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: PRODUCTION AND CHARACTERISTICS OF FISH PROTEIN HYDROLYSATE FROM PARROTFISH (CHLORURUS SORDIDUS) HEAD
Review round: 1
Reviewer: 2

Output:
Basic reporting: The work entitled “Production and characteristics of fish protein hydrolysate from parrotfish (Chlorurus sordidus) head” proposes to determine the characteristics of protein hydrolysates extracted at different pHs and hydrolysis duration periods. The information is not original, though if the species has important values of wastes, would be important to study, however, the authors don't discuss this topic.
Experimental design: The experimental design is incorrect because the authors don't include a control treatment, as "initial time" that corresponds to raw material without hydrolysis. It is important to have a control treatment to compare the results; sometimes, the raw material has high antioxidant activity for example and peptides of different molecular weights. In this work, there is no evidence of control treatment for then some results can’t be discussed.
Validity of the findings: Some results are too confusing. Figure 4, for example, has not well defined and is very incomplete. I think that is essential to show the electrophoresis for different hydrolysates and the raw protein without hydrolysis to compare, as control. Furthermore, I don't sure that proteins with molecular weight from 18.05 kDa to 75.89 kDa were the products after hydrolysis of 24 hs.
Additional comments: The manuscript is inconsistent. The principal error is the lack of initial control in the experimental design. Furthermore, the discussion section needs some argue about the importance of to use wastes of fisheries.


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: PRODUCTION AND CHARACTERISTICS OF FISH PROTEIN HYDROLYSATE FROM PARROTFISH (CHLORURUS SORDIDUS) HEAD
Review round: 1
Reviewer: 3

Output:
Basic reporting: 1- Topic of this research is important, since many different groups around the world are working on it, seeking how to use fish by-products, waste and efluents from the fishing industry as sources of bioactive compunds with potential value added. However, the writting of the manuscript is confuse and difficult to follow.
2- List of references is ok as the number and year of publicación. However, introduction is poor because discusión about previous research is short, lite and unsufficient.
3-The main goal and hypothesis of research were not mentioned.
Experimental design: 1- Material and methods are poorly described and needs better work, especielly in the statistical part.  Experimental statistical design do not mention análisis of variance for Figure 2 and Table 2, however this statistical tool do not apply, because the experiment design only has one repetition (n=1). Analyses were carried out in triplicate, but n=1. Overall, Figures and Tables are poorly described.
2- Nothing was said about endogenous enzymes and how experimental pHs and time of hydrolysis were chosen.
3- Información about initial conditions of fish heads were not mentioned.
Validity of the findings: 1- Difficult to have conclusive results, since the experiment has only one sample (There were no replicates. Besides, each time an hydrolisis is carried out, results could be different.
2- The Authors only describe results, and did not make any in deep análisis. For instance, how many polypeptides are in each hydrolizate? . Aminoacid profile is not useful. Primary estructure could be better. Conclusions are poor, if any.
Additional comments: Manuscript must be rewritten, with in deep análisis of data, and justify why only one sample was taken. Material and methods must be better described, mainly the statistical part.


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: EXPRESSION AND PROGNOSTIC ANALYSES OF ITGA11, ITGB4 AND ITGB8 IN HUMAN NON-SMALL CELL LUNG CANCER
Review round: 1
Reviewer: 1

Output:
Basic reporting: 1. This manuscript discusses the potential ability of ITGA11, ITGB8 and ITGB4 to act as diagnostic and prognostic biomarker for patients with NSCLC (LUAD + LUSC) and LUSC by using several online web sever including ONCOMINE, GEPIA, CCLE, EMBL-EBI, and STRING. This topic has great importance due to the clinical-oriented urgent need for discovering new biomarkers (either prognostic or predictive) in NSCLC patients. However, this study has several limitations. In summary, this paper needs major revision before it can be accepted.
2. Many sentences with grammatical errors are dispersed throughout the manuscript. It may benefit from an extensive editing by a native English speaking editor or a co-author.
Experimental design: The authors should download TCGA and GEO original data to analyze the expression, clinical significance of ITGA11, ITGB8 and ITGB4 in NSCLC, if not, the results are not solid.
Validity of the findings: 1.Integrins family has many members, why authors just concentrate on studying these three members? Author should discuss the reasons, and also need to add the research progress of known prognostic biomarkers of NSCLC in the introduction “Ann Surg Oncol. 2017 Dec;24(13):4017-4024; Mol Biol Rep. 2012 Jan;39(1):335-41; Sci Rep. 2019 Aug 23;9(1):12329; Oncol Lett. 2019 Aug;18(2):1723-1732; Genes (Basel). 2019 Jan 10;10(1) pii: E36. doi: 10.3390/genes10010036”
2.For the OS analysis, multivariate cox regression analysis should be performed based on TCGA data, rather than using GEPIA only. Moreover, it is better to further validate the prognostic value of ITGB4 in tissue microarray with patients’ survival information by immunohistochemistry (IHC).
Additional comments: 1.All images with low resolution are roughly made by online tools, and the quality of these figures should be improved.


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

Input:
Title: EXPRESSION AND PROGNOSTIC ANALYSES OF ITGA11, ITGB4 AND ITGB8 IN HUMAN NON-SMALL CELL LUNG CANCER
Review round: 1
Reviewer: 2

Output:
Basic reporting: 1. There are some errors in grammar. Check whether the singular and plural forms of nouns are used correctly. For example, in the conclusion section of the abstract, “biomarker” should be written as “biomarkers”. “was” should be written as “were” in line 68.
2. The horizontal axis of figure 3A-3C is not clear.
3. The format of genes should be in italics throughout the manuscript.
Experimental design: 1. Using bioinformatics analysis, the authors analyzed the expression of "integrin family ITGA11, ITGB4 and ITGB8" in human non-small cell lung cancer and its role in the prognosis of non-small cell lung cancer. Integrins are transmembrane proteins that mediate cell-cell and cell-extracellular matrix interactions. The authors should provide basic validation of ITGA11, ITGB4 and ITGB8 at the protein level. For example, is the ITGB4 protein correlated with its genetic level in NSCLC?
Validity of the findings: 1. The author discussed the functions of ITGA1, ITGA4 and ITGA8 respectively. Is there any relationship among them?
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: EXPRESSION AND PROGNOSTIC ANALYSES OF ITGA11, ITGB4 AND ITGB8 IN HUMAN NON-SMALL CELL LUNG CANCER
Review round: 1
Reviewer: 3

Output:
Basic reporting: In this manuscript, the authors investigated the expression levels of ITGA11, ITGB4, and ITGB8 in public cancer databases and found their applications as diagnostic or prognostic biomarkers. The results are clear and reproducible, therefore their findings seem to be useful for cancer research professionals. There are several points that need to be clarified:

1.	The methods are basically a series of database searches. And there are no in-house experimental validations. Thus, the originality of their approaches may be weak.
Experimental design: 2.	Why were these three integrins selected? There are 18 subunits of integrins as the authors mentioned. And omics datasets make it possible to ideally investigate all genes.
Validity of the findings: 3.	Figure 2B: ITGB4 showed lower expression distribution on stage IV. ITGB4 seems good diagnostic and prognostic biomarkers for NSCLC (Figure 2A) and LUAD (Figure 4A), respectively. So, it seems contradictory that the higher tumor stage group has a lower expression profile of ITGB4.
4.	The font size of Figure 5A is too small. It is difficult to understand which genes are co-expressed with three integrins.
5.	Is there any reason to change datasets for ITGA11 (Hou et al. 2010), ITGB4 (Build et al. 2006), ITGB8 (Yamagata et al. 2003)? (l.156-l.164, Figure 5A) If there are three datasets, I would like to know concordance or discrepancy of co-expressed genes among them.
Additional comments: 6.	Although several omics platforms are used to investigate cancer genome, traditional immunohistochemistry (IHC) approaches are still routinely used for cancer diagnosis. Therefore, it is recommended to check protein expression levels of ITGA11, ITGB4, ITGB8 as diagnostic and prognostic biomarkers. (optional 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: EXPRESSION AND PROGNOSTIC ANALYSES OF ITGA11, ITGB4 AND ITGB8 IN HUMAN NON-SMALL CELL LUNG CANCER
Review round: 2
Reviewer: 1

Output:
Basic reporting: 1. line 133, spaces before and after "<".
2. There are still some grammar mistakes, e.g.,line 145 and line 150, "are" should be "were".
3. In Figure 3, use the arrows to indicate where ITGA11, ITGB4, and ITGB8 are increased.
4. In line 174, delete "." after the word "results". Please check the punctuation in the manuscript.
5. Please explain what group the dotted line in Figure 5 represents
Experimental design: 1. The author analyzed the mRNA expression level of  ITGA11 and ITGB8 using GEO database. Has the author done q-PCR experiments to explore the mRNA expression of them in vitro?
Validity of the findings: no comments
Additional comments: no 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: EXPRESSION AND PROGNOSTIC ANALYSES OF ITGA11, ITGB4 AND ITGB8 IN HUMAN NON-SMALL CELL LUNG CANCER
Review round: 2
Reviewer: 2

Output:
Basic reporting: The authors have significantly improved the manuscript and have addressed all my comments. I have no additional comments.
Experimental design: no comment
Validity of the findings: no comment
Additional comments: no comment


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

Input:
Title: EXPRESSION AND PROGNOSTIC ANALYSES OF ITGA11, ITGB4 AND ITGB8 IN HUMAN NON-SMALL CELL LUNG CANCER
Review round: 3
Reviewer: 1

Output:
Basic reporting: no comments
Experimental design: no comments
Validity of the findings: no comments
Additional comments: The manuscript could be accepted.


