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: CO-CULTURING HYPHOMICROBIUM NITRATIVORANS STRAIN NL23 AND METHYLOPHAGA NITRATIREDUCENTICRESCENS STRAIN JAM1 ALLOWS SUSTAINABLE DENITRIFYING ACTIVITIES UNDER MARINE CONDITIONS
Review round: 1
Reviewer: 2

Output:
Basic reporting: Basic reporting
The article is generally well written, professionally structured and represents a self contained piece of work with relevant results. The introduction gives an excellent and concise background to the work, and referencing is appropriate throughout. However, it has minor English and grammatical mistakes  throughout. Fixing these would make it easier to follow. As a few examples:

L26 “Planktonic pure culturing of both strains were performed”. Suggest changing to something like: “Pure cultures of both strains were grown with”. Similarly on L271.

L37: Change to “in contrast to”. Furthermore, this sentence has two different tenses for the two organisms i.e. “JAM1 had a” vs “NL23, has a”

L59 “We have been studying for many years”. I am not sure its needed to explicitly state that you have been working on it for many years needed as you succinctly and approriately cite the work in the rest of the paragraph. Suggest changing to something along the lines of “This study focuses on a biofilm derived from….

L60; Change ”that contains” to “containing”

Throughout: Transitory is used instead of transiently

L472 “ran only with” change to “run only with”

Figures: The presentation of the data in the figures and tables could be improved. The  current layout makes it hard to interpret the results of the planktonic incubations and the biofilm study.  In particular:

Figure 3: I would strongly suggest splitting this into 4 panels,  the monocultures, and the two co-cultures. In its current format it is very hard to pull apart the different treatments. By separating this way, you could add other data such as the OD over time, and the ratios of the two organisms  (if available).

Figure 4: Scale bars are missing

Tables 5 and 6: A figure detailing the operation of the three reactor runs is essential to  interpret the results. Such a figure could incorporate nitrate and nitrite concentrations and when the media was changed, the proportions of the two organisms and the periods where rates were calculated. In essence this would be a visual representation of table 5  and 6, which contain a lot of information that is hard to compare. The tables could then be combined showing just the rates. Furthermore, please be more specific as to what “not measured” means… was there nothing to be measured or did no measurement happen?

Raw data: Where have the results from the RNA sequencing been deposited?
Experimental design: The manuscript investigates the nitrate uptake/reduction capacities of two methylotrophic denitrifiers (one which misses a nitrite reductase and one with a full denitrifying pathway) which, prior to isolation, dominated a biofilm in a reactor that treated a seawater aquarium. The authors overall aim was to enhance our understanding of how these two taxas co-existence might enhance denitrification systems treating saline/brackish waters. The main results of the study are that strain JAM1 is a better competitor for nitrate; it had a higher µmax, a lower ks and a better µmax/ks ratio, and it appeared to switch to nitrate respiration quicker. Furthermore, strain NL23 appeared to be sensitive to increasing salinity. As such, JAM1 dominated co-cultures both in planktonic and biofilm set ups and the authors conclude that its presence is “essential” for the operation of NL23 at NaCl concentrations more than 1%. There are some issues that need to be addressed in regards to the experimental design.

1)	The research question is currently not well defined/consistent throughout the manuscript. The introduction introduces the hypothesis that “that a tight relationship has developed between H. nitrativorans strain NL23 and M. nitratireducenticrescens strain JAM1 to achieve denitrification in the original biofilm”.  I would argue that this is not really a testable hypothesis and does not reflect the rest of the manuscript, it is not addressed explicitly in the conclusions. Furthermore the discussion introduces the hypothesis that “JAM1 has to be present for strain NL23 to survive under marine conditions”. I would suggest that the authors are more specific about the research questions that their experiments were designed to address at the end of the introduction.

2)	There are a number of details missing from the methods which made the results difficult to interpret. I think the most important omission is details regarding the planktonic culture experiments that inform figure 2. How many vials were set up per treatment? How often were they sampled, and for what?

3)	One of the few methodological details given is that the cultures were incubated for 1 – 7 days without shaking. Considering the biofilm capacity of both of these organisms how can you rule out that biofilms formed in the vials? The presence of a mixture of biofilm and planktonic cells in the vials could have a considerable impact on the apparent affinities due to diffusion limitation.

4) For all rates, please insert a table detailing the results of the linear regression used to calculate them with significance and error values. Similarly, for results from the bioreactors where estimates have been made based on two measurements i.e. denitrification rate per mg protein, what is the propogated error?

5)	Other details which should be added are:

L 142. Where did the material come from for the pre-cultures? Here it would be good to cite again the initial study describing their isolation and submission to relevant culture collections.

173. How was air added with a peristaltic pump?
How did you confirm anoxic conditions in the bioreactor, and are you sure that they were maintained. For example, how did you collect supports from the bioreactor without introducing O2? How did you change the media and ensure that the media added was anoxic?

What were the timings for sampling and when exactly was media exchanged in the bioreactors (see my comment about adding in a figure detailing the bioreactor performance and operation).

How was reactor 3 made anoxic from the start?

Considering the results of the planktonic experiments, why did you choose to inoculate the bioreactors with similar biomass of the two organisms?
Validity of the findings: Validity of the findings:

The major finding in the manuscript is that JAM1 is essential for NL23 to “operate” under higher salinity conditions. Currently this finding is not strongly supported. In the results (L355 onwards), it is stated that denitrification still occurred in reactor 3 (NL23 only), although at much lower rates as salinity was increased and before switching to IO media, therefore JAM1 is not “essential”. From the information given in table 5, it does appear that at 2.75 % NaCl, the co-culture had higher NO3- reduction and specific denitrification rates and I would suggest reformulating your discussion to reflect this more explicitly.

Secondly, I find your discussion about the differences in nitrate uptake/reduction from L443 a very convincing explanation for why JAM1 is a better competitor for nitrate and seems to outcompete NK23 in the planktonic experiments. Taking into account that NL23 also seems sensitive to salinity, I think an important question is therefore: How did these two organisms c-exist at high abundances in the original aquarium bioreactor film? Surely if NL23 is such a poor competitor for nitrate in terms of growth and affinity (if all other conditions are equal), there must have been another factor that allowed it to persist? If you do not address this, then you do not address your original intent to enhance understanding of how denitrification systems treating saline/brackish waters can be optimized.

Minor issues should also be addressed to make the interpretation of the results clearer:
What would have caused the initial difference in reactor 1 and 2 that lead to the higher rates and accumulation of nitrite?

How much gas would you expect to be produced based on the biomass and denitrification rate? Can you really guarantee that your system wasn’t leaking? What is the relevance of this result?

L433. I would suggest rephrasing this line. Why would an equilibrium have to be reached? Or did you assume an equilibrium was reached and here you assess how the physiological characteristics lead to the establishment of a biofilm with 2 species?

I do not think dynamics/dynamism is an appropriate term, their nitrate utilization characteristics? Alternatively, you must define what you mean by dynamism in the results and discussion.

What do you suggest that NL23 does in the lag phase? If it is not reducing nitrate as an electron acceptor, how does it avoid becoming “trapped in anoxia?”.

L438 I think that it means that its quickly induced, your results say nothing about its regulation.
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: CO-CULTURING HYPHOMICROBIUM NITRATIVORANS STRAIN NL23 AND METHYLOPHAGA NITRATIREDUCENTICRESCENS STRAIN JAM1 ALLOWS SUSTAINABLE DENITRIFYING ACTIVITIES UNDER MARINE CONDITIONS
Review round: 2
Reviewer: 1

Output:
Basic reporting: The manuscript was modified according to the reviewers suggestions (when possible). It is in a state to be published.
However, the reviewer suggests to correct all values with the erros in the manuscript:
an example: 0.428 ± 0.019 should be 0.43 ± 0.02.
Experimental design: nothing to report
Validity of the findings: nothing to report
Additional comments: No additional comments


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

Input:
Title: A NEW SPECIMEN OF SINOPTERUS DONGI (PTEROSAURIA, TAPEJARIDAE) FROM THE JIUFOTANG FORMATION (EARLY CRETACEOUS, CHINA)
Review round: 1
Reviewer: 1

Output:
Basic reporting: The article has a clear structure, with logical sub-sections, appropriate detailed, written in accessible/direct language and annotated figures complementing the text.
The raw data is shared well, the photographs are of high quality and focus on areas of interest described in the text. Specimen descriptions tend to lack detailed photographic pieces of evidence to back up the observations - but this paper does it really well, with well-annotated and outlined companion pieces which make reading more accessible.

I would suggest adding measurements of other elements (ie manual digits) to Table 1. Suggestions on additions to figures and literature used are included in the "Experimental design" & "Validity" sections.

The paper is self-contained, basing comparative description and conclusions only within clades of relation to the specimen.
Experimental design: The paper is tackling a hotly debated topic within pterosaur phylogeny, which is the Sinopterus taxonomic-over splitting, helping to solve this problem providing one of the more detailed osteology descriptions of Sinopterus. And it is refreshing and much needed to see a specimen description that is not another taxa assigned on poorly defined autapomorphies!

