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: ASSOCIATION OF HEAT SHOCK PROTEIN 8 WITH ATOPIC MARCH IN A MURINE EXPERIMENTAL MODEL
Review round: 1
Reviewer: 2

Output:
Basic reporting: In this manuscript by Jeong et al, the authors establish a modified murine model for studies on the atopic march and this model was confirmed by observation of increased airway inflammation and elevated allergic responses. The authors showed that compared to healthy control group, atopic march induced group exhibited augmented levels of IgE in serum and increased production of Th2 dependent cytokines such as IL-4, IL-5 and IL-13 in BALF. Moreover, using two-dimensional gel electrophoresis and mass spectrometry analysis the authors identified Hspa8 as one of the major differentially expressed proteins in BALF in atopic march induced group. Overall, the study addresses an important finding of Hspa8 as a possible candidate biomarker in atopic march.

The main concerns regarding the manuscript are the inconsistency throughout in describing the treatment duration (weeks) in murine model which creates confusion to the readers, experimental design, and the lack of some important controls. There are numerous minor errors in English expression and punctuation, and general typographic errors, throughout. I suggest the authors should check the entire manuscript carefully and make corrections.
Experimental design: The manuscript could be strengthened by addressing the below listed points.
There is a lot of irregularity in describing treatment duration that creates misunderstanding to the readers:

For example:
Line 27: twice a week for 5 consecutive weeks
Line 90: Once a week
Line 198: once a week for 4 weeks
Line 251: 8 consecutive weeks

Authors should describe the allergen doses and treatment duration in detail where required and keep it consistent throughout the manuscript to make it clear for the reader.

Authors should also represent by arrow AM induced group in Fig1.

Authors used Diff-quick solution to stain different population of immune cells, but it remains unclear how the counting was done and what do they mean by counting 200 cells in each of four different locations. Authors needs to explain in the manuscript how was the quantification performed?

Authors need to briefly describe the acetone precipitation protocol in BALF samples or should cite a reference for the protocol. Moreover, authors need to mention which method was used for protein quantification and what amount of protein were separated by 2-DE.
It was unclear how the treatment of methacholine was done in these experiments? In Fig 3A. authors mentioned that methacholine stimulation was done after 24h of final intranasal challenge, but it remains unclear how it was done for each different mice group, hence I suggest authors should describe in the method section how all the different mice groups (normal control group, AD induced group, AS induced group, and AM induced group) were stimulated with methacholine and at what time point. This needs to be clarified for the reader.

In Fig 3A both airway resistance and dynamic compliance were only significant at higher doses (50mg/ml) of methacholine between AS-induced and AM-induced group. This needs to be explained in the manuscript and authors should clarify the difference between the AS-induced and AM-induced group. While measuring dynamic compliance among groups, why there is so much variability in PBS treated groups. This need to be discussed in the manuscript.

In Fig. 3C how was the scoring performed on H&E-stained sections. Authors need to describe somewhere either in figure legend or in the method section.

Cell Infiltration in lung tissue when compared with NC control group looks very similar to AD-induced group. Any explanation? Authors need to discuss this point in the manuscript.
Did the authors perform an experiment to figure out the different cell types recruited in lung tissue in different mice groups? This would be helpful experiment to study lung pathology in different mice groups and would strengthen the manuscript.

Determining the RNA levels of Hspa8 in the serum or BALF would add more significance to the work. Additionally, comparing the Hspa8 RNA expression in serum/BALF between all four groups would strengthen the conclusions.
Validity of the findings: In Fig 2. Adding statistical significance between week 2 and week 4 of AD-induced group will add more relevance to the data. I suggest keeping consistent with figure legends, replace week 2 and week 4 by Day 8 and Day 22 respectively in the Fig 2B.

In Fig 3B. authors need to perform statistical analysis on different population of cell types between AS-induced group and AM-induced group. Further, authors need to discuss about the decrease observed in monocyte/macrophages and neutrophils in the AM-induced group compared to AS-induced group.

In Fig 4. Statistics is not uniform in all the graphs and hence need to do it in a more consistent manner. There are several places in graphs where statistics analysis needs to be done for example Fig 4 F statistics on AS vs AM induced group is missing. Also, authors should insert non-significant (ns) between the groups where applicable in the graphs.
Additional comments: Minor Concerns:

Line 70 ‘analysis’
Line 98 ‘administered’
Line 108-110 need to improve/reframe the sentence
Line 167 ‘Reference’
Line 213 mention Figure#
Line 220 mention IgE levels were measured in serum
Fig 2A Normal control group received PBS from 0-4 weeks?
Fig 2B Graph scale should be set to ~4000 ng/ml and not to 10000ng/ml since according to raw data provided IgE maximum levels were 3401.856ng/ml
Fig 3 legend insert *p<0.05 versus which group?


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: ASSOCIATION OF HEAT SHOCK PROTEIN 8 WITH ATOPIC MARCH IN A MURINE EXPERIMENTAL MODEL
Review round: 1
Reviewer: 3

Output:
Basic reporting: The authors have used professional English throughout the manuscript and the paper was very easy to read. A quick suggestion is that it will be beneficial for the readers to have the full form of the abbreviations spelled out in the figure legends as it makes reading the figures easier.
Experimental design: The authors have developed a schematic experimental protocol and have mentioned a few of the study's shortcomings in the discussion section.
1. The authors are suggested using ANNOVA analysis for Figure 3B which is the appropriate statistical analysis to be used for the particular figure.
2. In figure 3B, apart from eosinophils there were changes observed in macrophages and neutrophils populations in the normal group versus AM group. Authors should discuss this observation in the discussion section as immune cells interact with each other, especially as they are looking at Th1 and Th2 immune responses.
3. Il-10 (Th2) and THF-alpha (Th1) cytokines are well known to be involved with Th2 and Th1 responses respectively. Is there a reason that the authors didn't look at Il-10 and TNF-alpha in BALF for this particular study?
4. In the hub proteins and pathways study, it will be helpful to look at the GO terms and protein enrichment between AM and AD groups. This will provide further detail on proteins being differentially expressed that induce AM in AD groups and compare those proteins with AS group. This will help to understand whether there are any particular groups of proteins that are involved in the progression of AD to allergic asthma to AM.
5. For figure 5, authors should group the proteins (blue spheres) into the cellular pathways/activities they belong in. This will help the readers to understand whether there is any particular cellular pathway that may be involved in the progression of AM from AD.
Validity of the findings: The study showcases a new mice model for AM that addresses the challenges present in the current existing AM models. The authors did a good job of highlighting the shortcomings of the paper. Here are my suggestions to that needs to be addressed in the discussion section:
1. Authors need to discuss the change in the population of neutrophils and macrophages between AD and AM groups along with the eosinophils increased levels. What kind of implications can this have as a whole need to be addressed?

2. Since the GO and PPI analysis were not done between AD and AM groups, we don't know what proteins are involved in the progression. This data will add more value to the paper.
Additional comments: Jeong et.al developed a new mice model to study AM and their analysis showed that heat shock protein 8 is a potential candidate involved in the AM progression. Their mice model addressed some of the challenges present in other published AM models. Overall their study adds value to the AM research and this field.


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

Input:
Title: ASSOCIATION OF HEAT SHOCK PROTEIN 8 WITH ATOPIC MARCH IN A MURINE EXPERIMENTAL MODEL
Review round: 2
Reviewer: 1

Output:
Basic reporting: In this study, authors propose an improved murine model for atopic march and suggest HSP8 as a candidate biomarker. Using this improved methodology they have assayed IgE level, Th2 response cytokine and lastly have done proteomic analyses to find candidate biomarkers.

Authors have addressed my initial questions in this revised manuscript.
Experimental design: methodical and understandable
Validity of the findings: NO comment
Additional comments: None


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: ASSOCIATION OF HEAT SHOCK PROTEIN 8 WITH ATOPIC MARCH IN A MURINE EXPERIMENTAL MODEL
Review round: 2
Reviewer: 2

Output:
Basic reporting: Authors appropriately answered all the comments.
Experimental design: Authors appropriately answered all the comments.
Validity of the findings: Authors appropriately answered all the comments.
Additional comments: Authors appropriately answered all the 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: ASSOCIATION OF HEAT SHOCK PROTEIN 8 WITH ATOPIC MARCH IN A MURINE EXPERIMENTAL MODEL
Review round: 2
Reviewer: 3

Output:
Basic reporting: The authors have written the paper in professional English and the paper is very reader-friendly.
Experimental design: No comments. The authors have satisfactorily answered my questions
Validity of the findings: The study showcases a new mice model for AM that addresses the challenges present in the current existing AM models. The authors have addressed the shortcomings of the paper in their rebuttal very effectively. This study contributes new knowledge to this field.
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: PHOSPHORUS FLOW ANALYSIS OF DIFFERENT CROPS IN DONGYING DISTRICT, SHANDONG PROVINCE, CHINA, 1995–2016
Review round: 1
Reviewer: 1