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: EFFECT OF INDUCED HYPEROPIA ON FALL RISK AND FOURIER TRANSFORMATION OF POSTURAL SWAY
Review round: 1
Reviewer: 1

Output:
Basic reporting: The manuscript was written clearly. The results of abstract didn't clearly mention the results (difference and p-values etc.)
Experimental design: I am not expert on Hyperopia, but whether given spherical lens to the same person with 10-minute break will affect the results? Or will need reference on this.
Validity of the findings: The statistical methods used in the manuscript were correct and interpreted correctly. I would suggest adding p-values after multiple testing correction to make sure the significant findings still exist after multiple tests correction.
Additional comments: The discussion parts were clearly stated.


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: EFFECT OF INDUCED HYPEROPIA ON FALL RISK AND FOURIER TRANSFORMATION OF POSTURAL SWAY
Review round: 1
Reviewer: 2

Output:
Basic reporting: Reviewer read the manuscript “Effect of induced hyperopia on fall risk and Fourier transformation of postural sway” by Sang-Yeob Kim and co-workers with interest. This is a study the effect of refractive errors on their fall risk. The results indicate that fall risk index is more sensitively affected by uncorrected hyperopia than uncorrected myopia.
The topic is quite novel and results are very exciting findings.
Experimental design: Abstract
- If significant, p-values need to be provided.

Introduction
- It will be help to strengthen the purpose of this paper, if discuss how increase fall risk linked to poorer quality of life.

Subject and methods
- It would be better if you added the issue of the reliability of the measuring device.
- Line 134-135. What was paired t-test for, and what was repeated AVONA for? Additional explanation is needed.

Results
- Figures: what do the error bars mean? Additional explanation is needed in foot notes below the figures.

Discussion
- Line 228-230: In explaining the limitations of this study, you mentioned the problem caused by the difference between the induced hyperopia and the actual hyperopia… Do you think that the same results can be achieved between the induced hyperopia and the actual hyperopia?

- Line 195. 200, 215. ‘low-to-medium’ it is need to correct the letter style as ‘Time New Roman’
- Line 196. 199. ‘Peripheral’ it is need to correct the letter style as ‘Time New Roman’

References
- References do not have serial numbers, but there are reference numbers in the text.
Validity of the findings: The experiments are performed on satisfactory number of subjects and the manuscript is written in a good style. Reviewer still has some recommendations which would improve the quality of the work.
Additional comments: - This study is about the fall index in induced myopia or induced hyperopia. However, I think the fall risk index may be increased by the prism effect of the added lens, or the narrowing of the visual field on the trial frame. Do you consider this issues?
- This study is saying that the fall risk index rises because of the accommodative action in uncorrected hyperopia. However, people of higher age have little accommodative amplitude, so they cannot use accommodation even with uncorrected hyperopia. Doesn't the fall risk index rise for older people because of uncorrected hyperopia?


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: EFFECT OF INDUCED HYPEROPIA ON FALL RISK AND FOURIER TRANSFORMATION OF POSTURAL SWAY
Review round: 1
Reviewer: 3

Output:
Basic reporting: Your introduction needs more detail. I suggest that you improve the description at lines 64- 65 to provide more research results about proportion of hyperopia in Korea and why it was rarely studied before compared to myopia.
Experimental design: In Materials & Methods section, subjects are in 20’s and they averagely have myopic and astigmatic refractive error. The participants were accustomed to myopic condition, so it must be needed some time to adjust hyperopic condition, because it is not common that someone gets hyperopic changes all of sudden. Please explain did you have some time to adapt themselves to new circumstances.
Validity of the findings: Induced hyperopia has other problems such as limited vision and magnifying images. The authors should have mentioned in discussion section.
Additional comments: The authors have done some statistical testing which is good, however the results could be presented more clearly. The statistical testing results are presented in figure 5, however normally when reporting on ANOVA tests the degrees of freedom and F value should be reported as well. See http://www.yorku.ca/mack/RN-HowToReportAnFStatistic.html for an example of how to report ANOVA values. The authors should also report on the numerical scores resulting from post-hoc analysis and what kind of post-hoc analysis did you use.


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: CROWDSOURCING VISUAL PERCEPTION EXPERIMENTS: A CASE OF CONTRAST THRESHOLD
Review round: 1
Reviewer: 1

Output:
Basic reporting: 2.11: Basically - Do not state your opinion
2.13: There are articles discussing the issue of payment. Please refer to published resources.
2.14: 1000 - Based on your abstract, you had a smaller N. Why?
3.20: Impressive list. You may also mention studies that look into auditory stimuli, which relates to your scope. Also, there are more examples of studies made with jsPsych.
4.18: Add subheaders. Here: aim of the study
5.15: No questions on demographics, environment during the experiment (light conditions, input devices) were asked? Why not?
5.21: 138 - Nice. But surprising too, as crowdsourcing allows for much larger N, which is one of the biggest strengths.
6.6: 3 hours - How much time the experiments presented here took? Any control for the randomisation of the order of the experiments within 3h?
7.2: Add an illustration of the stimuli. Describe the timeline of the lab/online experiment
7.17: The method of constant stimuli was used. - More details?
9.10: Rather specific information in this section. Not sure if this is very useful. I'd rather focus on results more.
9.23: Rather small section. I would like to see an analysis from more angles. For example learning effects, a timeline of the experiment with means of results. Correlations?
  Please add anonymised results, code used to produce results, and if possible code used to create the study in the supplementary material.
  Average time spent on experiment online and in a lab? Are they similar? If not, why not?
10.1: only 80 participated - Why?
10.4: only 156 participated - Why?
10.7: rules detailed - Hard to follow. Please be more specific about who was filtered based on what
10.7: State dates of data collection for the online experiment
12.9: Could you take more measures for more selective recruitment? What about the design of the system in jsPsych? Without more details on the methods, it is hard to judge how universal your results are.
12.18: online non-repetition condition. Although it is difficult to interpret this result, the results of the online repetition condition may inform our understanding. - Not clear
13.11: At this time, these methods seem to require much effort from the participants and thus might not be effective. - Why exactly you did not
13.17: experiments - How do you know that? You did not ask any questions before or after the experiment?
13.18: situations - Not clear. In crowdsourcing experiments in general?
13.19: Satisficing - What?
14.5: another method - You can also filter bases on the time spent to complete.
15.4: Why was this study performed in the end? Are you planning to conduct more research using crowdsourcing? Expand future work.
16.1: Not clear why this needs to go into a footnote
Experimental design: More details on the timeframes of the experiments and illustrations of stimuli needed.
Validity of the findings: Results need to be supplemented by supplementary material. The results section is rather small.
Additional comments: Well-written text with good English. Please look into strengthening the methods and results sections.


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: CROWDSOURCING VISUAL PERCEPTION EXPERIMENTS: A CASE OF CONTRAST THRESHOLD
Review round: 1
Reviewer: 2

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: The contrast detection threshold as a function of the spatial frequency of the stimulus—the contrast sensitivity function (CSF)—is a fundamental measure of visual function. The study tries to assess it in the laboratory and online using crowdsourcing.

To measure CSF in humans, it is known that the intensity levels that can display the typical hardware used in behavioral sciences (256 intensity levels, 8 bits) are not enough (Allard and Faubert 2008; Lu and Dosher 2013). This problem is solved using hardware (Lu and Dosher 2013) or software solutions such as dithering (Allard and Faubert 2008).

It seems that the study did not use (in the laboratory and online) any solution to increase the range of possible intensity levels, which I think indicates that the CSF was not properly assessed. This might explain why the contrast sensitivity found in the study is much lower than the contrast sensitivity found in previous studies (the manuscript lacks literature about contrast sensitivity in humans) and why some participants had 0% contrast sensitivity.

