been used to assess whether this information helped the participants to correctly answer questions (hypothesis: people that feel well informed are able to answer more correct questions).

Line 186-190: you stated in the methods that a third aspect was added to the questionnaire but will not be used here. Why do you then report results for this?
Validity of the findings: I wrongly assumed (based on the introduction), that the impact of educational activities will be assessed in this paper, tested in questionnaires to evaluate their effect. However, this paper purely focuses on comparing answers to questions in 2012 and 2016 and the educational activities that took place in the four years between are used as speculative explanation for the difference. Overall, the discussion is purely based on speculation since none of the links between answers and educational activities were actually tested in the present study. Even though these links are plausible and those educational activities most likely had an impact, this has not been tested. To test these, the addition of a few more questions in 2016 would have been very helpful (for example, questions like “Did you take part in any of the listed activities…”; did you read about the NBI population in the newspaper; did you report bird sightings…). Without these questions, the questionnaire cannot be used to draw the here presented conclusions.

Line 277-278: The quality of reported data has not been mentioned anywhere in the manuscript (or problems with it). Why does this now appear in the conclusion?
Additional comments: Unfortunately, this study cannot deliver what the abstract promises - an analysis of the effect that educational activities with children as multipliers of knowledge have on the local knowledge about the resident ibis population. The only data that was collected where responses to knowledge questions in two different year and the differences between the years was described descriptively. The authors did not actually collect data to address their proposed questions and there was hardly any information and the type and amount of educational activities and how children actually functioned as multipliers of knowledge. The only link between the educational activities and knowledge change is purely based on speculation.


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: MONITORING PUBLIC AWARENESS ABOUT THE ENDANGERED NORTHERN BALD IBIS: A CASE STUDY INVOLVING PRIMARY SCHOOL CHILDREN AS CITIZEN SCIENTISTS
Review round: 1
Reviewer: 2

Output:
Basic reporting: The paper is interesting and well-written and my recommendation is that should be published with some amendments (subject to editor decision based on my comments below).

The case for doing citizen science and including children as knowledge multipliers is well made.
-	the structure of the written paper is sound
-	Figures and tables are adequate
-	data provided is sufficient to support what is written (nowithstanding some of the issues outlined below)
-	The referencing is generally ok in terms of the areas covered. There are some places where further literature could be used e.g. lines 86 – 89 could do with some reference to the education literature of in-filed or informal education complementarity to lived experience/informal education. Also there are many references which could have been used to support better design of the survey instrument (see comments in Experimental Design). Note: I have not been through and checked every reference I assume that you are asking for review based on technical expertise not my ability to cross-check in text citations and references which is an administrative task
Experimental design: The survey instrument is quite weak, which limits the analysis and makes the conclusions more speculative than they might have been with some  minor changes to the questionnaire and methodology.

It could be grounds to disqualify the paper, but I prefer that the paper is published with some acknowledgement of these flaws and suggestions about what to do better (rather than rejection of the paper) because there are some useful things to learn from the paper and it would be a pity to lose these learnings by not publishing at all.

I understand that the questions had to be kept simple for the children. Additionally, attribution of impact to particular research activities is always a challenge where there are multiple sources of influence. However I feel that some attempt should have been made in the interviews to ask the interviewees about the sources of their knowledge. At several points in the paper (for example lines 111- 115; 235 – 239) reference is made to other really important ways that the interviewees might have received information to improve their knowledge apart from the children as multipliers. I think that the interview should have been constructed to gather this information as a supplement to Q5 – this would help greatly with not only the attribution, but also with understanding where and how this particular community receives information effectively.

Other improvements might have been to include a complementary survey without the children – for example ask the same questions in a postal survey or on-line survey (which explicitly asked the respondent whether they have had contact with the children involved in the work). This would have provided some ‘control’ set of observations for the influence of the child interviewers as multiplier.

A further improvement would be to have some open ended question to supplement Qs 7, 8, and especially Q9. A simple question like ‘why’ would help greatly for interpretation of the ‘yes’/‘no ‘ answers.