Output:
Basic reporting: This manuscript is clearly written in professional, unambiguous language. The author focused on long-term P flow in Dongying district, a core region of the alluvial delta at the estuary of the Yellow River. It is interesting to know the status of P accumulation and PUE in a long period (22 years) at such a typical alluvial delta. However, this study mainly depended on data collected from different sources, the author should provide all the available raw data used for the analyses. There are too many separated figures, some of them (e.g. Fig. 4,5, and 8) can be shown together in one figure. Fig. 4 and Table 3 were not well labelled and described (Please see General comments),
Experimental design: This research is about environmental ecology which is within the scope of the PeerJ, and the research question about “long-term P flow of different cropping systems at alluvial delta” is also important to assess the impact of P fertilization on environment. However, some of the data is from interviews, questionnaire, and published data, the author should give details about how to get the data.
Validity of the findings: It is a common sense that fertilizer and crop yield are key factors affecting PUE, the PLS-PM model doesn’t provide more new findings. Because the author focused on the PUE of different cropping systems, it is interesting to know the relationship between P fertilization rate and PUE in different cropping systems over the 22 years, from which some practical guidance might be made to local farmers. The author should also discuss the limits of this study due to data uncertainties.
Additional comments: Line 102  reference   Yuan et al. (2011) , please delete Line 106 (Yuan et al., 2011)
Line 108  reference  same problem as shown in Line 102
Line 117  What does the author mean ‘gain crops’?   Should it be ‘grain crops’?
Line 119   doesn’t make sense  “ Why the soil characteristics and crop-planting structure forced excess P fertilizer usage?”
Line 135 – 139  This information is necessary, it should be removed.
Line 141  planning? Did you mean ‘planting’?
Line 143   gain?  ‘grain’
Line 150  Please indicate which four cropping systems.
Line 151-152  The English language should be improved to make it more clear.
Line 167  What do you mean ‘the risk model of P load’? More details are required to explain.
Line 180  ‘those’ should be ‘these’
Line 190  More details should be provided about how you collected data.
Line 196  The data indicated by author is not listed in table 1
Line 211-212 The author should explain how the straw return-to-field ratios were calculated?  The ratios should be related to the area the author focused on, not from the reference.
Line 337-338  This is a well-known conclusion about how to increase PUE. The author should edit this part.
Line 377-378  How does the author get this conclusion? The application of fertilizer and crop yield should have positive linear correlation at certain range, but the PUE might decrease along with the increased fertilizer.
Line 389  ‘fertilization’ should be ‘fertilizer’?
Line 413 ‘planning’ should be ‘planting’?
Line 432  need reference
Line 420-437  PSMs are not always effective, especially in field. You should mention this point. Screening effective PSMs is necessary for specific location.
Line 457-459 The P input is related to cropland size, so total P application rate should be more meaningful regarding to PUE.

Fig. 4   Why only three input factors were shown in the figure, but 7 factors were shown in the legend.
Too many separated figures, some of them (e.g. Fig. 4,5, and 8) can be shown together in one figure
Table 3  Classes I, II, and III should be indicated respectively.


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: PHOSPHORUS FLOW ANALYSIS OF DIFFERENT CROPS IN DONGYING DISTRICT, SHANDONG PROVINCE, CHINA, 1995–2016
Review round: 1
Reviewer: 2

Output:
Basic reporting: The article requires major revisions before it can be published in PeerJ. The authors need to clearly indicate the statistical design that was adopted in the study. There is also no proof of model validation which is very important to determine the accuracy of the current results. There are several minor errors throughout the manuscript that need to be corrected as indicated in the additional comments section. The results section also needs to be revised to distinguish it from the discussion section.
Experimental design: The experimental design is not quite clear and the authors need to clarify which design was adopted in the study. The authors also referred to different crops e.g. cotton, corn, etc as cropping systems. This should be corrected to crops and not cropping systems. It is important that the authors clarify the cropping systems (organic or convention) each crop was grown under and this will help point the factors that contributed to the differences observed in different crops with regards to PUE.
Validity of the findings: The authors indicated that this study was the first to use the PLS-PM to quantify the contributions of P inputs and outputs to PUE in the agricultural system of Dongying District. However, there is no proof of model validation to check the accuracy and performance of the model basis on the past data for which there are already actuals. This will also determine the accuracy of the results of the current study.
Additional comments: There are several other minor but several corrections required throughout the manuscript as listed below.
Line 26: Define P for the first time.
Line 28: Cotton is a crop and not a cropping system.
Line 29: Define PUE for the first time.
Line 64: What is the PLS-PM model?
Line 74: Remove the word flow after P.
Line 84: Remove the word respectively appearing before (Jiang et al...)
Line 88-98: Write PUE in full when appearing at the beginning of the sentence and it should be P fertilizer and not fertilizer P.
Line 91: research and not researchers.
Line 102: Write SFA in full when appearing at the beginning of the sentence.
Line 108: Li et al. (year) and remove the citation at the end of the sentence.
Line 116: Yields for which crop?
Line 146: Please insert the word ''year'' before 2000.
The results section reads like the discussion section. Please discuss your results in the discussion section and not in the results section.
Line 225 and 305: Write P in full when appearing at the beginning of a subsection or sentence.


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: PHOSPHORUS FLOW ANALYSIS OF DIFFERENT CROPS IN DONGYING DISTRICT, SHANDONG PROVINCE, CHINA, 1995–2016
Review round: 1
Reviewer: 3

Output:
Basic reporting: no comment
Experimental design: no comment, does not apply
Validity of the findings: The term “P Use Efficiency (PUE)” is used in literature for different levels and different methods of calculation as a way to calculate how efficient the P use is in a system. There is some unclearness about the calculation of the PUE in the study.
What I understand from the paper in Line 87-88:
In the manuscript it is suggested that in the paper the PUE that is used is defined as increase in crop yield by the application of P. For me this suggests that the yield without P is subtracted from the yield with P and divided by the P fertilization. This is a legitimate way to calculate the PUE and is often indicated as Apparent P Recovery (APR).
This is, however, not calculated in this paper. In this paper the PUE is calculated by input/output (in which output includes P losses by erosion and runoff). This is also a legitimate way of calculating the PUE but this is not what is described.
In this FAO bulletin the different methods of PUE calculation are described:
https://soil5813.okstate.edu/Spring2012/Syers%202008.pdf

Quote: “There are a number of agronomic indices and methods for measuring the efficiency of plant-nutrient use in agriculture. In summary, the methods and indices, based on those of Cassman et al. (1998), are: direct method; difference method; partial factor productivity index; physiological efficiency index; and balance method.”
“.... a balance method is used here, i.e. total P in the crop divided by the P applied (UP/FP)”

This is closer to what is calculated in this study.
However, in this study it looks like the losses are also considered as the output that is used to calculate PUE, line 157-158. If that is so: In my opinion this is not correct to use in defining the PUE: an efficiency would be the ratio of USEFUL output/input. If losses are included in the output the PUE would go up if the losses are higher. This is in my opinion not what you want to express in an efficiency indicator.
If losses are not used for output in the calculations of PUE: please mention that in Materials and Methods.
Additional comments: Line 103 and 107: please add year after references.
Line 143 : gain should be grain (I assume)
Line 156 : straw return to field: it is not clear from figure 2 what is the origin of this flow. Is it an internal flow? Harvested from the system and brought back into the system? The it should be also taken up as output. In the text in line 157 it seems to be correct, there all harvested straw is mentioned as output. Please make text and figure 2 correspond.
Line 163: dynamic : in what sense is it dynamic: in time?
Line 204: 0.45: what unit?
Line 206/207 and 208/209 : rates: what units? %?
Line 235: decrease of soybean is caused by decreasing area or yield per area? I presume area, please mention that.
Line 239: unbalanced: in what aspect?
Line 164: planting area: would “cropping area” be a better term? (not sure)
Line 323 – 331 move to Materials&Methods
Line 352/353: soil PUE: please define, what is the difference with crop PUE (line 342)
Line 391: how does excessive use of chemical fertilizers decrease soil fertility?
Line 398: P absorption: should be P uptake (I think)
Line 401: root P fertilization: what do you mean: fertilizer placement close to the roots?
Line 411: is more organic fertilizer available? How about other nutrients in organic fertilizer? Should there be warned for excess of other nutrients and thus decrease the use efficiency of other nutrients?
Line 430: dry matter: add “yield”


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: PHOSPHORUS FLOW ANALYSIS OF DIFFERENT CROPS IN DONGYING DISTRICT, SHANDONG PROVINCE, CHINA, 1995–2016
Review round: 2
Reviewer: 1

Output:
Basic reporting: The authors have answered my questions and comments. I agree to publish this paper in Peerj, once the following minor issues are addressed.