Also, the motivation of the study is not clear to me. The CSF has a characteristic bandpass tuning shape with a peak at 2-6 c/deg. The shape of the CSF could only be the same in the laboratory and online as long as the viewing distance is the same in both situations so the size of the stimuli is matched in the retina.

Allard, Rémy, and Jocelyn Faubert. 2008. “The Noisy-Bit Method for Digital Displays: Converting a 256 Luminance Resolution into a Continuous Resolution.” Behavior Research Methods 40 (3): 735–43.
Lu, Zhong-Lin, and Barbara Dosher. 2013. Visual Psychophysics: From Laboratory to Theory. MIT Press.


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: CROWDSOURCING VISUAL PERCEPTION EXPERIMENTS: A CASE OF CONTRAST THRESHOLD
Review round: 1
Reviewer: 3

Output:
Basic reporting: This manuscript reported the empirical study that investigated the viability of online experiments with crowdsourcing population in the domain of human sensory perception. Compared to studies of human higher cognition, it remains unclear whether the online experiment is also a powerful tool for studies in of low-level sensory perception, since it requires more precise control of physical environment.
The authors conducted an experiment that compared visual contrast threshold between participants from conventional student sample and online crowdsourcing sample. Results clearly indicated that data from the conventional laboratory experiment and the online experiment with the same number of iterations are equivalent, however data from online without repetition indicated significantly greater contrast threshold than other conditions. Results also exhibited that the data from online experiment may suffer from a high level of data exclusion partly due to lack of experimental control in the presentation of visual stimulus. From these results, the authors (seemed to) conclude that the online crowdsourcing experiment is useful tool for collecting data even in the visual perceptual study, and they also pointed out practical tips on conducting crowdsourced studies in this field.
The empirical study reported here is well-designed and results from the experiment seems to be beneficial for researchers studying human perceptual process. Unfortunately however, the manuscript as it is, failed to make an important contribution in the literature for two reasons. First of all, it seems to me that the central claim of the manuscript failed to show its distinctness. Second, procedures of the experiment (mainly about sampling and data exclusion) and displays of results cannot be understood easily, therefore should be revised accordingly.
Experimental design: Research question is well defined and the aims of the study is original. And the experiment reported here is well-designed and meets ethical standard. However, I would suggest the authors describe the criteria of data exclusion and the number of participants (or trials) excluded due to these criteria in detail.
Validity of the findings: The empirical findings from the present study is straightforward and seems to be statistically sound, although it seems that there’s room for improvement in the description of results for better understanding.
Unfortunately however, conclusion of the manuscript seems not to be explained clearly. I understood that the primary aims of present study is to exhibit the usefulness of crowdsourced online experiments for studies of low-level sensory perception. However, throughout the manuscript, the authors discussed about many cons of crowdsourcing usage. On the other hand, they did not discuss much about the pros of crowdsourcing. In fact, the results may suggest that the crowdsourced experiments on the low-level visual perception cannot be recommended due to its high exclusion rate. However, in the discussion section, the authors tried to conclude that crowdsourcing is a profitable tool for perception studies even if researchers should take into consideration of its high rate of exclusion. If the authors intended to argue benefits, they might want to describe pros of crowdsourcing more in detail throughout the entire manuscript. On the other hand, if they intended to give a warning to rash decisions to use crowdsourcing in low-level perceptual studies, the discussion section should be rewritten accordingly.
Additional comments: Please refer the attached document to see the detailed comments on the above 3 areas and other minor suggestions.


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: CROWDSOURCING VISUAL PERCEPTION EXPERIMENTS: A CASE OF CONTRAST THRESHOLD
Review round: 2
Reviewer: 1

Output:
Basic reporting: The replies of the authors have satisfied my concerns and answered my questions. I think the method of the presented study could be stronger, yet the received results are still worth sharing with the academic world. I support the publication of this article.
Experimental design: Thank you for improving this section and adding the timeline figure.
Validity of the findings: Thank you for improving the results section.
Additional comments: Thank you for considering my 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: CROWDSOURCING VISUAL PERCEPTION EXPERIMENTS: A CASE OF CONTRAST THRESHOLD
Review round: 2
Reviewer: 2

Output:
Basic reporting: see General comments for the author
Experimental design: see General comments for the author
Validity of the findings: see General comments for the author
Additional comments: My main concern has not been addressed. As I described, the measurement instrument used in the experiments does not have enough sensitivity to measure CSF in humans, which is the main aim of the study.


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

Input:
Title: CROWDSOURCING VISUAL PERCEPTION EXPERIMENTS: A CASE OF CONTRAST THRESHOLD
Review round: 2
Reviewer: 3

Output:
Basic reporting: The revised manuscript has been improved significantly from previous ones. I appreciate authors' extensive works in revising the manuscript.
Experimental design: Research question is well defined and the present study has successfully contributed to the current domain. In the revised manuscript, the criteria of data exclusion and the number of excluded participants has been described in detail. I found that the revision could enhance better understanding of the method.
Validity of the findings: In the revision, the results has been well displayed, and hence improved significantly in its understandability. The conclusions are well stated and compatible with present results. But, I also believe that it will enhance further contribution of this paper if authors discuss about any possible measures to resolve discrepancy issue, in other words, the method to prevent free-riders. For example, it might be possible to generate unique (different, I mean) random completion code to each participant. In this case, researchers should decide who is paid. I recognize that it might cause researchers additional work, but it also seems that it's worth the cost if we consider possible (and quite large number of) free-riders. Furthermore, readers might want to know whether other crowdsourcing providers suffer from similar high rate of free-riding. Any discussion (and suggestion) on this issue might be beneficial to other researchers.
Additional comments: As I mentioned previously, the revision significantly improves the quality of the manuscript. I admire the authors' extensive work on this revision. I thought that my comment on the possible measures to prevent free-riding will be beneficial to research communities, however it is not a requisite condition of acceptance since those measures and their effectiveness is speculative and might be dependent of environment of service provider.


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: CROWDSOURCING VISUAL PERCEPTION EXPERIMENTS: A CASE OF CONTRAST THRESHOLD
Review round: 3
Reviewer: 1

Output:
Basic reporting: The authors' response have satisfied my concern regarding free-riders issue. I think that the impossibility of preventing free-riders may restrict usefulness of the current system as a recruitment tool, so I am looking forward to the authors' further contributions on this issue. I support the publication of this manuscript in a current form.
Experimental design: No comments
Validity of the findings: The same comments as 'Basic reporting'
Additional comments: No 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: CROWDSOURCING VISUAL PERCEPTION EXPERIMENTS: A CASE OF CONTRAST THRESHOLD
Review round: 3
Reviewer: 2

Output:
Basic reporting: The manuscript is well written, and the subject is timely. However, it is important not only to show no difference in CSF between laboratory and crowdsourcing conditions but also to address discrepancies in anything other than CSF due to physical differences between the two conditions. Further analyses are required to determine the difference between the two conditions.
Experimental design: no comment
Validity of the findings: 1. In general, contrast measurements are significantly affected by gamma correction. In particular, the absence of gamma correction increases the likelihood of detecting stimuli at low contrast.
However, this manuscript did not show the difference in sensitivity between the two conditions (Laboratory vs. Online repetition) at low contrast. Is there a significant difference in mean % correct between the two conditions at the lowest contrast level? It would be appreciated if you performed 2-way mixed model ANOVA (4 cpd  x 2 conditions) and showed the results (main effect and interaction).

2. For a better understanding of the results, it would be good to provide figures that show the actual data points at each contrast (i.e., mean accuracy as a function of contrast level). Besides, is there a difference in the goodness of fit (R-square) between the two conditions?

3. The difference between non-repetition and repetition in crowdsourcing conditions can be learning (Fiorentini, A., & Berardi, N. (1980). Perceptual learning specific for orientation and spatial frequency. Nature, 287(5777), 43-44) or testing effect. I hope this possibility is discussed in the text.
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: CROWDSOURCING VISUAL PERCEPTION EXPERIMENTS: A CASE OF CONTRAST THRESHOLD
Review round: 4
Reviewer: 1

Output:
Basic reporting: I am satisfied that the authors have addressed all my comments and consider that the manuscript is ready for publication.
Minor comment: I recommend adding error bars in figure 2.
Experimental design: no comments.
Validity of the findings: no comments.
Additional comments: no 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: HOLE IN ONE: AN ELEMENT REDUCTION APPROACH TO MODELING BONE POROSITY IN FINITE ELEMENT ANALYSIS
Review round: 1
Reviewer: 1

Output:
Basic reporting: Generally fine, see specific comments below
Experimental design: I think some of the methods need slightly more detail to improve clarity, further comments below
Validity of the findings: The findings appear valid, but I think the study would benefit from using an alternative method of assessing the differences between models that is less sensitive to localized variation
Additional comments: This paper compares finite element models of cylinders of real mammalian trabecular bone with models of solid cylinders that have either had their stiffness (material) properties, porosity (physical properties), or both adjusted. The aim is to determine how these modeling choices impact the FE results (i.e. the study is a finite element sensitivity test).
Overall I think this is an interesting study that makes a good contribution to the field. Although the element reduction methods do not mimic the fabric of real bone, the results nevertheless demonstrate the material distribution has a more important effect than material properties when modeling trabecular bone. It is nice to see this quantified, especially as it runs somewhat counter to some of the largely untested conventional wisdom that’s been talked about in FE circles recently. Whether these differences are important enough to make the models more, or less valid, is not addressed, but that is not the point of this study.
I think the paper would benefit from increasingly clarity in some of the methods, and from an enhanced discussion of some of the points I raise above (and below in more detail).