At the very least, the Discussion should explicitly acknowledge that the questionnaire has some design flaws, and also suggest how the questionnaire might be improved in future.

There is good explanation of and compliance with ethics given working with animals and minors.
Validity of the findings: Currently the discussion around linking the children as knowledge multipliers and the tested knowledge in the community make sense, but is speculative only because the survey instrument did not collect sufficient information to give any evidence to strengthen the case. This could be more clearly articulated especially if include discussion on some of the points listed in 2. above
Additional comments: A well done to the authors for being innovative and inclusive in the way that citizen science and conservation are being linked. And well done to the children for participating!

Advice would be in this paper to discuss how to improve the survey instruments, explore the use of some open ended questions (acknowledging that this is cannot be made too complex with children - there could be ways to supplement this information gathering by use of supplementary interviews by adults, or by postal or on line surveys as discussed above). I don't think the issue can be fixed for this piece of work but is definitely worth considering for any future work of this sort. I recommend publishing with clear explanation of the shortcomings because there are useful and novel findings.

Minor edits
Line 71 should be e.g. not i.e.
Line 88 – not sure if ‘regional’ experiences is what is meant by ‘informal education’ – this line needs clarification. Perhaps you mean ‘lived experience’?
Also the sentence in.
Line 105 – manyfold rather than manifold
Line 146 – need additional ) after 200


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: MONITORING PUBLIC AWARENESS ABOUT THE ENDANGERED NORTHERN BALD IBIS: A CASE STUDY INVOLVING PRIMARY SCHOOL CHILDREN AS CITIZEN SCIENTISTS
Review round: 1
Reviewer: 3

Output:
Basic reporting: The manuscript ‘Monitoring public awareness about the critically endangered northern bald ibis: a case study involving primary school children as multipliers of scientific knowledge‘ provides a clear, unambiguous, professional English language used throughout. The literature is well referenced and relevant and the structure seems tob e conform with the Peer J standards. Figures are relevant and in adequate quality but might be a little more labelled and described in detail (s. below). The raw data is supplied. I checked for the authors‘ ethical approval statment and human participant/tissue checks and vertebrate animal usage checks are provided.
Experimental design: It might be helpful to provide some further information about the research design and the connection of the presented study to the general work of the KLF with the focus on beneficials due to integrating the idea of citizen science. More specific:
1) who exactly did why which kind of citizen science?
2) the asked questions in the questionaire - why this choice and not differntiating more the term science or the characteristics of the endangered species and its background?
Validity of the findings: Dependent from further information on the experimental design.
Additional comments: The authors of the manuscript ‘Monitoring public awareness about the critically endangered northern bald ibis: a case study involving primary school children as multipliers of scientific knowledge‘ present a highly innovative study that is of interest in the field of citizen science, conservation and education. They integrated primary school children into a long lasting conservation and reseach project on the endangerd northern bald ibis (NBI) at different stages by involving the general local public.
In my opinion it can be seen as a very innovative apporach integrating children as researchers to ‘interview‘ adults about a biodiversity topic. However due to some ambiguity in the experimental design and the validity of of the findings I would recommend a major revision on the manuscript to clearify the aspects mentioned below.

The heading suggests that the children acted as multipliers of knowledge in the sense of informing others about their work – maybe that was done when children learned about the NBI in school and told their parents – but that can not be understood from the text in the manuscript so far. Maybe the authors could further differentiate or clarify that in the manuscript. Furthermore the authors could show that over a time period of four years the knowledge of the public about the NBI increased – however I am really wondering why the sample was totally changed and the sample size increased more than four times – how can the samples be still compared? How do the authors exclude that the people asked in 2016 did not already know in 2012 or did not increase their knowledge over time? An explicit discussion about this issue would be really important and substantiate as this seem to be the main problem to understand the findings of this study at the moment.
Another issue that needs more explanation is the choice of the questions asked in the questionaire - why did the authors decide for this choice of questions? Is there any point why people could answer more often correctly due to a citizen science participation or was that recorded somehow? Here, I would like to ask the authors to precisely explain in this context who exactly did which kind of citizen science? Why it is a benefit of engaging the public? There are of course many important reasons, but I would ask the authors to mention and better explain some of them.
Another point that might need further explanantion is, why the authors suppose that the public after four years would know more about the endangered species – whether it was because of reading the local news, hearing of the project in the supermarket or because they were part of an active monitoring. Here, the reader definitely needs to have further information about how exactly the children or the public contributed by citizen science and more precisely to the conservation of the endangered NBI.