91:  police makers,     policymakers
249: Of,  In
313:  P, Phosphorus (Abbreviation normally is not allowed at the beginning of a sentence)
348-349:  Reducing fertilizer application rate might decrease harvest. It would be a better way to say “optimizing fertilizer application rate to maximize PUE”
352: betweenYellow    between Yellow
406:  What do the authors mean “the same”?
414: Supplementary figure 1 needs figure caption.
Experimental design: NA
Validity of the findings: NA
Additional comments: NA


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: PHOSPHORUS FLOW ANALYSIS OF DIFFERENT CROPS IN DONGYING DISTRICT, SHANDONG PROVINCE, CHINA, 1995–2016
Review round: 2
Reviewer: 2

Output:
Basic reporting: no comment after revision
Experimental design: no comment after revision
Validity of the findings: no comment after revision
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: ANALYSIS OF FUNGAL DYNAMIC CHANGES IN THE NATURAL FERMENTATION BROTH OF ‘HONGYANG’ KIWIFRUIT
Review round: 1
Reviewer: 1

Output:
Basic reporting: English must be revised.
Experimental design: The authors use a medium-enrichment and high throughput sequencing method to research the fungal communities during the natural fermentation process. However, fungal communities in wine and medium are different  because of the inhibitory effects of acid and alcohol.
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: ANALYSIS OF FUNGAL DYNAMIC CHANGES IN THE NATURAL FERMENTATION BROTH OF ‘HONGYANG’ KIWIFRUIT
Review round: 1
Reviewer: 2

Output:
Basic reporting: This manuscript studied the fungal changes in the natural fermentation broth of “Hongyang” kiwifruit. Overall, the manuscript should be well organized and written should be improved.
Experimental design: The experimental design was good. Detailed methods should be provided
Validity of the findings: The findings in this manuscript were good. But the results should be clearly demonstrated and improved.
1）The abstract needs to be simplified
2）L37-L49, lots of duplicate content, needs to be clear and precise.
3)  Is it necessary to have so many citations? L51, L53, L55
4)  L61-67. Please reorganize the sentences.
5) 2.1 Materials. Please provide more detailed information about the kiwifruit used in this study. Such as weight, color, it’s better to have image in the paper. Also, please detail the method and group assignment in the text.
6) Why the wash buffer different? L78-79 vs L87
7) Please add P value throughout the text.
8) L172, what does “the others” mean?
9) There are a lot of description “the most dominant were others”, if others indicated one fungus or yeast, please specify, if others mean a cluster of fungi or yeast, then it cannot be considered as the most dominant. This is important and should be clarify in the text.
10) Please check all the citations, the format should be the same.
11) Some description in the discussion should be moved to the introduction section. And the discussion should be improved.
12) Why only chose “Hongyang” kiwifruit, do you have some control samples in your experiment. It’s good to choose some regular sample as control so that we know it worth it to study “Hongyang” kiwifruit.
Additional comments: Please concise 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: ANALYSIS OF FUNGAL DYNAMIC CHANGES IN THE NATURAL FERMENTATION BROTH OF ‘HONGYANG’ KIWIFRUIT
Review round: 1
Reviewer: 3

Output:
Basic reporting: Overall the English language used is not bad, but there are some instances where the language has a “google translate-y” feel to it, and needs improvement to get it to an international standard. I suggest you have a colleague who is proficient in English and familiar with the subject matter review your manuscript or contact a professional editing service.

Some examples (there are many more) where the language could be improved include:

Line 12 – has high sugar content -> change to: has a high sugar content
Line 15 – the high-throughput sequencing method -> a high-throughput
Line 17 – in four stages such as one… -> change to: at four time points, day one (D1T), day three (D3T), day five (D5t) and day fifteen (D15T).
Line 23 – metabolic pathway -> change to metabolic pathways
Line 59 to 60 - ..which can understand the change process of colony structure and metabolic pathway and monitor these indexes -> I’m unclear what you are trying to say here, please make it more clear.
Line 106 – space between 2.4.3 and Species
Line 156 – the singleton -> change to singletons
There are many more examples…

Line 49 – Please provide literature references for the few studies that have been done.

Line 50 to 57 – I personally feel these lines can be deleted. This now well-known knowledge and does not contribute to the science of the paper.

Line 64 to 67 – Its different than lines 61 to 63, but still feels like repetition, I suggest keeping only one of the two sentences.

Line 154 – Results:  Results are to long and there is a lot of non-scientific descriptions of the figures. You don’t have to go “this is high and this is low but its higher than that but lower than this” we can see that on the graphs, you should just be giving the core message of the graphs as the importance will be discussed in the discussion.

Line 295 – Kiwifruit wine special yeast is needed to explore – I’m unsure what this heading is supposed to say, please write it better
Experimental design: The experimental design, how it is reported, aims and scope of the paper is very poor and has to be reworked.

Line 74 – What precautions were taken to not contaminate the samples? Where the outside of the fruit sterilized? Was a sterile surface and knife used?

Line 75 – It’s a bit unclear what was done here. Was the fruit just placed as is in the Erlenmeyer? So was something added? What was then sampled? Some liquid (how much) or pieces of fruit (how much)?

Line 76 – You can remove the word “named” before the sample names

Line 77 – What does “three replicates were set” mean? Did you take replicate samples. How does this fit in with the 500g fruit chopped? Where there multiple Erlenmeyers? Please expand on the experimental setup. How many samples were prepared? It seems like there was just one Erlenmeyer and you took multiple samples from that, which would not be triplicates.

Line 70 – What was then obtained? A cell pellet? Was this stored?

Line 81 – This whole section needs the same attention as I explained with the previous section. Was it just 500g in one Erlenmeyer? What volyme of YPD is in the flask? If its not 500g in one flask, but 500g was chopped and split into various flasks, into how many and how much was placed in each flask?.

Line 86 – I still do not understand what “replicates were set” means.

Line 90 – Section 2.4.1: This section needs a lot of extra info. You can look at the methods of this paper for some guidance: https://doi.org/10.3389/fmicb.2020.01451. You need to include the sequencing platform (e.g. Illumina MiSeq) (I see Line 97 at least says Illumina, but include specific machine), references for the primers, length of sequencing (e.g. 2 x 300) etc. VERY IMPORTANT reads need to be publicly available, so you need to upload them to a database (e.g. SRA) and then provide an accession number.

Line 99 – Cutadapt is for cutting adapters and primers and stuff, so not really for clustering.

Line 102 – change Qiime2 to QIIME2

Line 108 – R is a programming language, GGraph, GGplot, etc, are packages written in R. They also have references that you should include.

Line 112 – Out dots should probably be OTU dots

Line 112 – Qiime2 should be QIIME2

Line 106 to 142 – I recommend consulting literature to write this better. These are standard methods by now and any recent microbiome paper will help you to write it better. Too much information is given and too much repetition is present.
Validity of the findings: Overall the paper is a bit all over the place and can benefit from being more focussed. The title states “Analysis of fungal dynamic changes in the natural fermentation broth of ‘Hongyang’ kiwi fruit ”. This is presented, kiwi fruit is naturally fermented and samples are taken over time and results show a shift in the diversity. BUT then there is this whole second leg to the paper about how high-throughput sequencing will help the commercial fruit wine sector and how this paper has helped to identify yeasts that can benefit the commercial sector and that part of the paper falls flat. This paper should either be diversity changes during natural fermentation, but the literature should then support natural fermentation information and why that is important and then you will have to do additional analysis of the broth during fermentation to determine why the diversity shifts OR this paper should go with the commercial angle, assess the natural diversity in an attempt to isolate something that can improve fermentation and then isolate it, make a fruit wine with and without your isolate and determine if it adds anything. As is, Pichia is well known and referenced and present in fruit fermentations, and this paper as is, just does not add anything new what is already known.

Line 156 – This is a weird way to report the data. Singletons are OTUs consisting of only one read. So you would QC your reads with certain cut-off metrics and report the amount of reads you worked with. Then you would get OTUs during the analysis and filter out singletons and the report the amount of OTUs you have (removing singletons is usually just mentioned in Materials and Methods).

Line 159 – Species composition: I’m not going to go into too much depth here, but your reporting this wrong. During taxonomic assignment QIIME will attempt to assign taxonomy as best it can to a taxonomic level depending on the database, the reads, etc. So some OTUs it will be able to annotate down to species and some only to genus, some only to phylum (it happens), so it did not “find” X amount of genera and Y amount of species. Secondly, if it could annotate down to species, it had to have annotated down to genus before that. Thirdly, its generally accepted that we do not go down to species level with Illumina reads since the reads are so short, but some people do it, I’m personally against it. That is why people would rather do, e.g., PacBio sequencing if they want to go down to species level. If the other reviewers are fine with it, leave it, otherwise you’ll have to change it.

Line 164 – You should use your alpha diversity metrics to show a decrease in diversity.
Additional comments: Overall this paper feels like an attempt to "just publish" the work. I think it can be reworked into something publishable, but as is, I do not feel it is of sufficient quality to justify publication. With a focused aim and scope and results, discussion and conclusion (as well as a small amount of extra lab work) this can be made publishable.


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: ANALYSIS OF FUNGAL DYNAMIC CHANGES IN THE NATURAL FERMENTATION BROTH OF ‘HONGYANG’ KIWIFRUIT
Review round: 2
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: The authors use a medium-enrichment and high throughput sequencing method to research the fungal communities of kiwifruit as ‘CK’. But I think that's wrong. Fungal communities in medium and kiwifruit are different.
Validity of the findings: The results lack novelty. Pichia is well known in fruit fermentations, and this paper does not add any new findings what has been reported. The research results have no guiding significance for the production of kiwifruit wine.
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: ANALYSIS OF FUNGAL DYNAMIC CHANGES IN THE NATURAL FERMENTATION BROTH OF ‘HONGYANG’ KIWIFRUIT
Review round: 2
Reviewer: 2

Output:
Basic reporting: Overall, the manuscript was improved after the revision. The authors clarified some comments. As for the figures in the manuscript, all the font size are different, please make sure the figures are in the same format.
Experimental design: good
Validity of the findings: good
Additional comments: No additional comments


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

Input:
Title: ANALYSIS OF FUNGAL DYNAMIC CHANGES IN THE NATURAL FERMENTATION BROTH OF ‘HONGYANG’ KIWIFRUIT
Review round: 2
Reviewer: 3

Output:
Basic reporting: Overall English and writing throughout the paper was improved. Literature was added where needed.
Experimental design: Research question much better defined. Streamlining of paper also highlights the core better.
Validity of the findings: Finding are (and were) interesting.
Additional comments: Paper was improved and most comments adequately covered.