Main Comments
L132: “We defined the material properties to apply in all the meshes in the CC and PC experimental groups (Young’s Modulus: 20 GPa and Poisson's Ratio: 0.3). For the MC group, the Young’s Modulus is adjusted within a range (from 7 GPa to 22 GPa) that is linearly proportional to the density values of the control cylinder (actual species trabecular geometry) for that experimental group’s relative fill volume. Relative fill volume (mm3) was calculated using the species-derived 3D model that served as the standard (Wysocki and Tseng., 2018). The remaining boundary conditions for the MC group were set up as in the CC group.”
I think this needs more explanation. If I’m following this right, each CC has a percentage volume of bone, and this was used to adjust Young’s modulus for the corresponding MC? It took me a lot of re-reads, and the caption for Fig. 3, to wrap my head around this and I’m still not completely sure I get it. A worked example or a figure might be helpful, e.g. a CC with 50% bone by volume would get an E of 14.5 GPa, because 0.5(22 – 7) + 7 = 14.5. Is that right? I think it would help your readers if you spell it out. Also, how was the range of 7 – 22 GPa selected?

L153: Are the percentages here the percentage of the space occupied by bone, or by air? i.e. for Enhydra lutris, are you deleting 16.5% of the solid material, or deleting 83.5% of the material so that only 16.5% remains? From the way it’s written, I thought it was the second one (only 16.5% remains), but later (L169) you refer to “16.5% volume deletion”. 16.5% deletion makes more sense (these animals aren’t made of air!) but this needs to be clearer, especially in the context of figuring out what the material properties are.

Paragraph at L213: I agree that the numbers for PC are closer to the CC than the numbers for MC, but saying that they “replicate” the peaks and valleys is a stretch. In both PC and MC, you have what looks like a smooth curve with the PC line being slightly more jagged. It certainly cannot replicate the extreme peaks of the CC. The only real difference between PC and MC is the VM stress values, and I think that if you compared the gradients of the lines of the PC and MC curves from point to point, you would find them almost indistinguishable from one another.
This is unsurprising, because the peaks in the CC are almost certainly due to “hotspots” of stress developing around individual trabeculae (evident in Fig. 4). How do your transect lines match up with actual material in the CC models? Are they interrupted at all, especially in the lower bone volume cylinders? Are they passing directly through hotspots?
All of this make me wonder is transecting across the cylinder is the best way of measuring “equivalent” performance among your cylinders: although those hotspots are real (i.e. not artefacts of boundary conditions), they are still incredibly localized. In a whole bone model, you are unlikely to be interested in such localized effects and would probably care more about whether the overall deformations are correct. I wonder, if you were to look at something like the overall strain (by which I mean change in the height of the cylinder before and after compression), would this not be a better way of seeing which of the experimental cylinders match the controls? This will average out those hot spots and give you a better idea of how deformation is accommodated across the whole model. This would then allow you to comment on something else I’ve been wondering about: the arrangement of trabeculae within bones is presumably not random. If a fabric was present in the bones, it would be present in the CC models. Whether you’re loading them in a manner that matches how that fabric would be loaded in life is moot – if the fabric is important, I expect that it’d show up in the overall deformation, but not (or less) in the hotspots.
EDIT: you do acknowledge the measurement method later, in the discussion (L273), but I don’t think anything as complicated as plates is necessary. For a start, the plates could impose their own stiffness even with low Young’s modulus values. Overall deformation of cylinder length (and maybe width too) would be easy to measure as a starting proxy for relative performance, and I think it would be a useful addition to the paper. A good test of this would be in your repeatability study (Fig. 2) – does an alternative method mitigate the artefacts that distort the results from test IV?
EDIT 2: you also have mentioned the trabecular arrangements on L282. I got ahead of myself! If I were you, I’d move your paragraphs about these limitations forward in the discussion and end with the discussions of the useful advice (see next comment), because that’s the key contribution that this paper makes.

L255: This is a really good point. I wonder if you can highlight it more? I feel like you haven’t talked much about this combined effect aspect of the models, but the fact that porosity is more important than properties is arguably the key take-home message. This could be even more important in animals with lower trabecular volumes (e.g. birds and pterosaurs), or less important in marine mammals using ballast, and you could expand on this in the discussion. A further point (raised in L268 but could be expanded on) is that this potentially explains why so many FE models have been found to be too stiff when validated. From memory, most of them use “solid” approximations of trabecular bone with reduced stiffness, but hardly any account for the differences in material distribution that real trabecular bone produces.

Figs 3 and 4: The species here are organised alphabetically, but I think it would be more useful to arrange them in terms of bone volume, so that it will be easier to see how changing the bone volume alters the results. I wonder if it would also be worth plotting all of the CC models on one graph, and all of the PC models on another, again so the readers can more easily discern the effect of bone volume on the results. For instance, I am curious as to whether there is a linear relationship between percentage bone volume and performance.

Minor Edits
L73: “The pushing of the boundary for FEA and better modeling of bone structures have been continuous for the last decade or so to better understand skeletal form and function”
This sentence would be better if rearranged: “For the last decade or so, the boundaries of FEA have been pushed towards the better modeling of bone structures to better understand skeletal form and function”
L81: “When in the presence of this type of porous structures” Should be “this type of porous structure” or “these porous structures”
L264: I’m still not convinced that any of the experimental models capture the peaks of the controls, and think this phrasing should be reconsidered (in the results, too). Also, I think you meant to have a comma after the word “trajectory”
Fig 1 caption: should say “fixed nodes”
Fig. 3: Can you make the fonts bigger on the axes and legend? These need a lot of zooming on a computer, and will be tiny printed out. Maybe make the lines on the graphs thicker too, it’s not easy to discern the colors at this line weight. Also, the image in Fig 1 and the supplementary tables say that you took four transects per cylinder at 25% intervals around the circumference, yet each model only has one line of these graphs. Can you explain this?
Fig 4: Can you write which species and bone volumes the rows represent on to the figure?


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: HOLE IN ONE: AN ELEMENT REDUCTION APPROACH TO MODELING BONE POROSITY IN FINITE ELEMENT ANALYSIS
Review round: 1
Reviewer: 2

Output:
Basic reporting: The basic reporting is sound, with some possible improvements to the writing.
Experimental design: The research conforms to the aims and scope of PeerJ. The questions and implications are quite carefully defined. The methods for the innovative 3D erosion are a little unclear.
Validity of the findings: The research has highly important recommendations about assigning material properties to cancellous bone. Bulk/regional assignment is simply inferior to replication of trabecular networks through the authors' methods, and the approach will be a huge benefit for accurate replication of structural response of cancellous bone. The authors do well with negative controls, and convincingly explain the study as exploratory rather than statistical. The conclusions are well-stated and circumscribed.
Additional comments: This is an exciting study that will increase the accuracy of FE simulations for cancellous bone, at least at smaller size scales. I'm not certain at what scales the method will become impractical beyond the size range of cores the authors took from mammalian jaw joints. However, the erosion method is clever and may give results for back-calculating stiffness of large regions of cancellous bone. The figures could use larger font sizes, but are otherwise clear.

The main improvements to the manuscript will be to language. Pay attention to suggestions for more active writing and conciseness. For example, the heading "Element Reduction Script Verification Analyses" is all nouns for one concept. A more engaging heading would be "Does random element reduction deliver precise results?”. Some other passages also have a surfeit of gerunds. The acronyms (CC, PC, and MC) are hardly necessary, except in figures and explained in captions. PeerJ readers will be eclectic, and spelling out the terms the first time in each paragraph, or omitting the acronyms from the text, will prevent initial frustration.