Some further general recommendations to be checked:
Introduction
I think it is indeed a very good and important approach to introduce the public into the conservation of the NBI and communicating the background of the research. I am sure the authors are very well aware of the needs and benefits of doing that especially at the Konrad Lorenz Forschungsstelle (KLF), so I wonder if they could explain that a little more in the manuscript, additionally to the general monitoring and educational programs at the KLF.

Methods
I am wondering if the asked questions in the questionaire are too marginal to provide satisfying information on question i)-iii) by 50% of guessing propability – maybe the authors could explain a little more why they choose these questions.
I am wondering if question 7-9 seem to be a little blanket, however it might be helpful to explain the choice a little more –e.g. why do the authors not think that asking for science trust or understanding is a little vague in comparison of the big differences in research disciplines. Did the interviewed people knew that children were commissioned by a research institute? How to avoid social desirability of ‘trustwothiness‘? It would be helpful to learn more about the questions whether there was a specific intervention that was supposed to change attitudes to science or more specific the knowledge about the NBI project and background?
Did the interviewers capture any further information about the sample like where interviewed people got there informations from?

Discussion
It would be nice and might be helpful to find the structure of the introduction (i,ii,iii) in the discussion as well.

Here, the authors find some further issues in detail that might be checked to help future readers to better understand the study:
55-58: Maybe the authors could explain shortly the mentioned basic scientific concepts – did they play a role within the project in total, respectively in the communication with the public?
62-64: It might be helpful, if the authors could provide a source of that – which communication strategies?
64-66: Here the authors are talking about ‘several studies suggested by recent studies‘, but only one is cited – maybe some more would be helpful?
84-86: It would be nice if the authors could explain whether there was a control that had shorter training effects and therefore highlighting a possible citizen science benefit? Maybe the authors can provide some more information here.
88: It might be helpful to better understand, if the authors could provide a source of that.
105-106: It would be for a better understanding if the authors could explain which kind of activities were conducted in classrooms and provide some examples and an explanation how and why content knowledge should have increased due to the activities.
107-108: The idea of children as multipliers in the village seem to be very promissing – maybe the authors can explain or give the reader some mind picutres what it means to act as multipliers in the village in the name of NBI research?
110: It might be of interest for the readers what kind of acitivites?
111-112: It would be nice to receive some more informations from the authors about the issue which educational activities were supposed to be for whom.
112-113: Maybe the authors could give a short overview of the way of communication taking place between the KLF and the public.
112-115: I am wondering, if not all questions could possibly be answered after reading one informative press release, so maybe the authors can further explain why knowledge should be dependent from children as multipliers? The NBI colony at the KLF excists since 1997, so maybe people learned by time about the project – why do the authors suggest that citizen science explicitly increased people‘s knowledge here?
115-117: I suppose some more information should be provided by the authors how specifically knowledge was increased?
133-135: I am wondering if it might be better to use the term surveyed or somthing similar as the term interviewed is often connected to an oral survey. In this context maybe the authors could tell a little bit more about the details of the survey itself – was it a paper-pencil questionaire, did the children only collect the data or also help to type out them.
145-146: As mentioned before, it might be helpful when the authors could differentiate the science term – in methods and discussion, or explain why they did not in the questionaire.
154-155: Did the authors gather any information where from people had the information to answer the questions in the questionaire?
155-158: Can the authors surely exclude that the children from the same school did not influence each other by telling the right answers to the questionaire?
158-160: It might be beneficial for the study design if the authors could explain how the interviewed people were picked?
160-161: Even so that this issue was mentioned before – it might be helpful if the authors could explain why they interviewed different people? Where is the additional value of using different years when not comparing the knowledge of the same people?
162-163: It might be helpful for the wide readership to receive like two sentences about explaining the meaning of delta of proportions.
196-197: Sorry for that, but returning to the point oft he experimental design – how can the authors suggest that they would receive more right answers from the people that were interviewed in 2016, when other peoples were asked in 2012?
197-198: I would suggest to be more careful to mention that the findings support the communication policy (which is by the way not mentioned or explained in the manuscript) as the ‘research by the locals‘ is not mentioned or explained in detail in the manuscript nor obviously proved by any specific results? I would like to ask the authors to explain that issue to support a better understanding of that statement.
199-201: It might be helful for the understanding of the findings to know more about what was done in the workshops in the classroom respectively what kind of hand-on exercises were conducted?
202-203: I would highly recommend to search for some more literature here as this research topic has a long history already with more differentiated findings. Maybe the authors could cite some more and more specific studies.
206-207: Maybe the authors could explain what kind of change of attitude towards what they had in mind, here.
216-218: I would be careful to talk from ‚‘people’s knowledge to distinguish‘ as they had a 50% option to chose between correct or wrong with given patterns.
218-219: It might be helful if the authors could explain how this is relevant to the study as it is still not clear which role the public engagement plays in the conservation research mentioned in the study.
237-239: In my opinion this seem to be a very important issue, that should possibly be mentioned and explained earlier by the authors for example in the methods as it helps to better understand the manifold work of the KLF and therewith the here presented study.