There are minor corrections I would like to propose to increase rigidity of the observations.

As the paper relies on comparative description, more comparable numerical values would be useful and help with cross-referencing, in example 146 line “These vertebrae are relatively elongate slightly more so than in Tapejara wellnhoferi or thalassodromines”, if possible use of comparative ratios might be useful and more definitive. If feasible, comparative illustrations complementing the comparisons might be useful (ie wingspan and anatomy diagram to aid visualisation of ontogenetic variation noted on lines 336-340) or illustrating humeral morphology differences as described in lines 393-403. As of now, the most comparative section is qualitative, the quantitative approach might be useful (although I am aware this might not always be the best approach when describing pterosaur elements). Comparing pterosaur remains is hard, as you frequently need to resort to features that are preserved at different angles, in poor quality, in 3D or flattened – making a robust assessment of features (especially humeral or scapula-coracoidal) hard. But the paper and its well-annotated illustrated outlines of elements accompanying the photographs mitigate this problem (especially aiding resolution of trunk elements in Figure 3). Some of this uncertainty could be mentioned, especially when comparing elements poorly preserved like the scapula.

The manual claw preservation, especially the keratin is really interesting considering how rarely it is found (and how strongly accentuated it is in this specimen). Describe in more detail (to line 288-289) on how pedal and ungual claws differ, while line 318 states " manual claws have the same shape", manual phalanges have somewhat visibly larger flexor processes. While full discourse into the subject lies outside the aims of this paper, higher resolution in the description of these elements might be beneficial!
Validity of the findings: The concluding observations in assessment and comparison of the specimen are valid and robust, with minor suggestions for improvements on conclusive remarks.

As pneumatic openings are an important aspect of tapejarid taxonomic assignment, evolution and description of this paper – how are they assessed to make sure openings are indeed pneumatic? Especially with a severe degree of flattening in regions assigned as bearing foramina. How certain are you with the assignment of pneumatic opening over a fracture or non-pneumatic depression within the bone?

As ontogeny is an important aspect of this papers discussion body and I agree there is sufficient information (line 326-328) to show the specimen is indeed immature. The cited text is slightly out of date and sources used to assign ontogenetic stage disputed by the community – and in the case of this specimen – not fully transferrable. The ontogenetic stages proposed by Kellner (2015) applied to basal, much smaller non-pterodactyloid of the Triassic. Dalla Vecchia (2018) wrote a paper disputing this ontogeny sequence scheme (https://doi.org/10.13130/2039-4942/10099), to quote “Kellner’s six ontogenetic stages are an oversimplification of a very complex process. Ontogenetic features of different taxa that probably had distinct growth patterns have been grouped together into a sequence that Kellner (2015) considered being valid for all pterosaurs. (…) It is evident that the order of  fusion of composite skeletal elements during ontogeny differs in pterodactyloids and early pterosaurs and there is no universal pattern that can be extrapolated to pterosaurs in general.” There is also this fairly recent summary paper on this topic that might be of use (https://doi.org/10.1111/brv.12666). While Kellners OS is frequently used in literature, especially in the context of clade investigated in this paper, I would suggest refraining from using OS stages or at least providing a caveat on why this framework might be used with hesitancy.
Additional comments: I am eagerly awaiting a description of more than a dozen specimens currently in storage (mentioned on line 56), as these might bring much-needed clarity & look forward to more research on derived pterosaur phylogeny this paper will springboard!


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

Input:
Title: A NEW SPECIMEN OF SINOPTERUS DONGI (PTEROSAURIA, TAPEJARIDAE) FROM THE JIUFOTANG FORMATION (EARLY CRETACEOUS, CHINA)
Review round: 1
Reviewer: 2

Output:
Basic reporting: No comment
Experimental design: No comment
Validity of the findings: No comment
Additional comments: The present work is concerned with providing a detailed osteological description of a new specimen that, in my view and in agreement with the authors, is unequivocally a Sinopterus dongi. Overall, the article is solid and very well written.
I agree with the publication of this manuscript, since the description of these post-cranial elements has the potential to resolve biological, ontogenetic and taxonomic inconsistencies related to that genus. I do have a few comments that the authors should use as they see fit.

1 – Line 149 – I think that presenting a formula the length of a cervical series preserved in articulated and flattened might be inappropriate. What criteria were used for this measurement? from the most anterior to the most posterior point of the vertebrae?
Furthermore, according to Table 1, the cervical vertebra IV is not the longest in the series, as the length formula shows.

2 – Line 156 – Establishing a relation by height of the neural spine may not be appropriate due to the deformation of the flat material, since, according to Table 1, this seems to vary randomly throughout the cervical series due to preservation. Perhaps it is more appropriate to establish this relationship with the form of this structure.

3 – Line 165 – I have seen Thalassodrominae pterosaur vertebrae with 1, 2, or 3 lateral pneumatic foramina. The number of penumatic foramina often varies between individuals, vertebrae, and even sides of the vertebra. I think it would be more appropriate if the authors limited themselves to the presence or absence of foramina on the side and not if these are single or double foramina

4 – Line 192 – Here too I would not bother to include data on the height of neural spines of the dorsal vertebrae, which besides being crushed are apparently broken. The measures are also not present in Table 1

5 – Line 193 – The fossae appear to be present in a biomechanically appropriate region and closely resemble the pneumatic fossae found in the dorsal vertebrae of some Rhamphorhynchidae. However, due to the preservation of the material I find it inappropriate to state this in a description.

6 – Line 226 – Is the diameter measurement of the elements preserved in these conditions reliable?

7 – Line 342 – What do you mean "straight femoral shaft"? The femur is not described

8 – Line 413 – Here again I am concerned if the presence of two pneumatic foramina could be associated with an individual variation

9 – Figure 5 – In the figure legend, it says “A” and “B” where it should be “C” and “D”.

10 – Table 1 – Which unit of measure is being used in the values shown in Table 1? mm?

Review by Richard Buchmann


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

Input:
Title: A NEW SPECIMEN OF SINOPTERUS DONGI (PTEROSAURIA, TAPEJARIDAE) FROM THE JIUFOTANG FORMATION (EARLY CRETACEOUS, CHINA)
Review round: 1
Reviewer: 3

Output:
Basic reporting: Dear,

Below are my comments.

In this paper is presented in this the complete description of a new postcranial specimen of Sinopterus dongi (D3072) preserved on a single slab from the Cretaceous Jiufotang Formation. The skeleton is missing the skull, but shows confidential data, which permits the correct assignation to that tapejarine species.

The Tapejarinae represents edentulous pterosaurs with a considerable diversity during the Cretaceous. According the authors, for the Jiufotang Formation of China, a total of 7 nominal tapejarid species and 2 genera have been proposed. Despite the abundance of specimens (described and undescribed) and the relevant taxonomic problems involving of the tapejarine pterosaur Sinopterus dongi, detailed revisions of the matter were still lacking.

The manuscript is clearly written in professional, unambiguous language. It is very interesting, particularly because discuss about the problematic of the Sinopterus complex, with the addition of new information on the comparative osteology.

I hope that I can be helpful with this contribution.

Best regards,

Hebert Bruno Campos
Experimental design: Original primary research within Aims and Scope of the journal.

The investigation must was conducted rigorously and to a high technical standard. The research was conducted in conformity with the prevailing ethical standards in the field (Vertebrate Paleontology).
Validity of the findings: Conclusions are well stated, linked to original research question, supporting the results.
Additional comments: In addition, I suggest the authors to submit one single graphic with the main measurements of the limbs elements of the specimens of the Sinopterus complex, inclusing the new one, and another tapejarine species, to demonstrate the metric variation of the limb proportions.


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

Input:
Title: A NEW SPECIMEN OF SINOPTERUS DONGI (PTEROSAURIA, TAPEJARIDAE) FROM THE JIUFOTANG FORMATION (EARLY CRETACEOUS, CHINA)
Review round: 2
Reviewer: 1

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

The work is well written, direct and cohesive. The description of this material is extremely relevant to resolve taxonomic inconsistencies in the clade. All my doubts raised were clarified. I am satisfied with the modifications and responses given by the authors of the manuscript and suggest the publication of this work.


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: CONSTRUCTION OF A HIGH-DENSITY LINKAGE MAP AND DETECTION OF SEX-SPECIFIC MARKERS IN PENAEUS JAPONICUS
Review round: 1
Reviewer: 1

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

The article entitled “Construction of a high-density linkage map and detection of sex-specific markers in Penaeus japonicus” by Zhang et al. constructed a high-density genetic linkage map and identified sex QTL for kuruma prawn P. japonicus. This study not only provide a reference for further genome assembly and genomic selection for important economic traits, but also lay a research foundation for investigating the molecular mechanism of sex determination and differentiation of P. japonicus, which could provide theoretical support for unisexual breeding. The results obtained are beneficial both in theory and application. The tables are sufficiently organized, and the figures are well presented in the manuscript. Overall, the study deserves publication. However, there are still some issues needed to be corrected and clarified.

1.	Line 29: “an average inter-marker distance of 0.117 cM”. However, in the text, you presented that “an average inter-locus distance of 0.123 cM” on Line 153. Please clarify which is right?