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: UNCONSCIOUS PRIMING SHARES A COMMON RESOURCE POOL WITH THE MANIPULATION SUBSYSTEM
Review round: 1
Reviewer: 1

Output:
Basic reporting: The manuscript aims to distinguish the resource pools of the subordinate working memory systems, namely the maintenance and manipulation subsystems. The authors further employed a masked priming paradigm in a dual-task program to investigate the relationship between these subsystems and unconscious priming. They conclude that the manipulation subsystem interferes with unconscious priming and propose a two-pool attention resource model.
The manuscript provides a clear account of the motivation and reasoning of the experiments. The hypotheses formulated in the introduction are supported and derived from the literature that is provided. Overall, the manuscript addresses an interesting issue. However, substantial additional work is required to enhance readability and interpretation, especially concerning the literature review and descriptions of the design. Therefore, I conclude that the present manuscript is not ready for publication.

Overall, the English language should be improved (e.g. lines 63, 67, 80, 212f, 355-359). The current phrasing makes comprehension difficult, especially in sections concerned with the design and procedure of the experiments. I suggest that you improve the descriptions in lines 192-226. Statistical abbreviations should be italicized when appropriate. Moreover, the results section could be improved in terms of readability.

The authors provide an overview of the relevant literature, but the introduction requires more detail. Cognitive load theories, response priming, masking, and the employed tasks in general should be discussed in light of relevant publications (see for example Schmidt, Haberkamp & Schmidt, 2011; Klotz & Neumann, 1999; Klotz & Wolff,1995; Eimer & Schlaghecken, 1998; Sternberg, 1969, 1975). Extensive additions should be made regarding the concept of consciousness.

“Although previous findings are mixed, further analysis indicates that the load manipulation tasks utilized in those studies seem to involve different working-memory subsystems; thus, this could account for the paradoxical findings.” (line 81ff). Which tasks were utilized?

The structure of the article conforms to the required journal article conventions.
Raw data was not provided, but processed and averaged data, excluding subjects omitted from further analysis.
Overall, figures should be provided in higher resolution. Indication of significance in Figure 7 should be clearer (which effects are tested).

References:
Eimer, M., & Schlaghecken, F. (1998). Effects of masked stimuli on motor activation: Behavioral and electrophysiological evidence. Journal of Experimental Psychology: Human Perception and Performance, 24(6), 1737–1747. https://doi.org/10.1037//0096-1523.24.6.1737

Klotz, W., & Neumann, O. (1999). Motor activation without conscious discrimination in metacontrast masking. Journal of Experimental Psychology. Human Perception and Performance, 25(4), 976–992. https://doi.org/10.1037/0096-1523.25.4.976

Klotz, W., & Wolff, P. (1995). The effect of a masked stimulus on the response to the masking stimulus. Psychological Research, 58(2), 92–101. https://doi.org/10.1007/BF00571098

Sternberg, S. (1969). Memory-scanning: Mental processes revealed by reaction-time experiments. American scientist, 57(4), 421-457.

Sternberg, S. (1975). Memory Scanning: New Findings and Current Controversies. Quarterly Journal of Experimental Psychology, 27(1), 1–32. https://doi.org/10.1080/14640747508400459
Experimental design: The submission defines the research question and identifies a relevant research gap. Manipulation of the maintenance and manipulation subsystems required in the secondary task allows investigating their respective effects on the primary priming task. Through this, the subsystem involved in unconscious priming can be identified.
Overall, the experiment has been well-designed.
However, to ensure replication of the experiment or to make comprehension easier, some clarifications are needed:
1)	It is unclear to me how many trials of each condition were employed in exactly how many blocks across how many sessions. Because of this, it is difficult to assess whether enough data was acquired to make decisions on significance.
To reduce confusion, I suggest using the term ‘task’ only when referring to the two variations tested. Otherwise, you should follow the hierarchical order trial, block, session. Most importantly, avoid ambiguity when describing the procedure in lines 154-165.
2)	As mentioned in the previous section, extensive work is required in lines 192-226. It is especially difficult to follow the description of the high-load trials in the second paragraph. Specifically, you should be clearer when describing the change from memory probe to memory item. As I understand, the initial first memory item leaves the set and is replaced by the current memory probe. In the next trial, the previously second item is probed, and so on.
3)	The authors fail to thoroughly explain the employed N-back task. Typically, the N-back task requires a stepwise increase of N (first 1-back, followed by 2-back, then 3-back). Further, they never specifically state that they are using the 3-back as the high-load condition, and the 1-back as the low-load condition.
4)	It appears that the ethics approval of another experiment was submitted.
Validity of the findings: Data was only provided for the subjects that were analyzed. Excluded subjects are missing. Moreover, the provided data is already processed and averaged.
Overall, I commend the authors for linking their findings to the original research questions. However, in order to make a more compelling case, the following issues should be addressed:

1)	Exclusion criteria for subjects need to be justified and explained in more detail. For example, it is stated that subjects with accuracy significantly larger than chance were excluded. How exactly was this determined?
2)	It is unclear how the authors deal with the problem of multiple comparisons. Were corrections used? If so, which correction? Either way, the decision needs to be justified.
3)	The authors should be clearer in their definition of ‘unconscious priming’ in the reporting of their results. For example, it is not clear what they refer to exactly when writing “however, the distribution of unconscious priming under low load was not normal” (line 263f). For example, the priming effect can both be measured in differences in accuracy and reaction time.
Additional comments: The manuscript should also be checked for typos and spelling. Sometimes it seems that in the process of revising the manuscript multiple times, parts of sentences have been lost (e.g., lines 73-75).


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: UNCONSCIOUS PRIMING SHARES A COMMON RESOURCE POOL WITH THE MANIPULATION SUBSYSTEM
Review round: 1
Reviewer: 2

Output:
Basic reporting: 1. The majority of the manuscript is clear and easy to follow. There are some small inaccuracies in the English sometimes (e.g., prepositions, time), but it should be possible to resolve them easily. What was, however, very confusing to me was the usage of the word task/Task.
First, I would suggest that the authors pick more self-explanatory names than Task 1 and Task 2 (e.g. maintenance task and manipulation task or Sternberg task and n-back task would be more informative and one would not always need to keep in mind what 1 and 2 were referring to again). In the Abstract, it appears that the authors themselves confused the tasks, as they wrote that the working memory load did and did not decrease unconscious priming in Task 2 which is clearly contradictory (Results section of the Abstract).
Second, the word task was apparently used in several senses - the task to perform (Sternberg vs. n-back), a phase of the experiment? (practice task, main task, ...), as well as an equivalent of trial? (360 Sternberg tasks). This confusion is creating misunderstandings especially in the first paragraph of "Procedure". I highly recommend that the authors optimize their terminology in this paragraph and transfer the corresponding terms to the rest of the manuscript, so that the terminology is consistent and sufficiently distinct. In terms of consistent terminology, I also wanted to point out that the authors switch between retention attention resource pool and retention resource pool and between executive attention resource pool and executive resource pool. This should also be changed, so that the terms remain the same throughout the manuscript.

2. When referring to perspectives on automaticity in the 4th paragraph of the introduction, the authors should also cite at least one review article that describes different concepts and theories of automaticity (e.g., Moors & De Houwer, 2006).
Experimental design: The research question was developed and motivated very well and is very meaningful. However, there are some aspects of the methods description and the analysis that should be optimized before publication.

1. The authors should report a sample size estimation, so that the reader can evaluate whether their study was sufficiently powered to detect the effects of interest. As it appears that the authors did not compute a sample size a priori, they should at least elaborate on the power they were able to achieve for the comparisons of interest given the sample actually included in the analyses. This is especially relevant as the most essential effect the authors needed to find was a three-way interaction and a relatively large effect size is necessary to observe such a three-way interaction with sufficient power with a sample of only 22 participants. Therefore, more information on the authors' sample size considerations is essential.

2. Building on the previous aspect. A non-significant two-way interaction for Task 1 does not constitute strong evidence against an interaction especially given the small sample size. I think the authors' rationale and findings are very compelling, but they could improve their statistical foundation by additionally reporting corresponding Bayesian results, so that evidence against an interaction for Task 1 can also be assessed. For instance, the free JASP software provides easy opportunities for conducting such a parallel Bayesian analysis.

3. The high load n-back task did not become entirely clear to me. Based on the authors description, it seemed as if the first memory item was the only one relevant for later comparison. Then, however, the task would not have involved an increased load, in my opinion. Maybe I misunderstood something here, but in either case, the authors should explain the n-back task in a bit more detail to prevent misunderstandings.