Figures and tables

A picture of an adult and a juvenile NBI would be nice to get a better knowledge of the endangered species all the research is about.
Figure 1. The caption is very small – maybe the authors could size it up for a better visibility.
Figure 3. It might be helpful to have the figures out of the circles due to a better visibility.
Table 2. It might be helpful to note the sample size in the description.


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: GENETIC ASSOCIATIONS OF VITAMIN D RECEPTOR POLYMORPHISMS WITH ADVANCED LIVER FIBROSIS AND RESPONSE TO PEGYLATED INTERFERON-BASED THERAPY IN CHRONIC HEPATITIS C
Review round: 1
Reviewer: 1

Output:
Basic reporting: .
Experimental design: .
Validity of the findings: .
Additional comments: Title:
the title (Genetic Associations of Vitamin D Receptor Polymorphisms 2 with Advanced Liver Fibrosis and Response to Pegylated 3 Interferon-based Therapy in Chronic Hepatitis C) is informative with clear and relevant aim.

Introduction:
•	The research question justified given what is already known about the topic.
•	Please add the exclusion criteria and summarize the inclusion ones in a points rather than a paragraph.

Methods:
●	The process of subject selection clear.
●	Variables defined and measured appropriately.
●	We need enough details in order to replicate the study!

Results:
●	The data presented in an appropriate way.
●	Table 1: Table 1: Baseline patient characteristics according to response to PEG-IFN2 based therapy (no significant selection bias).
●	There is a huge selection bias in Table 3: Baseline characteristics, virological factors and liver fibrosis stage in 2 accordance to the bAt (CCA) haplotype!  Please try to make a comment on that to support your study because it might weaken your study significantly.
●	Five hundred and ten patients had data on liver fibrosis stage. Of these, 269 underwent liver biopsy before treatment with PEG-IFN and ribavirin=Please make a referral for any adverse events during the liver biopsies?

Discussion& Conclusion:
●	The conclusions answer the aims of the study.
●	The conclusions supported by references or results.