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: COMPARATIVE ANALYSIS OF BACTERIAL COMMUNITIES ASSOCIATED WITH HEALTHY AND DISEASED CORALS IN THE INDONESIAN SEA
Review round: 1
Reviewer: 1

Output:
Basic reporting: Overall, the manuscript lacks a clear narrative structure and the text is poorly written, quite prone to grammatical errors and frequently make sentences unclear to the reader. I suggest the authors to reduce wordiness, redundant expressions or phrases. Also, the authors have a tendency to overstate their ideas and/or dismiss references regularly. The connection between phrases and fluency can improve as well.
Experimental design: The amount of information you provide on the methods is not enough to reproduce your experiments. Please be more specific to help others following your protocols. For example, it is not clear for me how you attached your barcodes to your amplicons, did you make a second PCR? or was your PCR done with different primers for each sample? how many different barcodes did you use? Same goes for your sampling and nucleic acid extractions. On the other hand, data analysis was very well explained, thanks for that.
Validity of the findings: Title: no compare metagenomics, this is 16S rDNA amplicon diversity.

L80-85: The authors need to clearly make a distinction of what they are considering metagenomic analysis and analysis based on molecular markers. I suggest sticking to 16S-based diversity instead of calling it metagenomics.

Did you collect healthy tissues from diseased corals? otherwise, how can you take into account variation in individual-specific microbiomes?

Your results are mixing a lot of discussion and for me was hard to understand what you found and contrast it with what others have done. Please revise L165-173 in that regard.
Additional comments: L38: Please rephrase, coral reefs are not niches
L40: This needs references
L42-44: Consider rephrasing to: "The coral reef ecosystem is estimated to provide economic benefit worldwide, accounting for USD 29.8 billion per year from various sectors, e.g. fisheries, tourism, coastal protection, and biodiversity."

L50-52: is wordy and unclear
L59-61: Consider rephrasing to something along these lines: "Many of these diseases are prevalent in reef-builder corals (Order Scleractinia), leading to the deterioration of entire reefs structures"
L62: add "and" to "brown band, red band, and yellow band syndromes are.."
L63-64: This is a very strong statement, either reference it appropriately or tone it down.
L65-68: Try to be concise when giving examples and only emphasize on the most relevant ones. Otherwise, the statement turns lengthy and loses clarity.
L68-70: This statement needs references and examples
L72: remove "by microbiological methods" It is clear that Koch postulates involve microbiological methods. Also, consider replacing "Koch'shypotheis" to "Koch's postulates"
L73-74: Why some of the diseases are capitalized and others are in lowercase. Keep it consistent.
L75-76: either add "the" before "culture-depended" or replace "approach" by "approaches". Also, the phrase is long, is missing commas, and needs a grammar check.
L81-82: replace "take" by "make", replace "any" by "many", and replace "ecological niches" by "ecosystems"
L82: remove "the" and add "approaches" after "culture-independent"
L96: Consider rephrasing to "This work compares coral-associated bacteria in different host species and shows shifts in the bacterial community structure during the diseased stage. Our findings expand the current understanding on the microbiology of most prevalent coral diseases"
L111: "SDS-based DNA extraction" only makes reference to the lysis step you used. Please be more specific and state how you isolated nucleic acids (e.g. phenol-chloroform purification with ethanol precipitation) and importantly specify what modifications you made to previous protocols.
L129: What do you mean that sequences were determined? Do you mean: sequencing was done using the ION PGM platform?
135: rephrase to Data analysis was done in QIIME version 1.9.1
215: replace "as minorities" to "in lower abundances"

Table 2: replace "No. sequence (cleaned)" to "No. of sequences after QC"
Figure 6: it would be good to use colored text or colored boxes as a visual guide to better show your clustering. It's hard to remember all abbreviations, especially if they are not in the figure legend!


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: COMPARATIVE ANALYSIS OF BACTERIAL COMMUNITIES ASSOCIATED WITH HEALTHY AND DISEASED CORALS IN THE INDONESIAN SEA
Review round: 1
Reviewer: 2

Output:
Basic reporting: Overall, the writing needs to undergo considerable revisions for acceptance. The introduction and discussion should be more cohesive in delivering the message about the relevance of these diseases and their connection to the experimental design of the study (regarding bacterial taxonomy and the connection to studies that have been conducted previously). This is especially relevant because much work has been done on some of these various diseases, and their relation to observations in other geographical locations (previous work) is very interesting. While this connection is not completely absent in the text, the writing can be adjusted in order to better demonstrate the linkages to other previous observations.

I believe this study reveals very important information about the bacterial taxonomy associated with different disease states in various coral species. The overall structuring of the paper could use some revisions as well (see specific comments below). For example, lines 167-172 in the results section are not results from this study but descriptions of the diseases previously described. In addition, much of the grammar and word choices throughout the paper also need to be addressed. I offer some changes below in the general comments section that should be helpful. This study offers important new information in the field of marine disease ecology and I would recommend incorporating these suggestions and other reviewers’ comments for acceptance.
Experimental design: The experimental design is acceptable and research question is very clear. I would recommend using these suggestions to make this more effective in demonstrating relevance and meaning in the scope of connecting these results to the global information on bacterial diseases of the same type. The methods are described with sufficient detail and is replicable.
Validity of the findings: The analyses and results are impactful, especially in the context of the bacterial taxa associated with the four diseases in this study. Most of the statistics are acceptable for the analyses, but many of the specific statistical tests are not reported in the results (e.g. what statistic was run? Tukey, Wilcoxin, T-test?). These types of reporting need to be clearer (see below in general comments).
Additional comments: I suggest the term “bacterial microbiomes” should be changed to “bacterial communities” in the title

Abstract –

First sentence – “global warming” should be changed to “climate change”. “Endangered” is more specific to certain species, and should be changed to “impacted”. Other types of human impacts should be mentioned briefly here, such as overfishing, pollution, etc.

Second sentence – “infected stages” should be changed to “disease states”

The last sentence does not make sense grammatically. “relevant to the pathogenicity of the infected corals”. Maybe change to “pathogenicity of the microbes associated with infected corals”

Main text

Line 38 – Should be changed to “Coral reefs contain important ecological niche space harboring…”

Line 48 – “sponges with closed interactions to associated communities” needs to be elaborated on (i.e. what are these closed interactions?)

Line 51 – “phenomenon” should be plural, see “phenomena”

Line 60 – I would suggest using the word “infect” instead of “attack”, this applies to the rest of the text.

Lines 63-64 – Citations needed

Line 71 – “were” should be “ have been”

Line 76 – Needs to be reworded

Line 78-79 – The final sentence of this paragraph needs to be reworded for clarity

Line 80 – “environmental metagenomes” should be changed to “environmental communities through metagenomic sequencing”

Line 82 – “showed” to “have shown”

Line 86 – Change to “variation in the taxonomic composition”

Line 87 – Change to “existing coral diseases”

Line 93 -  Change to “our work”

Line 95-96 – This sentence needs to be restructured

Line 105 – Change to “from different colonies”

Line 160 – Citation needed for the human activities section of this line

Line 167-173 – These are not results from this study but descriptions of diseases that should be included in the discussion

Line 192 – Unclear what metric was used for genetic distance (units?)

Line 195 – What statistical tests are associated with this p-value, this applies to the rest of the text

Line 252 – What statistic was used to determine clustering? Is this just visual?

Figure 3 – Should include the type of disease associated with the corals in the figure, for visual reference. This applies to other figures that meet the same criteria.


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: COMPARATIVE ANALYSIS OF BACTERIAL COMMUNITIES ASSOCIATED WITH HEALTHY AND DISEASED CORALS IN THE INDONESIAN SEA
Review round: 2
Reviewer: 1

Output:
Basic reporting: I believe this study reveals very important information about the bacterial taxonomy associated with different disease states in various coral species.

The manuscript still needs minor revisions in my opinion, but the authors addressed most of the major changes I suggested in the first round of review. I would also recommend the other independent reviewer confirm this on their part, or another new reviewer be included in this round.
Experimental design: The experimental design is acceptable and research question is very clear. I would recommend using these suggestions to make this more effective in demonstrating relevance and meaning in the scope of connecting these results to the global information on bacterial diseases of the same type. The methods are described with sufficient detail and is replicable.
Validity of the findings: The analyses and results are impactful, especially in the context of the bacterial taxa associated with the four diseases in this study. The authors addressed most of the critique from the first round of review.
Additional comments: I am satisfied with most of the responses from the rebuttal. I recommend this paper be accepted with additional minor revisions outlined below.