4. For the sandwich masking task, it appears that the authors are reporting both ANOVAs per Task1/2 and an overall ANOVA with 3 factors. Only the 3-factor ANOVA should be reported as it includes all findings of the respective Tasks 1 and 2. Moreover, sometimes the authors only write that there was/ was no interaction. They should always refer to the respective interaction mentioning the involved factors to avoid misunderstandings. Moreover, as there are multiple analyses on the masked prime discrimination test, the working memory load induction, and the sandwich masking task, it would be easier to follow for the reader if the analyses per part were reported at the beginning of the result description of the respective part rather than in a - at present a bit hard to disentangle - joint paragraph at the beginning of the results section

5. The authors report that RTs that differed by more than 2 standard deviations from the mean were excluded. However, they did not specify whether the overall sample mean, the participant mean, or the individual cell mean was the reference. This information should be added. Furthermore, if the authors did not use the individual cell mean (i.e., the mean per participant and condition) as the reference, they should adapt their analyses to do so. Using the sample or participant mean distorts the data as fast/slow participants' data is more likely excluded or trials from a condition with faster/slower responses are more likely excluded. Thus, the individual cell means should be used as reference for computing the standard deviations.

6. In terms of terminology, the authors report that "responses were more correct". Responses can only be correct or erroneous. The phrasing should thus be changed to "fewer/more responses were correct".
Validity of the findings: The rationale of the study is clearly stated, timely, and interesting to the research community.

1. In their discussion, the authors state that "spare resources spill over involuntarily". I might have misinterpreted this sentence, but to me it seemed to contradict their description of their model elsewhere as well as its graphical illustration. I would suggest to change the phrasing of this sentence to avoid misunderstandings.

2. Furthermore, the atuhros write that "unconscious perceptual processing .... is considered an automatic process and is not affected by mental workload ...". This should be adapted to "... is considered an automatic process and therefore is assumed not to be affected by mental workload...". Otherwise, the factual statement at the end of the sentence would be contradicting other parts of the discussion.
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: UNCONSCIOUS PRIMING SHARES A COMMON RESOURCE POOL WITH THE MANIPULATION SUBSYSTEM
Review round: 2
Reviewer: 1

Output:
Basic reporting: The language has improved substantially. I especially commend the extensions with regard to the relevant literature.
If the following minor changes/corrections are implemented, I recommend the publication of this article.

I noticed a couple of minor issues still:

Line 93: AAAA are vowels
Line 95: it should be BOLD response
Line 100: COMPARED TO low loads
Line 138: a lower level of unconscious processES
Line 205-208: I think this sentence could be a little clearer with a small change:
and 6 blocks of either of the dual tasks (maintenance: nine sandwich masking trials were intermixed with nine modulated Sternberg trials per block; manipulation: nine sandwich masking trials were intermixed with nine N-back trials per block)
Lines 215ff: I think trial was more appropriate than block here
Experimental design: The description of the task is a lot clearer now.
Validity of the findings: Original review:
2) It is unclear how the authors deal with the problem of multiple comparisons. Were corrections used? If so, which correction? Either way, the decision needs to be justified.
Response:
2) Yes, Greenhouse–Geisser corrections were used. We added corresponding statements in the new manuscript “Importantly, Greenhouse–Geisser corrections were applied when sphericity assumptions were violated, although these corrections did not change any inferences” (Lines 539-541 in the Analysis section).

I commend the inclusion of Greenhouse-Geisser corrections. However, as the authors correctly state, Greenhouse-Geisser is a correction for the violation of the sphericity assumption.
My original concern regarded multiple comparisons. Given the large quantity of t-Tests, how did you deal with the multiple comparison issue? Similarly, which post-hoc corrections were used, e.g. line 378 (Bonferroni, LSD, etc.)?
Additional comments: Figure 1, Figure 2, and Figure 5 seem to be of low resolution still.

The ethics approval document (English translation) provided, appears to still be concerned with another experiment.


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: UNCONSCIOUS PRIMING SHARES A COMMON RESOURCE POOL WITH THE MANIPULATION SUBSYSTEM
Review round: 2
Reviewer: 2

Output:
Basic reporting: My prior concerns regarding basic report have effectively been addressed.

In the new version of the manuscript, the authors now refer to "the inhibition effect from the incongruent trials" in line 111. They did not introduce or explain this term and the reader can only guess what exactly they refer to. I therefore suggest that the authors adjust the phrasing of the corresponding sentence.
Experimental design: 1. I thank the authors for adding the requested Bayesian analyses which provide a clear-cut picture for the corresponding effects. Yet, now the authors offer more information on sandwich masking in lines 374-383. There, they also try to draw the conclusion that there is no difference between conditions. To do so, corresponding Bayesian anaylses would again be required. This also applies to other sections of the results where the authors state that there was no difference between conditions. Such a conclusion can only be drawn after a corresponding Bayesian analysis. Otherwise, it can only be stated that there was no significant difference, but not that there was no difference. Furthermore, there and elsewhere the authors now mention a 90% confidence interval. Why is a 90% confidence interval instead of the common 95% confidence interval reported?

2. However, I am not yet fully satisfied with the information on sample size estimation the authors provided. An ANOVA sample size estimation should always refer to the assumed (mean/singluar) effect size for the respective effect observed in one or multiple prior studies that the authors use as basis or postulated by the authors due to theoretical considerations. As the assume effect size is the essential basis of any sample size estimation, please add this information.

3. As requested by the editor, the authors added an analysis considering order. However, order was added as a covariate rather than a between-subject factor. Why was this done?
Validity of the findings: .
Additional comments: As Reviewer 1 already pointed out explicity in the first round of reviews, the authors did not make their raw data available so far. They only provided an aggregated version which contained the means per condition for only the participants included in the analyses. Furthermore, the authors did not react to R1's comment regarding raw data. In my opinion, the manuscript should not be accepted before the raw data is made available as required by PeerJ.


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: ASSOCIATION OF TOLL-LIKE RECEPTORS SINGLE NUCLEOTIDE POLYMORPHISMS WITH HBV AND HCV INFECTION: RESEARCH STATUS
Review round: 1
Reviewer: 1

Output:
Basic reporting: This is a review manuscript aiming to summarize data from TLR gene polymorphisms and HBV and HCV infection. In the Abstract, the authors start mentioning the high incidence of such infections in China, but the review apparently aims to cover studies made in diverse populations (although a clear bias to studies performed in China can be seen in the manuscript).

The Introduction brings general information about TLR and the involvement of these receptors in different pathways and diseases. In fact, sentences such as “The inflammatory signaling pathways mediated by TLRs are associated with a series of liver diseases, ...” are quite shallow since several pathways are involved in susceptibility or development of such diseases, therefore an actual link between these receptors and diseases should be made explicit.

Considering that this is a review, the methodology is correct, although a quick PubMed search revealed several studies not cited.
Only as an example, considering TLR2 and HBV infection, at least two other studies (not mentioned in Table 1) could be found:
Kim et al. Hepatogastroenterology. 2010 Nov-Dec;57(104):1351-5.
Chen et al. Vaccine. 2011 Jan 17;29(4):706-11. doi: 10.1016/j.vaccine.2010.11.023.

Extensive review of the English-language usage is needed.
Experimental design: Considering that this is a review, the methodology is correct, although a quick PubMed search revealed several studies not cited (see examples above).
Validity of the findings: The conclusion is somehow confusing and shallow. See these two contradictory statements in the same Conclusion paragraph: “More and more studies have shown that TLRs polymorphisms play an important role in HBV/HCV infection.” followed by “To date, few studies have explored the effect of TLRs single nucleotide polymorphisms on HBV/HCV susceptibility,...”

The discussion about “homogeneous individuals” should be revised, approaching “populations” instead of individuals, and highlighting also the need of inclusion of diverse populations in order to understand the potential involvement of TLRs in HBC/HCV infection.

Finally, there is some redundancy between the text and Tables. Therefore, I would suggest a reorganization of the manuscript, with data presented in the text and Tables just as a summary of data.
Additional comments: There are several incomplete sentences. For example, line 318 states “The main related SNPs in TLR7 gene summarized in Table 9.” Although Table 9 is not summarizing important TLR7 SNPs in general but solely those evaluated in the context of HCV infection.


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: ASSOCIATION OF TOLL-LIKE RECEPTORS SINGLE NUCLEOTIDE POLYMORPHISMS WITH HBV AND HCV INFECTION: RESEARCH STATUS
Review round: 1
Reviewer: 2

Output:
Basic reporting: - The expression “HBV/HCV infection” in the title and other parts of the text suggests that the authors will address the co-infection by HBC and HCV in the article, which is not the case. This expression should be replaced by “HBV and HCV infections” (or similar). I suggest using the expression “HBV/HCV infection” only to refer to co-infection.

- I understand that HBV and HCV infections are important problems in China. However, the article is not only focused on China, as the abstract suggests. This should be fixed to make the article more interesting to a wider readership.

- Genetic polymorphisms are just a few of the multiple determinants of susceptibility and clinical progression of viral infections. The effects of polymorphisms on these conditions must be interpreted in the context of environmental factors and other biological characteristics of the human host, in addition to viral factors. This information should be mentioned/better discussed in the introduction section. Some suggestions of references: Ellwanger et al. Infect Genet Evol. 2018; 66:376-391. doi: 10.1016/j.meegid.2017.08.011; and Ellwanger et al. Front Immunol. 2018; 9:1316. doi: 10.3389/fimmu.2018.01316
Experimental design: - This is a review article. The “Survey methodology” is satisfactory.
Validity of the findings: - The main body of the review is adequate. However, I suggest the authors point out that there is a discrepancy in the amount of information regarding Asian and non-Asian populations, with a greater amount of data for Asian populations. Further discussion of this discrepancy would make the review more relevant.