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: GENETIC ASSOCIATIONS OF VITAMIN D RECEPTOR POLYMORPHISMS WITH ADVANCED LIVER FIBROSIS AND RESPONSE TO PEGYLATED INTERFERON-BASED THERAPY IN CHRONIC HEPATITIS C
Review round: 1
Reviewer: 2

Output:
Basic reporting: 1.	For the treatment of HCV, the 2018 EASL and the 2018 AASLD recommendations for treating hepatitis C both consider DAAs as the first-line treatment. Therefore, did the authors consider the clinical significance and necessity of pegylated interferon-based therapy in the context of these recent recommendations?
Experimental design: 2.	Alcohol and obesity are also very important factors affecting the degree of liver inflammation, especially alcoholic liver disease and non-alcoholic fatty liver disease. Researchers did not exclude the patients with HCV who also had alcoholic liver disease or NAFLD. However, the use of alcohol as a variable could affect the conclusions.
Validity of the findings: 3.	Line 209, The advanced liver fibrosis was assessed using two criteria: liver histology and FIB4 index. Liver histology is the gold standard for evaluating the degree of liver fibrosis, while FIB4 is a non-invasive marker. Liver histology and FIB4 are essentially different. A potential measurement bias may be introduced if some assessed using histology and others using FIB4. Therefore, all patients should be assessed for liver fibrosis using either of histology or FIB4.
Additional comments: 1.	In results, lines 180-187, why not make a table which would show results clearer and more concise?
2.	Line 210, the P value of being male showed 0.08, which is not statistically significant. Please check it.
3.	In the introduction and discussion, there were a lot of repetition. Would authors mind to reduce them appropriately?
4.	There were a lot of ambiguity in the article. It is suggested that native English experts revise 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: GENETIC ASSOCIATIONS OF VITAMIN D RECEPTOR POLYMORPHISMS WITH ADVANCED LIVER FIBROSIS AND RESPONSE TO PEGYLATED INTERFERON-BASED THERAPY IN CHRONIC HEPATITIS C
Review round: 2
Reviewer: 1

Output:
Basic reporting: 1. For the treatment of HCV, the 2018 EASL and the 2018 AASLD recommendations for treating hepatitis C both consider DAAs as the first-line treatment. Therefore, did the authors consider the clinical significance and necessity of pegylated interferon-based therapy in the context of these recent recommendations?
It has been addressed.
Experimental design: 1.Alcohol and obesity are also very important factors affecting the degree of liver inflammation, especially alcoholic liver disease and non-alcoholic fatty liver disease. Researchers did not exclude the patients with HCV who also had alcoholic liver disease or NAFLD. However, the use of alcohol as a variable could affect the conclusions.
It has been addressed.
Validity of the findings: 3. Line 209, The advanced liver fibrosis was assessed using two criteria: liver histology and FIB4 index. Liver histology is the gold standard for evaluating the degree of liver fibrosis, while FIB4 is a non-invasive marker. Liver histology and FIB4 are essentially different. A potential measurement bias may be introduced if some assessed using histology and others using FIB4. Therefore, all patients should be assessed for liver fibrosis using either of histology or FIB4.
It has been addressed.
Additional comments: 1. In results, lines 180-187, why not make a table which would show results clearer and more concise?
It has been addressed.
2. Line 210, the P value of being male showed 0.08, which is not statistically significant. Please check it.
It has been addressed.
3. In the introduction and discussion, there were a lot of repetition. Would authors mind to reduce them appropriately?
It has been addressed.
4. There were a lot of ambiguity in the article. It is suggested that native English experts revise the manuscript.
It has been addressed.


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

Input:
Title: RISK ASSESSMENT OF FLT3 AND PAX5 VARIANTS IN B-ACUTE LYMPHOBLASTIC LEUKEMIA: A CASE–CONTROL STUDY IN A PAKISTANI COHORT
Review round: 1
Reviewer: 1