2.	Line 49-52: This sentence is not related to this paper, please delete it or replace it by other contents.

3.	Line 73: you used “,” in “9,289” but not in “3610.90”. Please unify them in the manuscript.

4.	Line 88: “Sex was determined by observing their sexual characteristics”, this is intricate to readers who are not very professional. Please state how to distinguish the sex of P. japonicus. For example, you can explain them by describing, pictures of sexual characteristics, or the references.

5.	Line 135-136, I am confused about this sentence “The feasibility of this pair of sex-specific primers was validated using PCR with samples from six females and six males”. I wonder which would you want to validate, primers or SNP markers? Besides, I think six males and six females are too few to validate the SNP markers. What’s more, where did these 12 individuals obtain, from the 200 offspring or from other groups? Please clarify.

6.	Line 150-151: “two LGs comprising only three SNPs and nine SNPs, respectively, which are relatively too short for one chromosome, were excluded from the map”. Is it logical and reasonable to remove SNPs and delete chromosomes from the genetic map just because the SNP markers are scarce? Please give the explanation.

7.	Line 160-163, you said that six SNPs on LG1 were obviously associated with the sex. However, you only presented one SNP marker in Figure 3. The sex-specific markers are very important for MAS breeding. Please show the detail information of these six SNPs, containing the genotypes of females and males and the picture of Sanger sequencing of every SNP marker.

8.	Line 172-174, this sentence is repetitive and useless, please delete it.

9.	Line 186-188, “9289 SNP markers” appeared twice in this sentence, please rewrite it.

10.	Line 190, “average inter-locus distance was 0.12 cM”, and “0.123 cM” was presented in Line 153. Please unify them in the manuscript.

11.	Line 212 and Line 215, “Jones et al., 2020b” and “Jones et al., 2020a” appeared, respectively. But I can’t find these two articles in the reference. Please clarify.

12.	Line 218-220, you said “One sex locus was located, supporting that the sex of the black tiger shrimp is determined by a WZ-ZZ chromosomal system”. I wonder that this sex locus was identified by three articles? Why did you give three references in this sentence?

13.	Line 220-222, please give the name of the species you described in this sentence.

14.	The discussion section is illogical, chaotic and simple. I suggest the authors revising this section carefully.


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: CONSTRUCTION OF A HIGH-DENSITY LINKAGE MAP AND DETECTION OF SEX-SPECIFIC MARKERS IN PENAEUS JAPONICUS
Review round: 1
Reviewer: 2

Output:
Basic reporting: no commont
Experimental design: no comment
Validity of the findings: no comment
Additional comments: The authors constructed a high-density genetic linkage map and develop a sex specific marker for Penaeus japonicus. This resources would benefit the basic research of sex determining mechanism and breeding in aquaculture. However, some issues must be explained more clear, especially the efficiency of the sex specific marker.
1.Line 31. delete “on LG1”
2.Line 41. please cite the book in references.
3.Line 64-65. please rephrase this sentence. The technology of AFLP is independent of GBS or RADseq.
4.Line 95-96. a library....the libraries
5.Line 99 rephrase this sentence. Two “using”.
6.Line 127. please rephrase this sentence.
7.Line 127 and 128. please briefly describe the method.
8.Line 136. are the six females and six males from the full-sib family? Please validate the sex specific marker in more population.
9.Line 149 vs line 124 “only four SNPs” vs “only two markers” are they inconsistent?
10.Line 160. what is the percent of phenotype that these marker explained?
11.Line 160. “obviously” is not suitable.
12.Line 162-163. How to explain the inconsistency of the genotypes between SNP and haplotype? Females are homozygous in haplotype genotypes while heterozygous in SNP genotypes.
13.Line 196. The choice of LOD is irresponsible. Actually, the lower limit of marker number of a group may vary from species to species. So the common feature would be the true linkage groups would contain relative more markers. Thus, the marker size distribution and the number of groups should be pay attention to at the same time. Usually, set the low limit of marker number infinity and observe the distribution of marker numbers with different LOD. When the best LOD chosen, the SNPs are expected to be assigned into stable number of linkage group. Thus, the discussion about chromosome rearrangement is not reasonable.
14.Table1. Please supplement the unique number of marker and change “Number of markers” to “Number of SNP”
15.Rephrase the legend of Figure 2


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: CONSTRUCTION OF A HIGH-DENSITY LINKAGE MAP AND DETECTION OF SEX-SPECIFIC MARKERS IN PENAEUS JAPONICUS
Review round: 2
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: 1.	Line 56: you deleted the sentence, but you did not delete the reference of “Thitamadee et al., 2016” in Line 462.

2.	Different forms of “WZ/ZZ” were presented in the MS. For example, “WZ-ZZ” in Line 103, and “WZ/ZZ” in Line 207. Please unify them in the manuscript.

3.	Line 175, please clarify the number of males and females in these 89 adult shrimps.

4.	Line 200: change “3481.982” to “3,481.982”. Please check the MS carefully, and unify them.

5.	Line 206: two “respectively” in this sentence.

6.	Line 237, please add “marker” before “density”.

7.	Line 399-404: first, the order of reference should be revised. Second, this is two references, should be separated.

8.	Line 446-450, this reference has been deleted in the context.


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: CONSTRUCTION OF A HIGH-DENSITY LINKAGE MAP AND DETECTION OF SEX-SPECIFIC MARKERS IN PENAEUS JAPONICUS
Review round: 2
Reviewer: 2

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: The authors have reported the genetic map and sex-lined SNPs for Penaeus japonicus. The manuscript has been largely improved after the first round of review. However,  there are also descriptions or analysis improper.
1. The validation of the sex-linked SNP is missing in Result.
2. The number of linakge group and the number of chromosome in karyotype analysis is still inconsistant. Even current genetic map is also of high quality and does not affect sex-linked SNP detection, it still can be improved. The authors has provided the relationship between LOD and number of linakge group in Figure S1. As the authors describe, the number of linakge group is 41 when the LOD set to 12. Check the Figure S1, when the LOD set to 17-21, the linage group would increae to 43, in which the number of liankge group is stabler. The threshold of site mumber in each linage group can be adjusted more according to the difference between the site number in the shortest linkage group and that in debris. Usually, the LOD can be selected according to the number of chromosome accroding to  karyotype analysis and is less related with the threshold of site bumber for those data with hundreds and thousands of SNP, which almost cover the whole chromosomes. Thus, adjusting the LOD is encouraged.


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

Input:
Title: CONSTRUCTION OF A HIGH-DENSITY LINKAGE MAP AND DETECTION OF SEX-SPECIFIC MARKERS IN PENAEUS JAPONICUS
Review round: 3
Reviewer: 1

Output:
Basic reporting: none
Experimental design: none
Validity of the findings: none
Additional comments: According to the authors's speculation that the chromosome rearrangements would be an interesting points. However, it is doubtful two chromosomes rearranged in such a short time. Comparing chromosome rearrangements, problems with data analysis, maybe  instrinsic to the species or the data, is more likely. This is still a high-quality paper, and the problem has no influence on sex marker develoopment.


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: IMPROVED ISOLATION AND DETECTION OF TOXIGENIC VIBRIO PARAHAEMOLYTICUS FROM COASTAL WATER IN SAUDI ARABIA USING IMMUNOMAGNETIC ENRICHMENT
Review round: 1
Reviewer: 1

Output:
Basic reporting: The manuscript is written with relatively good command of English. However, there are some grammatical errors in the manuscript. It includes relevant literature and presented in an organised manner. The authors included clear figures and tables shared in the manuscript.
Experimental design: The methods referred and used in the study, are well-cited. The description of methods are presented with sufficient information for future application.
Validity of the findings: The findings are presented in a clear and systematic manner. The authors has made a comprehensive discussion on robust data gained through their study.
Additional comments: Overall, the manuscript is good. However, the authors could provide some future recommendations on future works in the conclusion.


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: IMPROVED ISOLATION AND DETECTION OF TOXIGENIC VIBRIO PARAHAEMOLYTICUS FROM COASTAL WATER IN SAUDI ARABIA USING IMMUNOMAGNETIC ENRICHMENT
Review round: 1
Reviewer: 2

Output:
Basic reporting: General comments:
This paper focuses on the isolation and identification of toxigenic Vibrio parahaemolyticus from the coastal water in the Eastern Province of Saudi Arabia. This research is original, and the results are important.
The current manuscript’s writing is clear, and the research is novel. Based on the current manuscript quality, this manuscript should be accepted with minor revisions.

Introduction:
Generally, the goal of the study is clarified. There is sufficient background information about this research and the background information is well connected to the current study. The introduction could add more background knowledge related to this research.

Line 54-55 and Line 62-63: The authors give some basic backgrounds of Vibrio parahaemolyticus – it is a halophile and can survive a wide range of temperature and salinity. I suggest authors writing more details about this bacterium’s physiology. For example, the temperature ranges from XXX degrees to YYY degrees and the salinity ranges XXX to YYY, so the readers will have a clearer picture.