- The overall result of the review is very complete and interesting. However, the authors should also better explain to the reader why understanding the effects of genetic polymorphisms on infectious diseases is important. Some guidance: (I) it helps to develop better therapies based on the biological effect of genetic polymorphism (e.g., knowing the effect of CCR5delta32 was critical for the development of anti-HIV therapies; for this discussion/example, see: Ellwanger et al. Virus Res. 2020; 286:198040. doi: 10.1016/j.virusres.2020.198040); (II) it allows the identification of populations at higher risk for infection and that need more attention and prevention strategies; (III) genetic polymorphisms may help predict the clinical course of disease (for example, see: Lassner et al. J Transl Med. 2018; 16(1):249. doi: 10.1186/s12967-018-1610-8); among others. Creating a short topic covering these points can be interesting (place it before the conclusion section).
Additional comments: - The authors need to remind you that the name of genes must be written in italics. This should be reviewed and corrected throughout the article.

- The manuscript must be submitted to a professional English review. This point is critical for the improvement of the manuscript.

- Figure 1: authors must give credit for the platform used to create the figure (for this, use figure legend or acknowledgment section).


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: ASSOCIATION OF TOLL-LIKE RECEPTORS SINGLE NUCLEOTIDE POLYMORPHISMS WITH HBV AND HCV INFECTION: RESEARCH STATUS
Review round: 1
Reviewer: 3

Output:
Basic reporting: Manuscript ID: 67425
Name: “Association of Toll-like receptors single nucleotide polymorphism with HBV/HCV infection: research status”

In this manuscript (ms), Xu et al. reviewed the impact of genetic variants in the Toll-like receptor (TLR) genes in HBV and HCV infections. The review was divided into two major sections, HBV, and HCV. Overall, I found the ms difficult to follow at times as important information/background is missing or absent. Also, the lack of standardization and misuse of concepts are concerning. Therefore, the ms needs plenty of work to reach PeerJ readers, and my recommendation is to reject it. Please, see below my report.
Experimental design: 1.	The Introduction should be improved. Provide additional information regarding how differential expression of TLR may impact overall viral infections, disease progression, treatment, and downstream gene expression. In this context, there is no mention of downstream inflammatory response in Figure 1.
2.	L67-70. It should be clearly defined the aims of the review and the topics covered. Also, considering that SNPs are being reviewed it is fundamental to define how or which nomenclature will be used (e.g., position, amino acid change, according to the original study, reference sequence).
3.	Survey methodology. The search method needs to be more detailed. Inform the timeframe of your search, from the beginning (?) to the end (June 2021). How many studies were retrieved from in each database? Have the authors considered using MeSH terms?. If not, this might limit the range of search (PMID: 32912361). Please, include a flow diagram informing the total number of studies in each step (data gathering, exclusion, etc.). Lastly, what do the authors mean with “The role of SNPs is not described in detail” as an exclusion criterion? Although it is not a systematic review, one may benefit to follow the PRISMA guidelines (http://prisma-statement.org/).
4.	Section 3, 4, and 5. The lack of standardization (SNPs nomenclature) makes the overall comprehension of the review difficult. Also, when discussing the role of an SNP make sure to indicate the allele associated with the risk or protection. Also, there is no mention of how these SNPs contribute to the pathogenesis (regulatory SNP, coding region, etc.).
5.	Avoid new definitions. For example, chronic hepatitis B infection is traditionally defined as chronic HBV instead of CHB.
6.	Concept misuse. Mutation and polymorphism are not interchangeable. L126, what author mean by racial differences? I would strongly avoid this discussion and use ethnicity/population instead.
7.	Tables. Revise the concepts of correlation and association. It is not clear the meaning of “the genotype may be a protective genotype”. Was it associated or not? The sample size column is uninformative. Break it into cases and controls. I suggest including the minor allele frequency of SNPs observed in the control group. Lastly, standardize SNP nomenclature and define all acronyms used.
Validity of the findings: 8.	Conclusion. I would like to see a more detailed conclusion/discussion of potential TLR’ SNPs involved in HBV, HCV, or both. What is the impact of downstream signaling? Also, how functional studies may push forward the field?
Additional comments: Minor commentaries
1.	The ms would benefit from a language editing service.
2.	Make sure you list the corresponding author in the main text.
3.	L17, HBV, and HCV are major public health burdens worldwide. Unless you are reviewing the role of TLR in a specific population (i.e., China) it is wise to introduce it.
4.	Review in-text referencing of literature. For example, L39: replace “. [1-3]” with “[1-3].”.
5.	Define acronyms when first cited in the text. See, L54.


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: ASSOCIATION OF TOLL-LIKE RECEPTORS SINGLE NUCLEOTIDE POLYMORPHISMS WITH HBV AND HCV INFECTION: RESEARCH STATUS
Review round: 2
Reviewer: 1

Output:
Basic reporting: Although I acknowledge the efforts of the authors in order to answer all questions raised previously by this reviewer, and the fact that this is an actually better version of the manuscript, still some work should be done.

An extensive review of the English-language usage is needed. There are several typos (example line 218 “mete analysis”) and grammar errors (example: “The natural history of chronic hepatitis B and C are strongly influenced...”), but the most important is to review the manuscript to avoid several confusing statements.

For example, talking about TLRs, author’s claimed that “They recognize different molecular motifs, so called pattern recognition receptors (PRRs).” In fact “They recognize different molecular motifs, called PAMPS and DAMPS” .  PAMPs and DAMPs are the viral or bacterial, the external, molecules, although TLRs are PRRs expressed by the host (human) cells.  Please revise the text accordingly.

Another example: “For example, the TLR2 rs4696480 polymorphism is located in the promoter region, which may partially inhibit pro-inflammatory cytokines by reducing TLR2 transcription, suggesting that it may have the potential to reduce inflammatory damage in chronic HBV infection [42].” This sentence suggests that this specific SNP is capable to directly inhibit pro-inflammatory cytokines, although the reference cited is an association study. The authors are extrapolating the results from reference 42 . Please revise.

In the sentence “Furthermore, the polymorphism was significantly associated with the status of vaccine-induced serum anti-HBV response, and the TT genotype was a protective factor for anti-non-response.”, please define “anti-non-response”.  In the cited reference (number 47) one can read “…TT genotype of SNP rs3804100 (TLR2; Ser450Ser) was shown to be a protective factor against non-response”. Is that what the authors want to say here?
…
In conclusion, the present manuscript should be carefully revised both concerning English-language usage as well as concerning the interpretation of the results from the literature.
Experimental design: The review of the literature was well performed. Attention should be given to data interpretation.
Validity of the findings: As previously mentioned, authors should review the manuscript in order to avoid several confusing statements.
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: ASSOCIATION OF TOLL-LIKE RECEPTORS SINGLE NUCLEOTIDE POLYMORPHISMS WITH HBV AND HCV INFECTION: RESEARCH STATUS
Review round: 2
Reviewer: 2

Output:
Basic reporting: The authors responded sufficiently to my comments concerning the review's content.

I am still not fully satisfied with the English writing (some mistakes and typos are still present, see example below), but I would consider the manuscript with sufficient (not good) quality if the article is published in its current version.

Line 200 of PDF file: "mete analysis". Correct it in R2 or during proof (according to the Editorial decision).
Experimental design: The methods used for the selection of articles to be included in the review were significantly improved compared to the first version of the manuscript.
Validity of the findings: The new version of the manuscript presents the findings in a more organized way.
Additional comments: In general, the manuscript has been significantly improved compared to the first version presented by the authors. However, an additional grammatical revision would greatly improve the quality of the manuscript.


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

Input:
Title: ASSOCIATION OF TOLL-LIKE RECEPTORS SINGLE NUCLEOTIDE POLYMORPHISMS WITH HBV AND HCV INFECTION: RESEARCH STATUS
Review round: 3
Reviewer: 1

Output:
Basic reporting: This R2 is a truly improved version, although I would still suggest a few modifications.
First, the review of the English-language usage actually improved the manuscript, but a final revision by a fluent English speaker would eliminate some inadequacies. For example, at line 422, probably authors would like to say “According to in silico analysis” instead of “According to the silicon analysis”.
Also, authors present some opposing statements, and push too far some conclusions. For example, at line 522, they claim that “Clinicians may predict the clinical course of HCV-infected patients by TLR3 genotyping.”, which is an exaggerated conclusion based solely in association data, although at line 560, they acknowledge that “To date, the role of TLRs single nucleotide polymorphisms in HBV and HCV infection are still controversial.”
Experimental design: No comment.
Validity of the findings: No comment.
Additional comments: I suggest “Minor revision”, focusing in review of the English-language usage, without need to return the revised version to the reviewers, after these final corrections.


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: ASSOCIATION OF TOLL-LIKE RECEPTORS SINGLE NUCLEOTIDE POLYMORPHISMS WITH HBV AND HCV INFECTION: RESEARCH STATUS
Review round: 3
Reviewer: 2

Output:
Basic reporting: The manuscript underwent an English review, which resulted in some writing improvements. This version of the article shows sufficient quality for publication.
Experimental design: No additional comments beyond those already discussed in previous reviews.
Validity of the findings: No additional comments beyond those already discussed in previous reviews.
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: WATER STRESS AND NITROGEN SUPPLY AFFECT FLORAL TRAITS AND POLLINATION OF THE WHITE MUSTARD, SINAPIS ALBA (BRASSICACEAE)
Review round: 1
Reviewer: 1

Output:
Basic reporting: In my basic reports I, Helena Maura Torezan-Silingardi, want to congratulate the authors that chose to investigate a super relevant theme, which is up to date mainly considering the recent and gradual climate modifications we observe. The manuscript points direct and indirect consequences on plant species and their associated flower visitors and fruit production. The results obtained here to a cultivated species offer us consistent information. I think this manuscript soon will be an article cited by many other researchers, including myself. It was a pleasure to read this manuscript.