Line 17 – “Coral reef ecosystem” should be plural (e.g. “coral reef ecosystems are impacted by”)
Line 41 – “sea lives” should maybe be changed to “marine organisms”
Line 44 – did you mean “erosion” instead of “abrasion”?
Line 66 – should be “rising” instead of “raising”
Line 106 – consider adding “the Indonesian Sea” instead
Line 313 – “resulted” might be changed to “the result of”
Line 320 – “part of the world” should be changed to “regions of the world”
Line 322 – “finding” should be “findings”
Line 362 – add “,” after Gammaproteobacteria
Line 364 – “co-existed” needs to be defined. I’m not sure what is meant by this statement, or if it is just grammatically incorrect


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: SIMULATING MORE REALISTIC PREDATION THREAT USING ATTACK PLAYBACKS
Review round: 1
Reviewer: 1

Output:
Basic reporting: No comment
Experimental design: No comment
Validity of the findings: No comment
Additional comments: The current manuscript examines the response of Neolamprologus pulcher to a variety of cues of predation threat which avoid risk of harm to predators or prey. Specifically, the authors examine the response of N. pulcher to their natural predator Lepidiolamprologus elongatus in an adjacent aquarium, video of an L. elongatus in a relaxed state or behaving aggressively. The authors found that compared to controls, the N. pulcher tended to respond most strongly to the live predator or the video playback of the predator behaving aggressively. In a second experiment, the authors expose N. pulcher to chemical cues of predation risk (L. elongatus holding tank water, conspecific skin extract, or a combination or the two) in conjunction with a video playback of a predator. The authors did not detect any effect of chemical cues on the response of the focal fish to the video. The paper is well written, and the experiments are well conducted. I do have some questions and suggestions for the authors which may help to refine the manuscript for publication.

Is it possible that aggressive behaviours directed towards a conspecific and predatory behaviours are different in form? If so, then the method of stimulus production may not capture prey directed behaviours as accurately as the authors assume. Furthermore, because the predators are not directing their attacks at the focal fish, but rather in their general direction (at simulated conspecific opponent), these videos may not fully simulate the presence of an actively hunting predator.

L117 – How were the videos edited down to remove gaps in the apparent predator attacks without creating unnatural jumps or cuts in the video?

L124 – It seems to me a better control would have been to edit a square over top of an actual elongatus on video. This would have created a stimulus in which the object moves like a real predator, but without the visual identity information. Having the shape move randomly changes both the appearance and the behaviour of the “predator”.

Perhaps with a larger testing tank more avoidance behaviour may have been observed?

L133 – Feeding the chemical cue donors flake food rather than N. pulcher may have muted the strength of this treatment, as aquatic prey species have been shown to pick up on predator diet cues in their excrement.

L156 – please provide more details of how being “oriented towards” or “attending to” the stimuli were scored.

Throughout, “chemical” would be more accurate than “olfactory” because these cues may also stimulate taste receptors, not just smell.

In figure 2, the overall trends seem to indicate greater interest in more complex or unpredictable stimuli, rather than increasing fear of increasingly more threatening stimuli.

Were any other measures of antipredator behaviour taken such as freezing, darting, thigmotaxis, increases or decreases in overall activity level. Especially with reference to the chemical stimuli, some of these other measures could reveal as yet inapparent effects.

Minor comments:
-	L60: Given the lack of effect of chemical stimuli, perhaps it would be better to say “stimulate relevant sensory modalities”
-	L68 & 245: trout
-	L69: replace “acquire predator recognition” with “recognise predators”
-	L110: presumably cm
-	Please provide the full make and model of the tablet, along with its dimensions. It would also be useful to have some technical specifications such as the resolution, refresh rate, and colour gamut.
-	L154: citation not in the reference list
-	L188: more attentive
-	The experiment numbers are reversed in the figure captions.


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: SIMULATING MORE REALISTIC PREDATION THREAT USING ATTACK PLAYBACKS
Review round: 1
Reviewer: 2

Output:
Basic reporting: The manuscript entitled: Simulating more realistic predation threat using attack playbacks
investigated the eﬀectiveness of olfactory and visual predator cues in Neolamprologus pulche, which suggested that playbacks of attacking predator can induce similar effect with a real predator.

Abstract
Line 25 Why do you emphasize this fish “cooperatively breeding”? It seems irrelevant with your topic.
Line 34 What do you mean by “prey-directed behaviours”? Please clarify it.

Introduction
Some sentence especially the introduction of knowledge gap seems lack of logic. And the description of knowledge gap seems speculative.
Line 42 The authors may need to explain why “such experiments are not always logistically”?
Line 71-73 This sentence seems speculative.
Line 89-91 This sentence is ambiguous, and else where. Because the visual cues were present during exp 2.
Methods
N. pulcher lives in groups in nature, but in your study, the behavior of N. pulcher was measured individually. Can you explain why?
Line 100 The N. pulcher was juvenile? Stock were caught from Lake Tanganyika? Did they have predation experience? And where were the predators come from?
Line 113, 124, 132 Please provide the body size of the predator and the prey. Otherwise, we will not sure whether the predator can act real threat on prey.
Line 113-116 Where did you put the camera? please provide more details. For the prey fish, it may have different angle of view on the predator between in the tank (real predator) and in the screen (playback). E.g. the distance between predator and prey, the size of the predator.
Line 113, 117 Please provide the detailed size of each tank which used in your study.
Line 114-117 It is a good method to obtain the “live stimulus” through mirror test, while it still has a flaw that the response may be different between attacking their own reflection and prey. Why didn’t you make the predator attack their real prey and film the playbacks? Some predators may not have mirror response and may restrict the utilization of your method.
Line 136 Why didn’t you kill the N. pulcher with euthanasia to avoid ethical issue?
Line 146-148 Did you use the same playback with exp 1? Why the playbacks “from 9 different L. elongatus individuals” in exp 2, but in exp 1, only two L. elongatus individuals were used? Too many objects may decrease standardization. Additionally, why did you use the lateral predator playbacks (swimming calmly) instead of frontal predator playbacks?

Results
Line 188 Which group compared to frontal playbacks?

Discussion:
Some contents are speculative and need more background and reference to support the results.
Line 230-231 see Results Line 191: No difference was found between lateral and frontal playbacks (p = 0.98; Table 2).
Line 236-238 Do you have some data or observation that whether the predators never managed to harm the prey fish? This kind of explain seems speculative and needs deeper discussion.
Line 238-239 It isn’t be a subsection.
Line 239-256 Some contents are speculative and need more background and reference. e.g. I suggest that you provide more background of N. pulcher’s habitats. N. pulcher is active only in the daytime? Or it’s habitat with high water transparency, thus usually use visual predator cues? Following review may be helpful to improve your discussion. Kelley J L, Magurran A E. Learned predator recognition and antipredator responses in fishes. Fish and Fisheries, 4, 2003, 4: 216-226.
Line 250-251 This sentence seems suspect. Intuitively, olfactory cues may be also important for prey to response to their predator.
Line 252-253 Fischer et al. (2017b) have showed that olfactory and visual predator cues are used independently by N. pulcher in predator detection, it was different with your study. You may need to explain why.
Line 254-256 This sentence seems ambiguous and speculative, because no significant difference was found, we don’t know whether visual cues or olfactory cues played a role.

Figures
Figure 1 The annotation in the plot was different from the caption. Predator tank was present only during exp 1? Or exp 2?
The note of “Tablet screen (Present only during trials)” is ambiguous, please clarify it.
Figure 2 Time in attention: It seems that no significant difference was exist between lateral and live, but there was a * in the plot.
For figure 2 and 3. The sequence number (e.g. a, b, c, d) for each plot was missing.
Experimental design: Manuscript describes original primary research. More details of the design and methods need to be provided.
Validity of the findings: Data are robust, but the figures need to be checked carefully.
Additional comments: The experiment appears well designed (although the authors need to provide some missing details about the protocol). Although the manuscript is fairly well written, there are parts where it is somewhat difficult to understand what the authors are trying to say, and it should be edited for clarity.


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: SIMULATING MORE REALISTIC PREDATION THREAT USING ATTACK PLAYBACKS
Review round: 2
Reviewer: 1

Output:
Basic reporting: N/A
Experimental design: N/A
Validity of the findings: N/A
Additional comments: I think that the authors have done a satisfactory job of addressing my prior comments and I think the paper is now suitable for publication.

Note that the "olfactory" versus "chemical" terminology remains unchanged in the figures and captions and should be updated to match the main text.


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: SIMULATING MORE REALISTIC PREDATION THREAT USING ATTACK PLAYBACKS
Review round: 2
Reviewer: 2

Output:
Basic reporting: The authors have thoroughly replied to my comments and have incorporated the required information in the text. Many imprecise sentences have been reformulated and their meaning is now clearer. The missing details I mentioned have been provided in this version.
Experimental design: Research question well defined.
Experimental design was appropriate to solve the authors' question.
Validity of the findings: Results can support their conclusions.
Additional comments: No comment.