Line 93-105: From “ The US ….” to “ trh+ pathogenicity genes”. I suggest authors put all of these sentences into one individual paragraph, since all of these discuss the examples in the US.
Experimental design: Methods:

Generally, more information about water sample collection is needed.
Line 118: “a total of 192 sea surface water samples were collected”. It is not clear how these water samples were collected. Did the authors filter the water before measuring the pH? Did the authors rinse the tubes before collections or were these tubes sterilized? If the author used a standard protocol to collect water samples, I suggest authors citing this paper below. It used the standard USGS (United States Geological Survey) protocol to collect water samples.
Jiang, X., and Takacs-Vesbach, C.D. (2017) Microbial community analysis of pH 4 thermal springs in Yellowstone National Park. Extremophiles 21: 135-152.

Line 130: It is not clear about APW broth’s recipe. If this is the authors’ own recipe, the ingredients should be listed. If not, a citation should be given here.
Line 177-178: This sentence should be revised and made clearer about the purpose of finger printing and Eric-PCR.
For example, “In order to identify/study the presence of XXX, V. parahaemolyticus toxR positive isolates were fingerprinted using enterobacterial repetitive intergenic consensus (ERIC)-PCR as described elsewhere”.
Validity of the findings: Results:
The authors detailed research results and the results are clear.

Line 201: Revise to “were 8.22 and 31°C, respectively (Table 2).”

Discussion:
Some suggestions on the discussion:

Authors may discuss more at the end of the discussion to make their discussion more robust. For example, what is the significance of current results? Should we be aware of the potential food contamination of Vibrio parahaemolyticus, if the toxic genes are widely dispersed? Based on current research, should normal people be aware of this foodborne pathogen in the Eastern Province of Saudi Arabia? Do the authors have any suggestions and advice on the future study of Vibrio parahaemolyticus.

Line 263-263: “with several studies conducted worldwide that have reported the….”. This sentence is too long to express clear ideas. Revision is needed.
Line 281-283: “disagrees with findings on the unsatisfactory performance of TCBS agar in selecting …….”. It should mention more details about the previous research results here, so readers can understand the differences between current research results and previous results.

Tables and Figures:
In this study, 192 seawater samples were collected from 5 locations in the Eastern Province of Saudi Arabia. I suggested a basic map should be included as a figure. On the map, 5 sample locations should be marked, so it is easy for readers who are not familiar with the geography of Saudi Arabia.
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: IMPROVED ISOLATION AND DETECTION OF TOXIGENIC VIBRIO PARAHAEMOLYTICUS FROM COASTAL WATER IN SAUDI ARABIA USING IMMUNOMAGNETIC ENRICHMENT
Review round: 1
Reviewer: 3

Output:
Basic reporting: In this study, the author isolated tdh positive V. parahaemolyticus from sea surface water of the coast of the Eastern Province of Saudi Arabia by cultural, IMB, and molecular techniques. They first reported genetically diverse pathogenic V. parahaemolyticus from the coastal water in the Eastern Province of Saudi Arabia. The study was conducted in a short time-period and failed to provide supportive approaches to build up the study. Extensive revision is required in the method and writing for clarification and explanation. The English writing of the work needs to be improved. Some of the sentence structures need to be improved as they are not clear enough to understand. Specifically, lines 25-27, 129-131, 160-161, 302-306, 310-313 are needed to be revised. In this study, the authors mainly compared the isolation of Vibrio parahaemolyticus using IMB on TCBS and CaV agar but the title used here is ‘Isolation and molecular characterization of toxigenic Vibrio parahaemolyticus from the coastal water in the Eastern Province of Saudi Arabia’ which does not match with the study.
Experimental design: The method section needs improvement particularly the sample collection and isolates selection.
Please clarify how the V. parahaemolyticus isolates number was determined? Are multiple isolates taken from the same sample?
Please comment why did you choose one particular month of sample collection instead of throughout the year.
Validity of the findings: In the environment, the prevalence of tdh gene in V. parahaemolyticus is less than 5%. But in this study, 80.77% (21/26) of the toxR confirmed V. parahaemolyticus are positive for tdh gene, which is quite abnormal.
Additional comments: In this study, the author isolated tdh positive V. parahaemolyticus from sea surface water of the coast of the Eastern Province of Saudi Arabia by cultural, IMB, and molecular techniques. They first reported genetically diverse pathogenic V. parahaemolyticus from the coastal water in the Eastern Province of Saudi Arabia. The study was conducted in a short time-period and failed to provide supportive approaches to build up the study. Extensive revision is required in the method and writing for clarification and explanation. The English writing of the work needs to be improved. Some of the sentence structures need to be improved as they are not clear enough to understand. Specifically, lines 25-27, 129-131, 160-161, 302-306, 310-313 are needed to be revised. In this study, the authors mainly compared the isolation of Vibrio parahaemolyticus using IMB on TCBS and CaV agar but the title used here is ‘Isolation and molecular characterization of toxigenic Vibrio parahaemolyticus from the coastal water in the Eastern Province of Saudi Arabia’ which does not match with the study.
Table 3
Primers targeted for the toxR gene are well studied for V. parahaemolyticus species identification, but in this study, only 2.5% (Isolated without IMB) and 26% (Isolated with IMB) of the isolates is confirmed as V. parahaemolyticus. So misidentification of the 120 V. parahaemolyticus may occur here.
Line 39: “17.5% and 2.5% are positive for thermostable direct hemolysin……..” please mention the number of isolates along with the percentage. Are they from 3 and 26 isolates or 120 isolates? If 120, then looking for tdh among them is not logical as they are not even V. parahaemolyticus.

In the environment, the prevalence of tdh gene in V. parahaemolyticus is less than 5%. But in this study, 80.77% (21/26) of the toxR confirmed V. parahaemolyticus are positive for tdh gene, which is quite abnormal.
Introduction:
Please mention if there are some reports of V. parahaemolyticus incidence in the middle east or Saudi Arabia. And the relevance to conducting this study in this area particularly.
Method:
The method section needs improvement particularly the sample collection and isolates selection.
Please clarify how the V. parahaemolyticus isolates number was determined? Are multiple isolates taken from the same sample?
Please comment why did you choose one particular month of sample collection instead of throughout the year.
Line 152: “supernatant was discarded”

Discussion:
Line  263-265: Mentions some references for the previous reports

Please use standard --%(--/--) format in line 34, 39, 210-212, 215,217, 234, 245, 278,280, 287 to make the results clear to the reader.


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: IMPROVED ISOLATION AND DETECTION OF TOXIGENIC VIBRIO PARAHAEMOLYTICUS FROM COASTAL WATER IN SAUDI ARABIA USING IMMUNOMAGNETIC ENRICHMENT
Review round: 2
Reviewer: 1

Output:
Basic reporting: Dear Editor,

	The manuscript has been significantly improved after the revision. The current manuscript’s writing is clear, and the research is novel. The authors have addressed all of questions from my first review. Therefore, I recommend the Editor to accept this manuscript.
Experimental design: The authors have addressed all of questions from my first review.
Validity of the findings: The authors have addressed all of questions from my first review.
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: MULTIPLE VIRULENCE FACTORS REGULATED BY ALGU CONTRIBUTE TO THE PATHOGENICITY OF PSEUDOMONAS SAVASTANOI PV. GLYCINEA IN SOYBEAN
Review round: 1
Reviewer: 1

Output:
Basic reporting: 1.	A little confuse of this description. “For Psg gene expression profiles, bacteria were grown in HS medium optimized for COR production (HSC; Palmer and Bender, 1993) for 3 and 48 h, adjusted to an OD600 of 0.1, and grown again in fresh HSC medium for 3 h.”
2.	No figure legends for Supplementary figures.
3.	The efficiency of the complementation of algU seems not good, as shown in Supplementary Figure 1.
4.	The importance and function of AlgU in Pseudomonas has been studied widely. Authors should discussion more with their results.
Experimental design: That is ok.
Validity of the findings: That is ok.
Additional comments: That is ok.


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

Input:
Title: MULTIPLE VIRULENCE FACTORS REGULATED BY ALGU CONTRIBUTE TO THE PATHOGENICITY OF PSEUDOMONAS SAVASTANOI PV. GLYCINEA IN SOYBEAN
Review round: 1
Reviewer: 2

Output:
Basic reporting: In this paper, the authors found that the algU mutant showed significantly reduced virulence together with reduced bacterial populations in planta. They showed that AlgU regulated algD, fliC, and hrpL expression and also demonstrated that expression of coronatine-related genes and coronatine production were down-regulated in the algU mutant. The molecular mechanism of the pathogenicity of Pseudomonas species are a recent interesting topic. In particular, this work suggests that stomatal-based defense and flagellin-based defense may contribute to the reduced pathogenisity of the algU-mutant, which will provide important insights into the interactions between plants and pseudomonas in molecular level.
I think the manuscript is suitable for publication in PeerJ, but suggest the following several comments for a higher quality. Additional experiments are not always necessary. If the authors already have the data, adding them will make the paper even better.
Experimental design: In Line 192, the authors demonstrated that the algU mutant showed dramatically reduced bacterial populations in planta. But it may be necessary to investigate whether the bacterial growth ability of the algU mutant on the medium is the same as that of WT.