Now I will present my comments about each part of the manuscript. The language is unambiguous and professional, the literature is adequate. The abstract is concise and points the most relevant information, but the keywords are missing. The introduction is very strong and organized, with recent, important literature and clear and well-defined objectives. The experimental design is interesting, even so I suggest improving it adding some few information in the methodology, as I point in detail below. The results are described with high quality figures and tables, all of them very informative and clear and well explained in the text, so I consider the validity of the findings are very good and clear. The discussion is extensive and contain relevant comparisons with many other studies. The conclusions are concise and valid.

General comments: I indicate minor revision as the manuscript is relevant, well planned and executed.
Confidential notes to the editor: The manuscript is relevant, very good, and will be cited by many researchers. The modifications I ask are few and simple to be done.

Here I present my suggestions for each line numbered:
Line 32 – Authors wrote the “nitrogen availability played an important role in nectar production”, what is presented also in lines … and in Figure 2c. This information seems to be imprecise, as Figure 2c presents a higher nectar volume restricted to just one treatment and one season: low water/high N in Spring, but neither in any other treatment nor in Winter or Summer time. So I suggest the authors to make the text more precise, defining the exact treatment and the season which presented the distinct benefits of N supplementation.
Line 122 – If the main insects visiting the flowers are Apis mellifera, bumble bees and solitary bees in Europe, were the observations were made, I expected to have these same kind of visitors observed. But bumble bees were absent. Could you explain?
Lines 127-128 – Please, explain the reason you used a distinct number of plants (30, 60 and 45) in each of the seasons investigated. Are the distinct numbers related to diseases and aphids, as mentioned in line 150 ?
Lines 134-135 –I think all the seeds used in the same season received water in the germination trays in same day. Is this supposition correct? Could you detail a little bit more the germination process, please?
Lines 144-145 – I suppose the N quantities should be described in the same way, and not as “0,242 g N pot-1 (~100kg N ha-1)” and “0,121 g N pot-1 (~50 kg N pot-1)”. Please, check them.
Line 153 – I think the authors intended to write ‘floral traits’, but accidently they wrote “flora traits”.
Lines 155-156 – Could you explain your decision about counting the number of leaves only in the main shoot and not in the entire plant, please? Is it possible that all the distinct treatments had plants with the same number of shoots? The steam diameter was measured in just one shoot, I suppose the higher one, is it correct? This information could be detailed better.
Line 171-172 – Please, indicate the number of flowers used in each treatment.
Line 173 – Please, indicate how many days you had to wait from pollination to the completion of seed development.
Line 223 – Observing Figure 2A it seems wrong to say that the onset of flowering was “slightly delayed also by high nitrogen supply”. I suppose there is not a significative statistical difference on the flowering time.
Line 225 – The number of flowers produced in winter does not seems to be “slightly negatively by nitrogen supply” with high water.
Line 273-274 – Please, inform that in Spring the plants grown under higher nitrogen availability produced a lower number of seeds per flower, but the results were not the same in Summer.
Lines 294-296 – Nectar production was significatively increased in Spring with higher nitrogen and low water supply. But not in any other treatment.
Line 339 – Please, consider removing the ‘and’ from “plants grown in the summer 2018 revealed that and in both cases…”.
Lines 352-353 – There was not a different composition of flower visitor conditioned by water supply.

______________________________________________________________________
Experimental design: The experimental design is interesting and extensive, performed with rigourous care. Even so I suggest improving it adding some few information in the methodology to make possible to replicate it perfectly, as I pointed in detail in the basic reporting.
The research question is relevant, clear and meaningful. The results are properly presented with great figures and the discussion is extensive.
The manuscript do not fail to meet the PeerJ standards.
Validity of the findings: The findings are clearly presented and discussed, they are linked to each of the objectives proposed. The discussion presents many studies properly related to the present one. After some minor ajustements in the text the experiment can be repeated by any other reseacher in the word.
Additional comments: I suggested minor modifications in the manuscript to improve it's quality, They will be done easily by you. The manuscript is very interesting and will be readed by many of researchers, including in my laboratory.


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: WATER STRESS AND NITROGEN SUPPLY AFFECT FLORAL TRAITS AND POLLINATION OF THE WHITE MUSTARD, SINAPIS ALBA (BRASSICACEAE)
Review round: 1
Reviewer: 2

Output:
Basic reporting: English needs improvement
Some important references are missing (v.g. Majetic 2017 Flora, Rushman et al. 2019 Plant Cell Environ, Gomez et al. 2020 Nat Comm.)
Experimental design: It seems that experiments were performed in different years: spring 2017, winter 2017-2018 and summer 2018. This is unfortunate, since it means that authors may be confounding years with season. This is specially true for that part of the study performed outside the greenhouse. So, for example, it is hard to tell how much of the seasonal variation in visitor abundance and identity is due to season itself or, on the contrary, is due to yearly variation (what it is well known to occur in many other systems, including pollinator-generalized crucifers). I am afraid that this fact precludes any possibility of considering season a main factor in the experiment.
Overall, statistical analyses are fine. However, the experimental design suggests that a different staristical modelling should have done. So, season was not manipulated, and if I am right plants were all of them located in the same greenhouse. If this how the experiment was done (it is not clear from the description if there were on or several greenhouses), I think that the rightful way of analyzing the data is by means of a split-plot model (or a repeated-measure model, they are analytically similar), where greenhouse is the main plot and the two manipulated factors are nested into it. Consequently, no triple interaction is possible, and no conclusion about the effect of season can be reached.
Putting together these two concerns, authors should consider removing from their narrative any mention to season. The design of the experiment impedes any possibility of considering season as a factor at the same explanatory level as water and nitrogen.
Validity of the findings: There were different number of experimental plants in each season.mThis may also have consequences for how data should be analyzed.
Additional comments: Review of manuscript entitled ‘water stress and nitrogen supply affect floral traits and pollination in the white mustard, Sinapis alba (Brassicacea).

In this experimental work, authors checked the effect of altering water and nitrogen on some plant traits and the interaction with pollinators. Results were interesting, since they found that these two factors impacted flower visitation rate and affected seed production. Overall, the study is well conducted and analyzed, and the conclusions are interesting, highlighting the importance that the abiotic context has on the pollination process in many plants. I have however some concerns on both the methodology and the analysis.
Main concerns
It seems that experiments were performed in different years: spring 2017, winter 2017-2018 and summer 2018. This is unfortunate, since it means that authors may be confounding years with season. This is specially true for that part of the study performed outside the greenhouse. So, for example, it is hard to tell how much of the seasonal variation in visitor abundance and identity is due to season itself or, on the contrary, is due to yearly variation (what it is well known to occur in many other systems, including pollinator-generalized crucifers). I am afraid that this fact precludes any possibility of considering season a main factor in the experiment.

Overall, statistical analyses are fine. However, the experimental design suggests that a different staristical modelling should have done. So, season was not manipulated, and if I am right plants were all of them located in the same greenhouse. If this how the experiment was done (it is not clear from the description if there were on or several greenhouses), I think that the rightful way of analyzing the data is by means of a split-plot model (or a repeated-measure model, they are analytically similar), where greenhouse is the main plot and the two manipulated factors are nested into it. Consequently, no triple interaction is possible, and no conclusion about the effect of season can be reached.

Putting together these two concerns, authors should consider removing from their narrative any mention to season. The design of the experiment impedes any possibility of considering season as a factor at the same explanatory level as water and nitrogen.

Other concerns
Introduction can be shortened. For example, the sentences “For example, several studies on fruit and vegetable plant species showed that pollinator preferences for their flowers strongly depended on nectar volume or sugar concentration (Jabłoński et al., 1984; Schmidt et al., 2015; Roldán-Serrano and Guerra-Sanz, 2005). A study on Citrus plants also showed that honeybee visitation rate was positively correlated with nectar volume (Albrigo et al., 2012). These investigations indicated that both the quantity and quality of nectar influences flower preferences of pollinators” can be deleted.

Why were there different number of experimental plants in each season? This may also have consequences for how data should be analyzed.

Authors should consider mentioning some recent studies published on floral plasticity and its consequences on floral visitors, such as Majetic 2017 Flora, Rushman et al. 2019 Plant Cell Environ, Gomez et al. 2020 Nat Comm.