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

Input:
Title: HEATSTROKE-INDUCED HEPATOCYTE EXOSOMES PROMOTE LIVER INJURY BY ACTIVATING THE NOD-LIKE RECEPTOR SIGNALING PATHWAY IN MICE
Review round: 1
Reviewer: 1

Output:
Basic reporting: First, the manuscript contains numerous counts of unclear, ambiguous, and unscientific English writing, which is reflected by inconsistent, misleading, exaggerating, and/or inaccurate statements as well as grammatic errors and misspellings.

Examples include but not limited to:
Line 223 describes “Fig.2A showed the top 10 high-expressed proteins..” whereas in the corresponding figure (erroneously Fig4), top 20 most abundant proteins were shown.
HS is defined as “heat stress” in the abstract (line 42) but “heatstroke” in the introduction (line 71).
In line 217, does “three exosomes (should be “exosome”) samples for each group” mean three independent experiments or three technical repeats in one experiment?
In line 192, authors stated that “The trafficking of exosomes in vitro and in vivo were observed with ….”  However, the static images of fluorescence staining cannot indicate the movement, i.e. trafficking, of the exosomes.

Second, the introduction fails to provide sufficient background to validate the rational of the study due to inaccurate citation of references. None of the first 5 references cited in the first two paragraphs of the introduction support the statements.  For example, authors stated “A multicenter epidemiological 78 survey of inpatients showed that approximately 31.9% of HS patients developed ALI [3],” in lines 77-78, when Reference#3 is a case report of a single human subject.

Third, the figures are in a different order than described in the text.  Figure 4 and 5 in the figures section should be Figure 2 and 3 based on the text.
Experimental design: Authors intended to investigate whether exosomes are induced by heatstroke and then contributed to heatstroke-induced liver injury by combining in vitro and in vivo assays. The strategy described in the manuscript add strengths to the study; however, the method section largely lacks important and necessary details and information to confirm the validity of the results or to replicate. Major points include:
1.	iTRAQ based proteomic profiling and KEGG pathway enrichment were major analyses used to explore differential protein expression in the exosomes caused by HS; these procedures should be described in the method with respect to the experiment protocol, the definition of “kinds/groups of proteins” and significance.
2.	How many mice were used in each group of the in vivo study? Had the animals been acclimated before use?  Were the experiments reviewed and approved by the Instructional Animal Care and Use Committee (IACUC)?
3.	Many commercial kits (eg, “a Odyssey kit”) and equipment (eg, “a fluorence microscope”) lack specific name or model description.
Validity of the findings: The validity of the results cannot be verified, mainly due to a) the lack of detailed description and information for the experimental procedures; and b) unclear and ambiguous description of the results.
Additional comments: Authors should improve the overall writing of the manuscript on both the scientific and English aspects to ensure accuracy and clarity.


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: HEATSTROKE-INDUCED HEPATOCYTE EXOSOMES PROMOTE LIVER INJURY BY ACTIVATING THE NOD-LIKE RECEPTOR SIGNALING PATHWAY IN MICE
Review round: 1
Reviewer: 2

Output:
Basic reporting: The figures are out of order. Otherwise, the writing is clear and effective, but could use grammar check.
Experimental design: Overall, the study is design and execution are acceptable/
Validity of the findings: The data are sound and the conclusions are mostly supported by the data, though I'm concerned about possible mix-ups in samples or otherwise poor labeling of their data.
Additional comments: Here, the authors tested a novel hypothesis in an understudied field (heat stress-induced liver injury). The experimental design is adequate. However, there are several issues to address:
Major issues...
1) All figures: The figures are out of order.
2) Current figure 2 (hepatocytes treated with exosomes): The blots and the densitometry in panel A do not match. The blots shower lower abundance of inflammasome markers with HS-exo treatment. The authors need to be certain their data are correct and they know which sample is which on the blot, and then correct their labeling.
3) Current figure 3 (immunohistochemistry for inflammasome markers): The "positive" staining just looks like diffuse background staining to me. The authors should support these data with western blots.
4) Current figure 5 (uptake of exosomes): It appears that the authors tested uptake of exosomes by hepatocytes in control mice. What about in mice with hyperthermia, which is what they are really interested in? It is entirely possible that uptake differs in hyperthermic mice.
Minor issues...
1) Overall, the manuscript is well-written, but the English grammar could use some minor improvement.


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: HEATSTROKE-INDUCED HEPATOCYTE EXOSOMES PROMOTE LIVER INJURY BY ACTIVATING THE NOD-LIKE RECEPTOR SIGNALING PATHWAY IN MICE
Review round: 2
Reviewer: 1

Output:
Basic reporting: No additional comments.
Experimental design: No additional comments.
Validity of the findings: No additional comments.
Additional comments: It took me considerable time to locate new reference 3 due to the obscurity of the journal (Med J Chin PLA). I understand that the authors cannot control where this paper was published, but it would be preferable if the authors could provide more accessible references in the early Introduction.


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: BACTERIAL MICROBIOTA COMPOSITION OF IXODES RICINUS TICKS: THE ROLE OF ENVIRONMENTAL VARIATION, TICK CHARACTERISTICS AND MICROBIAL INTERACTIONS
Review round: 1
Reviewer: 1

Output:
Basic reporting: Basic Reporting

The manuscript is well written and uses well-referenced publications which make the manuscript coherent, comprehensive and theoretically solid. The manuscript structure accomplishes with Peer J standards norms. I am happy to recommend acceptance however some aspects needs to be considered before its publication. I offer a specific line by line suggestion below:

Supplementary File

1. I found that some paragraphs are repetitive with results (i.e: in page 5 " Using a variance partitioning approach…explaining on average 14.3% of the total variation (Figure 2 in the main text)"; and in page 6 and 7: "The effect sizes varied…to 27%44% at north-facing sites”). I suggest the authors to compile the information in just one section preferably in results.

2. Figures and Tables are relevant and high quality, however, I have found some details that need further revision before publication:

- Figure S3, needs to be re-label. I have found in the supplementary file references to Figure S3a-i, and Figure S3e, but these labels are not included in the Figure S3. I also recommend to include this labeling information in  Figure S3 Footnote.

- The authors should check the labeling of Table S2 in the main manuscript. As you can see in Line 289-290 it is referenced the Table S5 instead of Table S2.

3. The reference Lemoine et al. (2018) is included in the narrative but not in the reference list. However, it is included in the reference list of the main manuscript.

Main Manuscript

1. The authors included several times across the manuscript the importance of ticks vectoring pathogens to humans and wildlife. However, I suggest to include the term "domestic animals" as well.

2. In line 177: I recommend to use “reads” instead “amplicons”, and so on across the manuscript.

3. In Line 203, add the web associated with the oligotyping pipeline software (http://oligotyping.org).

4. The paragraph from Line 221 to Line 231 it would probably fit better as part of a conclusion or a discussion. Thus I recommend the authors to consider deleting it from Mat and Met section.

5. In Line 249, please explain which criteria were used to exclude OTUs from the analysis.

6. To facilitate readers the understanding of the analysis and the results I recommend the authors to keep uniformity between the names of the variables in the main manuscript and the Table S1. For example:

- Line 275, instead of Topography use Aspect
- Line278, instead of Tick density use tick abundance since abundance estimates and density estimates are different.

7. In Line 412 and 413, delete the extra parenthesis ).

8. In Line 314 correct Ross et al. (2017), the correct reference is Ross et al. (2018), and in the Reference list as well.

10. I have found an incongruence in the manuscript that needs to be clarified in the Discussion section. In Line 311 to 314 the authors discussed that  " This substantial microbiota variation across individual ticks…may be partly explained by some of the bacteria present in ticks being no resident (i.e., bacteria that accidentally obtained from the environment)". However according to the pipeline explained in the Mat and Met section, the authors have previously used methods to get rid of rare OTU (Line 249 "after excluding rare OTUs) and potential environmental contamination and potential environmental contamination (Line 143-144: "To avoid environmental contamination…".). Following this assumption, the method utilized to avoid rare OTUs and potential environmental contamination does not work?.