In Fig 3 and Fig 4, the authors investigated gene expression profiles involved in the virulence and COR quantification of WT and the algU mutant. But did those of algU-complemented strain recover to the same level as the wild strains?
Validity of the findings: The authors investigated disease symptoms on soybean leaves inoculated with WT and mutant strains in Line 181 and Fig 1. But is the area of chlorotic region inoculated with the mutant strain the same as that of the wild strain? The authors should put a scale bar in the figure.
Additional comments: Line 267
It is interesting to note that AlgU may suppress stomatal-based defense. The authors observed the bacterial populations of Psg by spray-inoculation in Fig. 2, but how about the bacterial populations when inoculated by syringe infiltration in Fig. 1?  If there is no difference in the bacterial populations of WT and the algU-mutant when inoculated by syringe infiltration, it is considered that stomatal-based defense may greatly contribute to the reduction of pathogenicity of the algU-mutant.


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: MULTIPLE VIRULENCE FACTORS REGULATED BY ALGU CONTRIBUTE TO THE PATHOGENICITY OF PSEUDOMONAS SAVASTANOI PV. GLYCINEA IN SOYBEAN
Review round: 1
Reviewer: 3

Output:
Basic reporting: No comment - my comments are in my review.
Experimental design: No comment - my comments are in my review.
Validity of the findings: No comment - my comments are in my review.
Additional comments: PeerJ 62630v1

Multiple virulence factors regulated by AlgU contribute to the pathogenicity of Pseudomonas savastanoi pv. glycinea in soybean

This manuscript reports the results of a transposon insertion screen to identify mutants with altered virulence phenotypes of Pseudomonas savastanoi on host plants.  From screening 1000 transposon insertion mutants the authors identified 28 mutants with reduced virulence.  The majority of these insertions were in genes affecting coronatine phytotoxin production.  A mutant with an insertion in algU, a global transcription regulator, was also identified.  The authors took advantage of this to test the function of AlgU in regulating virulence in this strain.
	This is a concise study that adds to the emerging understanding of AlgU as having a prominent role in helping P. syringae transition to pathogenic growth – including regulating coronatine, hrpL and fliC expression.  The paper presents a clear central message and is noteworthy for people interested in P. savastanoi biology specifically, and AlgU function in general.  The paper is fairly well written but could use some careful editing to improve readability.
	In terms of criticism – the conclusions about alginate production are weakened by the biofilm experiment.  For example, the authors use their observation that biofilm formation is reduced in the algU mutant to conclude that alginate biosynthesis is reduced.  As presented, this is a circuitous, uninformative (and unnecessary) way to obtain information about alginate production.  First, alginate is not a major or necessary component of Pseudomonas biofilms, which suggests that this assay does not inform about alginate production. Second, AlgU regulates on the order of ~1000 genes, additional work is needed to make claims on which of those is responsible for altered biofilm formation. Third, the paper reports results of qRT-PCR analyses that show AlgU regulating the algD gene (which encodes the enzyme responsible for the first committed step in alginate production).  With that result and because there is a deep literature on AlgU’s role in regulating alginate production, I would think that would be sufficient for this study to claim that the biosynthetic genes are AlgU dependent.  Showing that AlgU regulates algD is basically a positive control, showing that AlgU has the same role in P. savastanoi as it does in almost all Pseudomonads.  However, the focus of this paper is on P. savastanoi virulence, considering that, the result is not important because alginate is not considered to be a virulence factor for this strain.  With that said, demonstrating that alginate affects either biofilm formation (by comparing wt vs alginate-minus strains) or virulence (WT vs alginate-minus) would be a noteworthy contribution.

Specific comments:

Lines 80-81 – I think it is fair to say that the relative importance of specific virulence functions may vary for each pathovar or for each specific pathovar-host interaction.  The observed relative importance of individual virulence factors may also be a function of the type of read out used to assess virulence.  For example, in this ms, the authors “screened for mutants with less…chlorosis”.  This might explain the disproportionate numbers of mutants that were identified with defects in coronatine production.

Table 1 – Please list the locus tag for the affected gene and the genomic coordinates for the transposon insertions.

Lines 86-88:  Which EPS is being referred to here?  If the authors are refering to alginate, please consider https://doi.org/10.1099/mic.0.2007/012864-0, which shows that AlgU, but not alginate are important for virulence.  Additionally, there are not any results in this MS demonstrating a role of EPS in plant virulence.

Line 31-32, Line 231 -232 – Finding reduced biofilms with the algU mutant is not evidence that
AlgU regulates alginate biosynthesis.

Figure 1. Scale bars would help in comparing virulence of mutants.

Line 252-253 – This reference does not discuss alginate in biofilms.  There is evidence that
alginate is not a significant (or necessary) component of biofilms, see: https://doi.org/10.1073/pnas.1231792100

Line 274 – AlgU also regulates hrpL expression in DC3000.

Line 275 – This phrasing is confusing – the genes are in the hrp cluster, but they are not regulated by the hrp cluster.

Line 282-283 – These references (Bao et al.  2020; Lam et al. 2014) are not the original or recent papers dealing directly with how flagellin or the flg22 epitope trigger PTI.


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: MULTIPLE VIRULENCE FACTORS REGULATED BY ALGU CONTRIBUTE TO THE PATHOGENICITY OF PSEUDOMONAS SAVASTANOI PV. GLYCINEA IN SOYBEAN
Review round: 2
Reviewer: 1

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


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

Input:
Title: MULTIPLE VIRULENCE FACTORS REGULATED BY ALGU CONTRIBUTE TO THE PATHOGENICITY OF PSEUDOMONAS SAVASTANOI PV. GLYCINEA IN SOYBEAN
Review round: 2
Reviewer: 2

Output:
Basic reporting: The authors have improved all the points I have previously suggested, and the additional experiments were sufficiently conducted. I think the manuscript is suitable for publication.
Experimental design: no comment
Validity of the findings: no comment
Additional comments: No additional comments


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

Input:
Title: MULTIPLE VIRULENCE FACTORS REGULATED BY ALGU CONTRIBUTE TO THE PATHOGENICITY OF PSEUDOMONAS SAVASTANOI PV. GLYCINEA IN SOYBEAN
Review round: 2
Reviewer: 3

Output:
Basic reporting: no comment
Experimental design: no comment - The experimental design is appropriate.
Validity of the findings: no comment
Additional comments: I reviewed the first version of this manuscript and would like to commend the authors for addressing the reviews.   I think this paper helps to expand understanding of AlgU’s role in virulence.  I still have an issue with using the reduction in biofilms to conclude that EPS production was reduced (see lines 253, 273-274).  This is a reasonable hypothesis, but based on the data the most that can be said is that biofilm formation was reduced in the algU mutant, possibly because of reduced EPS production.  AlgU is a global regulator, and as such regulates many genes.  I don’t think that it’s possible to say which are involved in biofilm formation without testing them.  There are tests for EPS production if the authors are interested in demonstrating that EPS was reduced.  I should also add that demonstrating reduced biofilms is an important result on its own.


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: FUSARIUM HEAD BLIGHT IN THE RUSSIAN FAR EAST: 140 YEARS AFTER DESCRIPTION OF THE ‘DRUNKEN BREAD’ PROBLEM
Review round: 1
Reviewer: 1

Output:
Basic reporting: The article is clearly written, intelligible, and provides a coherent narrative. GenBank accession numbers are provided; authors' sequences are not yet available on GenBank but that is common practice (to not release sequences until publication) and the authors provided a FASTA file with the sequences and other raw data in supplementary files.
Experimental design: The paper reports original primary research which falls within the Aims and Scope of PeerJ. The detailed analysis of species and mycotoxin composition of organisms causing Fusarium head blight of wheat and barley in the Russian Far East is a valuable contribution to the growing body of literature detailing Fusarium species distribution, especially as those distributions are changing in many parts of the world. Methods used are standard in the field, and are described and/or referenced such that they can be repeated.
Validity of the findings: Findings and conclusions are justified by the data and the methods.
Additional comments: Here are a handful of minor edits and suggestions:
Lines 27, 245: Values like “6089 x 10^3” and “2102 x 10^3” are not standard; more commonly I would expect to see “6.1” or “6.1 x 10^0”
Line 88: suggest “endemic Asian species”
Line 128: probably not necessary to specify “self-made”
Line 148: suggest “normalized” for “aligned”
Line 179: “SecurityGuard” (no space; capital “S” and “G”); this is a trade name for the brand of cartridge. “Security guard” (with a space; lowercase) has a different meaning.
Line 230: “verified that infection by Fusarium fungi was the primary cause” or “verified that Fusarium fungi was the primary causal agent”
Line 241: suggest leaving out “The representatives of” and starting the sentence with “Cladosporium spp.”
Lines 253-256: rephrase; suggest “exceeded the maximum permissible limits (MPLs) in grain for food (700 ppb for wheat grain, 1,000 ppb for barley grain) and for fodder (1,000 ppb for cereal grain) (TR TS 015/2011; TR TS 021/2011), by up the 13 times.” As written, it sounds as though all samples had 13x as much DON as MPLs, while what you want to say is that all samples exceeded MPLs, and the highest levels were up to 13 times MPL.
Line 327: suggest “Using morphology” instead of “The morphological recognition”
Figure 1: “were drawn”
Figure 2: suggest “expulsion of asci and ascospores”


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: FUSARIUM HEAD BLIGHT IN THE RUSSIAN FAR EAST: 140 YEARS AFTER DESCRIPTION OF THE ‘DRUNKEN BREAD’ PROBLEM
Review round: 1
Reviewer: 2

Output:
Basic reporting: No comments
Experimental design: No comments
Validity of the findings: No comments
Additional comments: At some places language needed to be improved for clarity and in some others the run on sentences needs to be broken down into shorter sentences for better clarity to the readers.