Output:
Basic reporting: 1. The article is not written in clear English. It contains many mistakes. Sentences are partly wrong in grammar.
2. references are not up to date. Citations are either old or not appropriate. Some important studies on ALL are missing at all. Please, go and check pubmed.
3. The format is acceptable. However, the authors mention the education of the parents was taken into account with reference to Table 1, but there is no such information.
4. The authors give statements on the prevalence of SNP's genotypes/alleles. This is clearly wrong. If at all, it should be "frequencies".
5. A part of the introduction (lines 43-55) seems to be simply copied as the language is completely different from the rest of the manuscript.
Experimental design: The aims are clear, but the statistical analysis was not performed on a high standard.
Education of the parents is missing in the results, even though mentioned in the manuscript. The material could have been analysed with a multivariate model with covariates included.
Besides the methods are described in a bad way.
Validity of the findings: The data are not analysed in an appropriate way. There should be a more general table showing the material used. The table 3 with the results is not clear to me. Many numbers are missing. For instance, 16.77 % have a positive family history, 52.9 have a negative family history. What about the remaining 30%?
Thus, the conclusions are not sufficient and clear enough.
Additional comments: First of all, the idea of the manuscript is fine, given the overall importance of B-ALL in Pakistan.
However, the statistics should be approved by a statistician. Multiple testing has not been considered (3 SNPs). The testing should have been performed on a multivariate level with alleles/genotypes and other covariates included. More recent literature should have been added.


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

Input:
Title: RISK ASSESSMENT OF FLT3 AND PAX5 VARIANTS IN B-ACUTE LYMPHOBLASTIC LEUKEMIA: A CASE–CONTROL STUDY IN A PAKISTANI COHORT
Review round: 1
Reviewer: 2

Output:
Basic reporting: The authors evaluated the association between DNA single nucleotide polymorphisms (SNPs) in PA5X and FLT3 genes with B-ALL in a case control study with equal number of cases and controls. The study is appropriately designed but the article is poorly written and the results lack clarity.
1-	The whole article needs a thorough proofreading as it contains many spelling errors /misspelled words and grammatical mistakes that are at times changing the context. Few examples:
I.	Title: line1 says coharts” instead of cohorts
Risk assessment of FLT3 and PAX5 variants in B-acute lymphoblastic leukemia: A case-control study in a Pakistani cohart
II.	Abstract
Prevalence, association and frequency have been used interchangeably which gives the impression the authors don’t know what they are talking about.

Abstract RESULTS: Risk allele frequency ‘A’ of FLT3 missence  (missense??) polymorphism rs35958982 showed significantly more prevalent (should be prevalence) in B-ALL subjects (p=0.00526)
proginator

III.	Introduction (words in parentheses)misspelled or grammatically incorrect)
Line 56=>In hematologic (melangices), 70% to 100% increased expression of FLT3 in acute (myloid) leukemia (AML) and acute lymphoblastic leukemia (ALL) are reported previously
These studies signify the critical role of FLT3 expression in (proliferaltion) of blast cells (Carow et al., 1996).
B-cell-specific activator protein (PAX5) encode transcription factors that are the member of paired box domain. PAX5 plays imperative role in the (commintment) of B-cell lineage from blast cells as it controls the differentiation

(proginator cells) has been used several times instead of progenitor cells
IV.	“Figure 1 -3”

DNA ladder has continuously been referred to as “Leader”
(Moreover figure 2 is not properly labelled)
And countless other examples like this are present within the body of the manuscript showing that the article has not been proofread.
Experimental design: 2-	Materials and Methods:

I.	Discrepancy in the number of patients between abstract and M&M
155 patients and 155 controls =310 patients not 300.

II.	What was the criterion used for the selection of SNPs for the study?
III.	Was the association analysis adjusted for the family history, smoking and Consanguinity?
IV.	Descriptive stats with respect to age sex and family history should be clearly explained in the form of a table
Validity of the findings: 3-	Results
Table 4 is confusing; one cannot comprehend whether the ORs presented are for allelic or genotypic model. 95% CI in the case of rs35958982 is very wide (For OR=3.67, CI= 0.75-18.10) what could be the cause of it? Was it inadequate sample size or Software failure?

4-	Discussion
I.	Ultrasounds are not radiations
II.	In general a more detailed discussion of the results would be valuable including the presentation of hypotheses linking the SNPs identified in the study and increased risk of B-ALL. The biological plausibility leading from the selected SNPs to the ALL should be stressed.
Additional comments: No additional comments