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: WATER STRESS AND NITROGEN SUPPLY AFFECT FLORAL TRAITS AND POLLINATION OF THE WHITE MUSTARD, SINAPIS ALBA (BRASSICACEAE)
Review round: 2
Reviewer: 1

Output:
Basic reporting: I want to thank the authors for their thorough review and for having added all our suggestions. The ms is much improved now. I have nothing to say on this point.
Experimental design: I think authors have done a good job in discussing their results in this new version. However, i still think authors have not analyzed the experiment using the most appropriate statistical model. I am sorry for previous misunderstanding. I did not mean using a repeated-measure model, as authors seemed to understand (if you go to my first review, you can see clearly that i included rm-model within parenthesis, just to indicate that they are analytically similar to other more appropriate statistical models). I still think that authors could do a better job using any type of model that assume some kind of constrain in the random assignment of experimental units, rather than the fully-factorial model they have used (with all the consequences this have for the interpretation of the results, that although two different issues they are tightly interlinked). I suggested split-plot analyses in my previous review, no rm analyses, but other models including nested variables could be appropriate too. Nevertheless, if authors consider 3-way GLM is appropriate to analyze their experiment, it is OK with me.
Validity of the findings: I think the findings are very interesting, and well discussed. I do not have anything else to add.
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: CONCENTRATION AND QUANTIFICATION OF TILAPIA TILAPINEVIRUS FROM WATER USING A SIMPLE IRON FLOCCULATION COUPLED WITH PROBE-BASED RT-QPCR
Review round: 1
Reviewer: 1

Output:
Basic reporting: The purpose of this study is to detect tilapia tilapinevirus (TiLV) in water collected from fish farming environments. Tilapia tilapinevirus is a novel RNA virus that affects tilapia production worldwide. Thus, the study is helpful in managing tilapia farms before and during the virus outbreak. The testing of TiLV in water collected from tilapia ponds or cages suggested that TiLV could be detected in the water.
Experimental design: - Although the objective of the study is to detect the virus in water, nonetheless, the main part of the manuscript focus on the development of a new probe-based real-time PCR method. Previously, numerous real-time PCR and RT-PCR protocols have been developed to detect this virus. Any justification why the authors decide to develop a new assay? If the reason is the matter of sensitivity of the previously developed assay, however, to come to that conclusion, different PCR methods should be tested with the same samples or laboratory.
- In this study, samples were collected from two fish farms. Additional samples taken from different farms/outbreaks will help confirm whether the viral load in the water reflects the amount of virus in the fish.
Validity of the findings: - The inconsistency of virus detection in sick fish, healthy fish, and water collected from the specific ponds raises the concern of the accuracy of the data. More testing of water samples (both from the outbreak and no outbreak situation) is likely to increase the reliability of the findings, which should support the conclusion that TiLV detection in water could be used as an indicator to forecast the outbreak status. In detail, the level of TiLV in disease fish ranges from undetectable to low and high detectable levels (Cq as low as 10). However, the viral loads in the water collected from the close/open cages were in the same range (31 to negative), no matter the viral load in the fish. One should expect that a high viral load should be detected in the pond water during the outbreak. In contrast, low detectable or absence of TiLV is present in water with apparently normal fish.
Additional comments: - Delete extra “,” in line 150


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: CONCENTRATION AND QUANTIFICATION OF TILAPIA TILAPINEVIRUS FROM WATER USING A SIMPLE IRON FLOCCULATION COUPLED WITH PROBE-BASED RT-QPCR
Review round: 1
Reviewer: 2

Output:
Basic reporting: The article is very useful for TiLV disease management and biosecurity. The article is very  clear writing in all parts with professional English used. The methodologies used were in a professional standard level. I have only few general comments to the authors as follow :

Line 113 ; What is actual number of sequences used for multiple allignment ? 25 or 27 or Both ?
Line 269-271 ; Please write it more clear for this sentence.
Experimental design: The research question was well defined and meaningful.
The investigation have been conducted with high technical standard.
The methodologies used were professional and standard.
Validity of the findings: The article is very useful, impact and novelty for TiLV disease management and biosecurity.
Additional comments: I have encourage the authors to further improve the viral concentration method or find new methods for concentration of virus in the future. It will be more impacted and very useful to fish farm biosecurity in the case that they have very low viral concentration in rearing water.


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: CONCENTRATION AND QUANTIFICATION OF TILAPIA TILAPINEVIRUS FROM WATER USING A SIMPLE IRON FLOCCULATION COUPLED WITH PROBE-BASED RT-QPCR
Review round: 1
Reviewer: 3

Output:
Basic reporting: Ln 3.1 TiLV is an RNA virus. The notion that it can be detected from eDNA is misleading
Ln 113 ’25 or 27’ the context needs more explanation as to how it was applied
Ln 118 use of respectively not clear
General note: need for sentence construction refining
Experimental design: Ln 134 authors need to describe in more detail the preparation of the standard curve and the plasmid that was used
Ln 137 this section should have been used as a validation step for the developed assay as concordance testing. Why did authors use a subheading that could mean their assay when not?
Ln 160 the authors have not mentioned how virus titration was done but still go ahead to give the viral titer (107 - 108) how exactly was this done?
Ln 164-165 the reason for subjecting to different treatments should be explained at this stage
Ln 186 the manufacturer of the kit used should be indicated
Ln 194 Cq of 38.24 is not very reliable!
Ln 170-183 the description seems not to be in line with the main study indicated in the title and the objective. Why are the fish samples being included? The objective should be adjusted to include this or this component should be removed. In addition, the relationship between the submitted pond/cage water and assay conducted is not clear. Was it used immediately after submission? Was the assay developed earlier and then applied to screen the water samples? Was it kept prior to analysis? What were the conditions? How long should a water sample be kept before analysis? What storage conditions should be used?
These questions should be answered by the authors to guide the prospective users of this screening technique
Ln 205 all the standard deviations used in this manuscript are so wide! Authors need to crosscheck their analysis.
Validity of the findings: no comment
Additional comments: General note: authors are mixing clinical disease investigation and with assay development. It’s advisable that they concentrate on clear description of assay development without mixing it with clinical investigation (diseases fish tissues). Ln 213-230 shows clear deviation from the title and objective set out by the authors.

Compliment: I have been struggling to raise TCID50 of 107 by cell culture in E11 cells. Its impressive how the authors could get 108. It would be nice to include the quantitation method that was used in these assays and how the procedure was conducted to guide readership.
Table 2: not clear, crowded and hard to understand
Fig 1 legend does not correspond with subtitles in the figure. Read ‘a’ in the figure and ‘a; in the legend. Communicating different information.
Fig2: as observed previously, the process leading to the generation of the standard curve should be described in detail.


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: DIFFERENTIATION PATTERNS OF EMPEROR MOTHS (LEPIDOPTERA: SATURNIIDAE: SATURNIINAE) OF A CONTINENTAL ISLAND: DIVERGENT EVOLUTIONARY HISTORY DRIVEN BY PLEISTOCENE GLACIATIONS
Review round: 1
Reviewer: 1

Output:
Basic reporting: The authors of "Differentiation patterns of emperor moths (Lepidoptera: Saturniidae) of a continental island: divergent evolutionary history driven by Pleistocene glaciations" present an interesting paper on the genetic distances between moths in Taiwan and their mainland sisters, with dated phylogenies to place the timing of splits in the Pleistocene. The molecular data consisted of three genes (of which one was mostly invariable), which is considered a small dataset in todays world of genomic sequencing options, but the findings are worth publication regardless, at the least as a basis for further research.

The manuscript at present needs a fair bit of 'polishing'. Quite strikingly, the english language needs to be checked for grammar and spelling - there are sentences that are incomprehensible. That said, I was able to perform the review to find the following issues that should be addressed.

It is worth pointing out in the introduction that Saturniidae, despite their large size, are poor flyers and will have to rely on land bridges to disperse, as opposed to some other large moths - e.g. sphingids. This is an important underpinning for the study.

Lines 187-8: Neighbor-joining is a 'quick and dirty' method for phylogenetic inference, not suited for phylogenetic publications because repeats can give different outcomes. Use a maximum likelihood approach instead.

For data retrieved from GenBank: please include how you ensured data integrity, how can you be sure of the accuracy of the identifications?

This study by Merckx et al. 2015 covers a quite similar topic and is perhaps worth comparing the author's data against:

Merckx VSFT, Hendriks KP, Beentjes KK, Mennes CB, Becking LE, Peijnenburg KTCA, Afendy A, Arumugam N, de Boer H, Biun A, Buang MM, Chen P-P, Chung AYC, Dow R, Feijen FAA, Feijen H, Soest CF, Geml J, Geurts R, Gravendeel B, Hovenkamp P, Imbun P, Ipor I, Janssens SB, Jocqué M, Kappes H, Khoo E, Koomen P, Lens F, Majapun RJ, Morgado LN, Neupane S, Nieser N, Pereira JT, Rahman H, Sabran S, Sawang A, Schwallier RM, Shim P-S, Smit H, Sol N, Spait M, Stech M, Stokvis F, Sugau JB, Suleiman M, Sumail S, Thomas DC, van Tol J, Tuh FYY, Yahya BE, Nais J, Repin R, Lakim M, Schilthuizen M (2015) Evolution of endemism on a young tropical mountain. Nature 524: 347–350. https://doi.org/10.1038/nature14949

Given the source and size of the data used for the study, the discussion should incorporate a section with recent papers on the dangers of using small (mitochondrial) datasets and molecular dating. See e.g:

Ebdon S, Laetsch DR, Dapporto L, Hayward A, Ritchie MG, Dinca V, Vila R & Lohse K (2021) The Pleistocene species pump past its prime: evidence from European butterfly sister species. Molecular Ecology (published online ahead of print). DOI: 10.1111/mec.15981

I support that the authors did not use their data for any formal nomenclatural changes.

With the aforementioned issues addressed, I recommend this manuscript for publication.
Experimental design: no comment
Validity of the findings: no comment
Additional comments: no comment