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: FUSARIUM HEAD BLIGHT IN THE RUSSIAN FAR EAST: 140 YEARS AFTER DESCRIPTION OF THE ‘DRUNKEN BREAD’ PROBLEM
Review round: 1
Reviewer: 3

Output:
Basic reporting: The author tried to explain very well regarding writing, but the message delivery was not precise; for example, the research objective was not clear, the writing is not in flow, and information was scattered all around.  The material and methods and result section should be clear and consistent.

This article needs to rewrite with a clear objective, hypothesis, methods,  result, and discussion.
Experimental design: I think the experimental design was not enough to support for aims and scope of the journal. No clear objective or hypothesis was given. Sampling methods, sample location, or distribution pattern were not clear. The morphology is not enough to identify the Fusarium species complex. Only 29 samples collected in a single season are not enough to represent the whole story of the genetic relationship in this complex pathosystem.
Validity of the findings: Finding is valid for the amount of data collected. However, it is not clear how sampling was done, how isolates were selected, and which isolate was used for which purpose. For example, grain/isolate used for mycotoxin analysis, the sample used in DNA extraction from grain or the mycelia,  selection criteria of  29 isolates, etc.
Additional comments: Dear Authors,
I enjoyed reading your manuscript and understand the problem of Fusarium Head blight in Far East Russia for such a long time. You have collected nine spring wheat and four barley samples from the Amur region of Russia (Far-east) in August 2019. Samples used for mycotoxin analysis, isolate collection, or species characterization based on morphology. qPCR was done to quantify F. graminearum and F. avenaceum or 3-AcDON and 15-AcDON chemotypes of F. graminearum. You have also performed a genetic analysis of 29 isolates using TEF, URA, RED, and Tri101 gene sequences.

However, this article lacks a clear objective or hypothesis; the abstract is too long, the sampling method is not clear, the identification of species based on morphology is not enough, the number of isolates used in the genetic analysis is insufficient, and data based on a single season lacks actual representation. I think this article doesn't meet the requirements to be published in PeerJ with such flaws.
Thanks


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: FUSARIUM HEAD BLIGHT IN THE RUSSIAN FAR EAST: 140 YEARS AFTER DESCRIPTION OF THE ‘DRUNKEN BREAD’ PROBLEM
Review round: 2
Reviewer: 1

Output:
Basic reporting: English is not that of a native speaker but is clearly understood.
Experimental design: No comment
Validity of the findings: No comment
Additional comments: The authors have addressed the concerns I raised in the previous review. Further, I agree with their argument for retaining their proposed title, and they have bolstered their identificatiosn with appropriate DAN studies (EF1A, in particular) for identifying morphologically-indistinguishable members of the Fusarium graminearum species complex, and/or used "Fusarium graminearum sensu lato" when discussing taxa idenitfied based on morphology.


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: THE MORPHOLOGY AND INTERNAL STRUCTURE OF DOGWOOD (CORNUS L.) ENDOCARPS IN THE TAXONOMY AND PHYLOGENY OF THE GENUS
Review round: 1
Reviewer: 1

Output:
Basic reporting: This manuscript provides excellent documentation of the variation in features useful in the identification and systematic assessment of many species of Cornus fruits and gives good support for the value of certain suites of endocarp characters for taxonomic determinations.  It will be useful to paleobotanists as well as systematists and is a good example of the kinds of analyses that would be usefully applied to other clades of angiosperms.

In the annotated copy of the manuscript I made minor corrections to English and a few critical comments.   I consider the manuscript acceptable following minor revision.  Here are some specific comments.

I was confused by the use of the term “anatomy” to refer to features usually thought of as morphology. Line 40:  “With regard to the anatomy of endocarps, the most important features were the germinating valve thickness, the ratio of the endocarp wall thickness divided  by the endocarp diameter, the septum width and its structure, the presence or absence of crystals in the outer endocarp, the number of cell layers in the transition sclereids zone and the primary and secondary sculpture”

Some of the characters listed here are traditionally considered morphology rather than anatomy:  germination valve thickness, ratio of the endocarp wall thickness divided  by the endocarp diameter, the septum width and its structure.  Only the presence or absence of crystals in the outer endocarp, the number of cell layers in the transition sclereids zones are characters of anatomy/histology.

In my opinion, the acronyms (such as GVT, SMW and WTP) are unnecessary in the main text, but useful only in the figures and captions.  The article will be much easier to read if the actual words are used.   But if you do want to use them, each acronym should be explained at its first occurrence in the text (this has not been done consistently in this manuscript).
Experimental design: It may be appropriate in the material and methods to indicate which species of Cornus were not available or not studied.  Although all major clades were sampled, not all of the species were presented.
Validity of the findings: Line 420:   “ the presence of secretory cavities in the endocarp wall was a
species-specific trait. It occurred only in C. mas and C. officinalis fruit stones.”

I am curious why C. sessilis is not mentioned as another example.  Were the cavities missing in your collections of C. sessilis?   Figure 2 of Manchester et al 2010 shows cross sectioned stones with cavities present in C. mas, C. officianalis, C. sessilis, C. volkensia, C. eydeana, C. chinensis and this is thought to be a feature of all the species of subgenus Cornus (Cornelian cherries), so it might be a subgenus trait rather than species-specific trait.
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: THE MORPHOLOGY AND INTERNAL STRUCTURE OF DOGWOOD (CORNUS L.) ENDOCARPS IN THE TAXONOMY AND PHYLOGENY OF THE GENUS
Review round: 1
Reviewer: 2

Output:
Basic reporting: In general, the manuscript contains valuable and interesting descriptions and analysis of Cornus endocarps.  There are problems with the grammar and sentence structure and I’ve tried to make some corrections.  The data set is complex.  The tables do not maintain character lists in any order, so it is difficult to compare among tables and text.  The characters are not well described in some places and there are problems with calling characters and character states “variables”, “variants” and “traits”.  References are appropriate.  Structure is problematic.  There is a need for better organization to help the reader keep track of the characters.  Table 6 description states that “(for trait description [sic] see Table 5)”, yet there are no trait descriptions in Table 5.  Maybe Table 2?  Discussion of traits does not follow in order of Tables 9 and 10.  This would help the reader understand the results and how they inform our understanding of the phylogeny.  Figure 8.  A dendrogram is presented, yet there was no discussion in the manuscript of how this was generated.  What data were used?
Experimental design: The manuscript is evaluating phylogenetic studies and asking if the presented data supports or does not support the phylogenies, yet the data are not presented in a character and character state way that allows the reader to evaluate the value of the character for phylogenetic analysis.  Many of the character states are variable, yet those that vary within taxa are used interchangeably with those that do not.  It would be useful to differentiate those characters or character states that are invariant, recognize these as synapomorphies, and then ask the question of whether these synapomorphies support the published phylogenies.
Validity of the findings: The statement “based on the morphological features of their endocarps exactly reflect their phylogenetic relations BW(CC(DW-BB)) shown by Fan & Xiang (2001) and Xiang et al. (2006) was verified positively” is not supported by the results presented.  It appears that data are picked for discussion that support certain phylogenies, but those that do not support the published phylogenies are ignored.  For example, Figure 2 shows overlap of CC and BB, but this is ignored in the discussion.  In another example, Figure 9 circumscribes the BB and DW groups, but ignores the overlap with the BW taxa with DW and BB, but not with CC.  No outgroups were examined.  Traits such as lack of apical cavity are pleisomorphic, so have no value in analysis of the phylogeny of the genus.  Traits were described as supporting particular phylogenies when the trait was not uniform within the taxa.   Many of the character states described are variable within taxon, yet the phylogenetic utility of the described characters is not discussed.
Additional comments: I have made comments on 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: THE MORPHOLOGY AND INTERNAL STRUCTURE OF DOGWOOD (CORNUS L.) ENDOCARPS IN THE TAXONOMY AND PHYLOGENY OF THE GENUS
Review round: 2
Reviewer: 1

Output:
Basic reporting: This article is much improved following the previous round of review.
Experimental design: no further comment following earlier rerview
Validity of the findings: Yes, well presented.
Additional comments: I have accepted your rebuttal to some of my earlier recommendations.   In the annotated pdf, I have indicated a few minor corrections an places for clarification.


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: OBESITY PHENOTYPES AND THEIR RELATIONSHIPS WITH ATRIAL FIBRILLATION
Review round: 1
Reviewer: 1

Output:
Basic reporting: .
Experimental design: .
Validity of the findings: .
Additional comments: The paper is solid on an important topic , so this should be publishable in a solid Journal. A limitation that should be mentioned is only minimal data on physical activity (PA) and none on Fitness , and Fitness may be more important than weight ( Barry VW et al. Prog Cardiovasc Dis 2018; 61: 136-141) and even PA/Fitness even may markedly impact the MHO ( Ortega FB et al. PCVD 2018; 61: 190-205.) The authors adjusted for WC and showed their data by WC-They could combine both high WC and high TGs , as this is a group who has more marked visceral adiposity. Also, they could consider including 2 highly cited major JACC State of the Arts , one on Obesity and AF ( Lavie CJ et al. JACC 2017; 70: 2022-2035) and one on the whole idea of Healthy Weight including MHO( Lavie CJ et al. JACC 2018; 72: 1506-1531.)


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: OBESITY PHENOTYPES AND THEIR RELATIONSHIPS WITH ATRIAL FIBRILLATION
Review round: 1
Reviewer: 2

Output:
Basic reporting: Tsai et al. reported a normative study to explore the relationships between obesity phenotypes and atrial fibrillation. The background was well organized, and the prior literatures were approprately referenced. The results found that the risk of Afib was elevated in the MUNW and MUOW/MUO groups (without statistical significance), but these findings lack of innovation. The major hypothese about MHO and atrial fibrillation was Still unproven. The authors should derectly point that whether people with MHO would have higher risk of Afib is unknown, and need further studies.
Experimental design: This cohort was well designed, and the outcome assessment was credible. But there are some deficiencies.
1. According to the Inclusion criteria, there were 4000+ people excluded because of various reasons, please discuss possible selection bias from this progress.
2. Line 95,  why you further excluded participants younger than 20 years old?
3. Suggest to add a flow chart figure to show the participants selection, and to report the rate of lost to follow-up, these is important for cohort study.
4. Line 142: the author used "The time-dependent covariate regression method was used to assess the proportional hazards assumption", what is the results? Whether all the Covariates meet the "proportional hazards assumption"? If not, how do you solve this problem?
5. The event count is so small in some groups (From Table 2), I doubt that whether the Cox regression model is suitable for HR estimations. Please demonstrate its applicability. Whether possion regreesion is more suitable?
Validity of the findings: 1. From the Abstract, all the results you stated were Not statistically significant, but the conclusion is "manage metabolic components and obesity to mitigate the risk
of Afib". It is inappropriate. The results were not robust, it suggested futher studies.
2. The authors should think more carefully about Model C and Model D.  The valvular heart disease, myocardial infarction, and heart failure are all associated with atrial fibrillation? All of these covarites were added to Cox model, and the HRs were weakened (insignificant). How to explain these results? you should consider clinical and statistical correctness.
3. The authors selectively reported the results about MUNW, MUOW/MUO, and MHO groups, even though the HRs were without statistical significance. Why not explain the results from "Metabolically unhealthy Underweight" group? It seems intersting
Additional comments: 1. Some typo, eg Line 254, "he"?


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: OBESITY PHENOTYPES AND THEIR RELATIONSHIPS WITH ATRIAL FIBRILLATION
Review round: 2
Reviewer: 1

Output:
Basic reporting: The authors have adequately addressed my concerns.
Experimental design: None.
Validity of the findings: The authors have addressed the limitations that were pointed out and added the appropriate disclaimers to the findings.
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: THE INCONSISTENT MICROBIOTA OF BUDU, THE MALAYSIAN FERMENTED ANCHOVY SAUCE, REVEALED THROUGH 16S AMPLICON SEQUENCING
Review round: 1
Reviewer: 1

Output:
Basic reporting: Article is logically consistent and clearly written.
Experimental design: Methods are well described, most of samples have biological replicates. The only drawback is the lack of negative controls that can help to catch possible contaminations (for example potential contamination with Chromobacterium during sampling). However microbiome composition is very specific and do not look as suffered from contamination.
Validity of the findings: no comment
Additional comments: No additional comments


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

Input:
Title: THE INCONSISTENT MICROBIOTA OF BUDU, THE MALAYSIAN FERMENTED ANCHOVY SAUCE, REVEALED THROUGH 16S AMPLICON SEQUENCING
Review round: 1
Reviewer: 2

Output:
Basic reporting: A manuscript describes an analysis of the microbiota of a Budu, a Malasyan sauce produced by fermentation of anchovies. Because fermentation, in this case, is a microbial-based process, the structure of the microbiota may be crucial for the features of the final product, including its impact on consumer health.
The manuscript is quite interesting and devoted to a rather unusual topic, as like food-producing microbiota. The work is well done and the stated goals are achieved successfully.
The text of the manuscript is written clearly and it was interesting for me to read it.
Finally, I recommend accepting this manuscript after some minor revisions.
Experimental design: The experimental design is correct and well thought out. The workflow includes sample collection (60 samples from 20 bottles of the sauce), amplification and analysis of the 16S rRNAs, the definition of the taxonomical structure of the samples, description of the positive and negative correlations for different species of microbes, and prediction of the overrepresented pathways.
Validity of the findings: The results of this research can be interesting for microbiologists and biochemists, as well as for specialists in the food industry.
Additional comments: Minor comment:
Lines 59-60: "The fish sauce microbiota can change the fish sauce metabolite content, potentially altering its health and gastronomic properties." - Absolutely unclear what are there "health properties" of the sauce. Please change this sentence to make it clear.

Desires:
1. I would like to see a more detailed explanation for why is it important to study the microbiota of the Budu sauce. I mean, why this sauce is so important to study its microbiota?
2. I would like to see a more detailed description of functional and pathway metagenome prediction. What pathways were also enriched, in addition to the listed ones? How different are the analyzed samples on the level of enriched pathways? Is a taxonomic variability of samples correlate with a pathway variability?


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

Input:
Title: A NOVEL ROLE FOR APATINIB IN ENHANCING RADIOSENSITIVITY IN NON-SMALL CELL LUNG CANCER CELLS BY SUPPRESSING THE AKT AND ERK PATHWAYS
Review round: 1
Reviewer: 1

Output:
Basic reporting: The authors use a clear and unambiguous, professional English throughout the manuscript.

Literature references, sufficient field background/context provided?
1.	In the first line of the introduction the authors mention that “Lung cancer is the leading cause of morbidity and mortality worldwide “, and they refer to Siegel et al. 2013, which is a paper on cancer statistics published in 2013.  The authors must include a recent reference to support their statement. They could also refer to: https://gco.iarc.fr/.  According to this database, Lung cancer is the leading cause of mortality, but the second and forth in incidence and prevalence, respectively.
2.	In lines 49-52 the authors mention that the strategies currently used to improve NSCLC are not very efficient, and that “…current research focuses on enhancing the radiosensitivity”, however they cite three references (Cao et al. 2013; Milas et al. 2005; Seidlitz et al. 2020), which are not related to NSCLC, there are many recent reports in the literature reporting different approaches to improve radiosensitivity in NSCLC, it will be better to refer to some of the most recent reports. Some examples are:
•	Kim SY, Jeong EH, Lee TG, Kim HR, Kim CH. The Combination of Trametinib, a MEK Inhibitor, and Temsirolimus, an mTOR Inhibitor, Radiosensitizes Lung Cancer Cells. Anticancer Res. 2021 Jun;41(6):2885-2894. doi: 10.21873/anticanres.15070. PMID: 34083279.
•	Lewis CD, Singh AK, Hsu FF, Thotala D, Hallahan DE, Kapoor V. Targeting a Radiosensitizing Antibody-Drug Conjugate to a Radiation-Inducible Antigen. Clin Cancer Res. 2021 Jun 1;27(11):3224-3233. doi: 10.1158/1078-0432.CCR-20-1725. PMID: 34074654.

•	 Xiong L, Tan B, Lei X, Zhang B, Li W, Liu D, Xia T. SIRT6 through PI3K/Akt/mTOR signaling pathway to enhance radiosensitivity of non-Small cell lung cancer and inhibit tumor progression. IUBMB Life. 2021 May 25. doi: 10.1002/iub.2511. Epub ahead of print. PMID: 34033225.
•	Wrona A, Dziadziuszko R, Jassem J. Combining radiotherapy with targeted therapies in non-small cell lung cancer: focus on anti-EGFR, anti-ALK and anti- angiogenic agents. Transl Lung Cancer Res. 2021 Apr;10(4):2032-2047. doi: 10.21037/tlcr-20-552. PMID: 34012812; PMCID: PMC8107745.

Professional article structure, figures, tables. Raw data shared.
•	The article is presented according to Peerj standards.
•	The figures are relevant to the content of the article, and they are in general of sufficient resolution, however the bar graphs in figures 3 and 5 have poor resolution, the resolution must be enhanced.
•	The title in Table 1 is “Radiation biological parademeter”, Shouldn’t it be “Radiation biological parameter”?. I suggest including a brief description of the content in Table 1 (the parameters).
•	In the graphics in Figure 1 and Figure 2, the authors do not indicate if they performed any analysis to show that the differences are statistically significant. Asterisks should be included to indicate where statistically significant differences were found, and the statistical methods used should be mentioned in the figure legends. They should also indicate if the data are from three or more independent experiments.
•	In figure 2 A and B, one of the conditions is labeled as “blank”, were these cells untreated (blank), or were they treated with DMSO?  This should be clarified in the labeling and figure legend.
•	The same comment as above is for figure 3, one of the conditions in all the graphics is labeled as “blank”. It should be clarified if the cells were untreated or treated with DMSO.
•	In figures 3, 4 and 5, the p value for *, ** or *** should be mentioned in the figure legends.
•	In figure 4, the label for the Y axes in the bar graphs is “Percentage of positive gH2AX foci cells (%)”. According to the material and methods section what the authors counted was the number of cells showing positive nuclear staining for gH2AX, as far as I understand they didn´t quantified the gH2AX foci.   I suggest changing the label to “Percentage of gH2AX positive cells”
•	The authors present supplemental materials containing the raw data, however for the colony formation assay they show the images of the assay but there is no excel file with the quantification data.  The data for cytotoxicity are presented for the two cell lines in separate excel files, but the data in both excel files are not organized in the same manner making it difficult to verify the data.
•	The raw data for ERK WB (LK2) shows only two blots, and in the blot labeled as E1, the pattern of the bands is very different to the pattern in the E2 blot.
•	p-AKT blots for A549, there is only one cropped blot shown.
The paper is presented as an appropriate ‘unit of publication’, and it includes all results relevant to the hypothesis.
Experimental design: -	The authors clearly define the research question, which is relevant and meaningful. The knowledge gap being investigated is whether AP may play a novel role in the radiosensitivity of NSCLC cells. The authors suggest that the knowledge generated may provide a theoretical basis for the clinical combination of radiation and AP.
Rigorous investigation performed to a high technical & ethical standard.
-	The investigation was conducted rigorously and to a high technical standard and in conformity with the prevailing ethical standards in the field.
-	Methods are in general described with sufficient information to be reproducible by another investigator. I suggest adding the following information to the methods section:
a)	The number of cells that were seeded for proliferation and apoptosis assays
b)	The amount of RNA used for cDNA preparation and the amount of cDNA used for qPCR
c)	The amount of protein that were used for SDS-PAGE, and the catalog number for the primary antibodies
Validity of the findings: All underlying data have been provided; they are robust, statistically sound, & controlled.
The conclusions are appropriately stated, they connected to the original question investigated, and are supported by the results. However. I suggest modifying the sentence “accelerating cell cycle arrest in the
329 G2/M phase”, as the authors didn´t evaluate the speed of the cell cycle arrest. The experiments in figure demonstrate that there is an increase in the number of cells arrested at G2/M, but there is no evidence that this occurs because the arrest is accelerated.
Additional comments: No additional comments


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

Input:
Title: A NOVEL ROLE FOR APATINIB IN ENHANCING RADIOSENSITIVITY IN NON-SMALL CELL LUNG CANCER CELLS BY SUPPRESSING THE AKT AND ERK PATHWAYS
Review round: 1
Reviewer: 2

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: A novel role for apatinib in enhancing radiosensitivity in non-small cell lung cancer cells by suppressing the AKT and ERK pathways by Li, L, et al. show results of the radiosensitive effect of apatinib on  NSCLC cell lines. The results are interesting, however, authors should respond to some questions.

1. Why the authors didn´t use H226 cell line to evaluate the effect of apatinib and radiotherapy? H226 cell line express higher levels of VEGFR2. The effect of apatinib must be evaluated in H226 cells.

2. LK2 cells express lower levels of VEGFR2 (almost imperceptible), these cells must represent the negative control of the effect of apatinib, however, LK2 cells behavior are similar to A549 cells, in fact, LK2 cells are more sensitive to apatinib and radiotherapy (LK2 cells showed higher levels of apoptosis and lower clonogenic survival). Radiotherapy is able to induce the expression of several membranal receptors to promote survival, its important that authors evaluate the expression of VEGFR2 after treatment of radiotherapy, apatinib or both (as figure 5) to explain the similar results between both cells. These results must explain the effect of apatinib on NSCLC cells.


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

Input:
Title: A NOVEL ROLE FOR APATINIB IN ENHANCING RADIOSENSITIVITY IN NON-SMALL CELL LUNG CANCER CELLS BY SUPPRESSING THE AKT AND ERK PATHWAYS
Review round: 2
Reviewer: 1

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


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

Input:
Title: FLPS 2.0: RAPID ANNOTATION OF COMPOSITIONALLY-BIASED REGIONS IN BIOLOGICAL SEQUENCES
Review round: 1
Reviewer: 1

Output:
Basic reporting: 1. Overall, clearly written, nice figures.

2. The program fLPS is updated, but this term itself was not defined clearly. What do the letters mean- I assume "fast low-probability sequence regions"?

3. Lines 70-81. It is not fully clear why this new version of fLPS is better or an advance over the prior version, or with respect to other programs. Are the updates enough to warrant a new publication? Explain more clearly why these updates are essential, and why they warrant a new publication.
Experimental design: Lines 94-111 appears to be repeating the program methods from the author's 2017 paper. Could be much shorter, and emphasize that this is not new to the present paper.

Lines 114-143 provides a description of the updates in the fLPS program. Some of these updates appear very minor such as providing an output file name option "-O". Or a "restriction list" with a "-r" option. Again, please make more clear why these updates are considered major and important enough to warrant a new paper.

Lines 229-232. Similarly, is this enough benefit to justify publication of the update? Same for lines 234-244. It may be enough, but there should be a better explanation of why.
Validity of the findings: The findings appear valid. The author should improve explaining why researchers would find this program useful for them. The patterns may exist (e.g. Fig. 4), but why are they important, or why should one be interested in studying those patterns found using the updated program.

I am wondering why the author does not provide a web server, or user friendly version particularly for researchers interested in this analysis, that may prefer a more user-friendly version, and better visualization (graphical display) of results. This may be a substantial drawback, as some biologists would like to conduct such analyses, but in a more user-friendly way.
Additional comments: Overall, the manuscript is well written and clear. I suggest improvements in explaining why this update should be considered crucial and worth a new publication over the original, and why researchers should be interested in the outputs and patterns detected only from the update. Also, why a user-friendly version appears to not be available (if it is, then the author needs to state this clearly). I suggest the manuscript would be improved by addressing these points, and can be published at PeerJ.
This is between a major and minor revision. I will assume the author can address these points and thus selected minor.


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: FLPS 2.0: RAPID ANNOTATION OF COMPOSITIONALLY-BIASED REGIONS IN BIOLOGICAL SEQUENCES
Review round: 1
Reviewer: 2

Output:
Basic reporting: The proposed MS is largely a technical text decribing update details of the fLPS software and their impact on application of fLPS in sequence analysis and annotation efforts. At the same time, it is more than a software application note as it dives into review aspects such as the definition of sequence bias/low complexity/etc., the impact for biological function, comparative mentioning of some of the most used software tools and of a few major insights achieved with sequence bias studies. This rather enhances the value of the text that, generally, provides a smooth, mature impression and seems to be correct in its critical statements. The application examples seem useful.
Experimental design: n/a
Validity of the findings: There are a few questions that remain in the mind of the reader such as (1) how does fLPS fair with problematic sequence elements such as TM regions or signal leader peptides that sometimes pop up as disordered/low complexity (PMID 20686689, 24890864) or (2) can fLPS recreate the list of regions in some databases for these specific sequences types (e.g., PMID 27899601).
Additional comments: The author can be proud of his contributions to the field; yet, the reviewer feels that the author goes overboard in certain aspects.

The statement “The fLPS program is optimal for analyzing CB regions since ...” (line 70) is a no-go and should be toned down, for example by “The fLPS program is especially useful for analyzing CB regions when ... the following aspects are at the center of attention:”.

The author has managed to include all of his previous papers into the reference list, even those with (though justifiable) distant relationship to the matter. This is fully appropriate if ... but, essentially, no other literature is mentioned except if technically inavoidable. It appears that the author does not exactly follow the publications in the field. Otherwise, he would have noticed that the Section Editor of this journal has a history in sequence disorder (e,g, PMID 11784292, a review). It is not the reviewer’s attempt to provide a full list here, just a few pointers: recent reviews (PMID 30698641, 33875885), a method to make the disorder definitions from several tools more comparable (PMID 20158872), other methods (PMID 32702119, 32535960) or a meta-server (PMID 32421769). There are databases of disordered regions/IDPs (e.g., PMID 29385418), etc. The article would benefit from putting the updated software tool into the real context of the state of the art.


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: FLPS 2.0: RAPID ANNOTATION OF COMPOSITIONALLY-BIASED REGIONS IN BIOLOGICAL SEQUENCES
Review round: 2
Reviewer: 1

Output:
Basic reporting: The MS is a straightforward, pleasant reading. The revisions have further improved the MS.
Experimental design: no critical comment
Validity of the findings: no critical comment
Additional comments: n/a