In this regard, I recommend to include biological explanations about these results and biological expectations can be made from "non-resident bacteria" driving the microbiota variation across individual ticks? I suggest the authors some literature that can be evidenced the importance of the environment in the internal shape and assemblage of tick microbial communities due to bacteria can be eventually acquired from soil and plants from the environment through the spiracles, the mouth and the anal pore, or even during copulation (paternal transmission route) and on each blood-feeding event (skin microbes, pathogens).
Horner-Devine, M.C. and Bohannan, B.J.M. (2006) Phylogenetic clustering and over dispersion in bacterial communities. Ecology 87, S100-S108
Engel, P. and Moran, N.A. (2013) The gut microbiota of insects-diversity in structure and function. FEMS Microbiol. Rev. 37, 699-735
Zolnik, C.P. et al. (2016) Microbiome changes through ontogeny of a tick pathogen vector. Mol. Ecol. 25, 4963-4977
Zolnik, C.P. et al. (2018) Transient influence of blood meal and natural environment on blacklegged tick bacterial communities. Ticks Tick Borne Dis. 9, 563-572
11. In line 42 it should be added “Similarly, the host´s immune system can influence colonization success of microbes… I suggest the author to consider microbes-derived factors in the colonization success of bacteria. It is demonstrated that microbes have mechanisms that participate in the host selection process, (e.g:  microbial associated signaling patterns (MAMPs)). (see references)
Chu, H. and Mazmanian, S. K. (2013) Innate immune recognition of the microbiota promote host-microbial symbiosis. Nat. Immunol. 14, 668-675
Lee, S. M. et al. (2013) Bacterial colonization factors control specificity and stability of the gut microbiota. Nature 501, 426-529
12. I suggest the author be more specific about the co-occurring concept (Lines 427-430) What are co-occurring individuals? Where are they co-occurring and why does this matter? it remains complexity. Microbes occurrence with other microbes is based on the assumption that coexisting species are ecologically similar (Leibold and McPeek, 2006, Barberán et al., 2012). But it is relevant to note that co-infections may results when hosts are independently infected by different microorganisms at the same time, or when interaction among parasite species facilitate co-occurrence.
Experimental design: Ticks are well-known as vectors of a great variety of infectious agents but recent surveys have also underlined that the structure of ticks microbial communities shows obvious co-occurrence patterns of non-infectious microbes and pathogens. This interesting because some of these microbes are even essential for tick survival and pathogen competence. Aivelo et al. interestingly discuss the intervention of ecological factors, host characteristics, and interactions among microbes shaping ticks microbial communities. The authors are providing a conceptual framework, which I found sufficiently interesting for publication. I especially liked that the authors included the supplementary file and the raw data with information to be reproducible by other authors. However, I offer specific line by line suggestion below that could help to improve the manuscript:

1. I thank the authors for providing the metadata ("ticksampledata_figshare.csv") available in FigShare, needs to include detailed information of metadata identifiers.
In this regard, I recommend the authors to include details about meaning, and metrics on each case to easily replicate the methods. For example:
 - site_masl= Elevation of gradient (meters above sea level).
 - Tick_het= tick individual heterozygosis

5. In the Supplementary File, I suggest the authors include in Table S1 more information to be useful to future readers, for example in can be improved as follows:

 -  Indicate which components and variables are considered "fixed effects" in the same way that Latent variables are considered "random effects".

- Within the “Full variable set”, indicate which variables are considered at “tick-level” or “site-level”.
Validity of the findings: 1.While the paper needs improvements, as various definitions and concepts are diffuse and need to be fully developed to finally validate the findings (as I have mentioned above in the Basic Reports).

2. If there is a weakness, it is the small number of ticks samples in some sites, thus a low number of samples and replication, Considering inter- and intraindividual differences and high variation of microbiota the statistical power of analysis for any comparative analyses deserves some important discussions.

3. I suggest the authors revise the "ticksampledata_figshare.csv" file and the ID codes concordance with the column "tick-stageF" , and Table 1, and the total number of Females and Males. If I assume that 0 correspond to males, and 1 to females (similarly to the column tick-stageN in which 0 is adult and 1 nymph). In the column (tick-stageF”) is assigned 0 to nymphs (HXX) and thus considering nymphs (0) as males (0) when filtering the column by 0. This was very confusing to me, and since this was the input matrix used for the HMSC, I am concerned this fact could have influenced the results and conclusions
Additional comments: No additional comments


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

Input:
Title: BACTERIAL MICROBIOTA COMPOSITION OF IXODES RICINUS TICKS: THE ROLE OF ENVIRONMENTAL VARIATION, TICK CHARACTERISTICS AND MICROBIAL INTERACTIONS
Review round: 1
Reviewer: 2

Output:
Basic reporting: The manuscript entitled “Bacterial microbiota composition of Ixodes ricinus ticks: the role of environmental variation, tick characteristics and microbial interactions” may be of interest to the readership of PeerJ journal. The research quality and statement conform to the standards and scope of the journal. The study is focused on the identification of the factors driving the taxonomic composition of the microbiota of Ixodes ricinus, including the presence of tick-borne pathogens, which constitutes a frontier of knowledge, with relevance for the prevention of tick-borne diseases. The text is written clearly, with adequate structure. In the introduction, statement and references are correctly used to describe the scientific problem and its background. The methods are clearly described. The data is available online, with the appropriate description. However, I consider that some aspects should be revised:

Mayor comment

1-The introduction should be shortened for greater conciseness.

2- Figure 1 (sampling sites) should be improved. The elevational gradient is the main reason to choose the sampling areas, however, it is not possible to identify it without observing Table 1. To solve this problem, the authors could use different shapes or colors to differentiate low, medium and high elevations points within each location). Remove the unidentified black point at location 1.

3- The major aim of this study is to evaluate the taxonomic composition of microbiota in individual ticks in different conditions, and this is the variable studied in the model. However, only the most abundant OTUs and their distribution are described and included in the results.  Better characterization and visualization of all the bacterial diversity should be achieved. For example, a barplot with the taxonomic composition of the samples, grouped by sex and life stage across the elevation gradient should be included. Other compositional traits analysis of the microbiota could be added to complement the analysis, such as alpha and beta diversities.
Experimental design: The study sought to determine how do large-scale abiotic factors and small scale tick-level variables affect tick microbiota composition, and which large-scale abiotic and small-scale tick-level variables predict pathogen occurrence. The authors conducted a well-designed observational study, consistent with the research questions. They chose an elevation gradient that allowed them to work on a well-characterized environmental gradient. They selected standardized framework (JSDM model) to determine the effect of the predictive variables on the taxonomic composition of the microbiota tick. The identification and use of the predictors were well-executed, and individual ticks were used as a random effect in the model which is a strength of this study. As response variable they used the taxonomic composition of the microbiota, assessed through high-throughput amplicon sequencing of the 16S rRNA gene. They conducted a considerable sampling effort, repeated three times (monthly) in each area. However, some aspect of the analytical procedures should be reviewed and improved as possible, specifically related to the variable response on analysis.

Mayor comment

1-The taxonomic composition matrix of all the ticks was used as the response variable (95 total) in the study. However, analyzing the distribution of samples across life stages, sex, sampling area, and elevation, in many of these conditions there were no biological replicates (Table 1), or only two replicates, which could be collected at different times (June, July, August). This suggests that this dataset lacks robustness to support the predictive model used, how did the authors address this limitation? This is an important point because it affects the strength of the conclusions.

2- It was intended to evaluate the effect of the predictors on the taxonomic composition. However, as the authors referred, it is well-known that the microbiota of Ixodes ricinus, as other ticks, is highly variable in taxonomic composition. Even the results of this analysis indicated that more than 60% of the taxonomic composition was explained by the random effect of each individual tick. Perhaps the authors should analyze, at a previous stage, other features of the taxonomic composition of the microbiota less sensitive to changing taxa (i.e. the alpha and beta microbial diversity metrics), this would improve the results.
Validity of the findings: The results are consistently presented and discussed in context.

Mayor comment

1-In the conclusions it is argued that the JSDM framework can contribute to a better understanding of the factors shaping bacterial communities. However, there was no analysis for the evaluation and/or validation of this model. Although its main limitations (in materials and methods) are described, its performance in the analyzed data is not addressed and discussed.
Additional comments: In consequence of the sampling effort made and the importance given to the taxonomic composition in this study, a higher resolution taxonomic classification should be achieved. That is using Qiime2 pipeline since it works at the level of a single nucleotide and each amplicon variant (ASVs) can be identified, improving the taxonomic classification.


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: BACTERIAL MICROBIOTA COMPOSITION OF IXODES RICINUS TICKS: THE ROLE OF ENVIRONMENTAL VARIATION, TICK CHARACTERISTICS AND MICROBIAL INTERACTIONS
Review round: 1
Reviewer: 3

Output:
Basic reporting: The manuscript by Aivelo et al. investigates the effect tick-specific and environmental variables have on the microbiome composition of Ixodes ricinus. In particular the manuscript looks at the impact of these variables on selected endosymbiont and pathogenic bacteria. The authors are to be commended on the inclusions of extensive supplementary methodology and details on modelling. While it is clear the authors had a large amount of data to work with they have done well to be selective in their inclusive of results to include data which makes the most sense from an ecological point of view. The manuscript is well written with clear aims.

The authors have made the raw data available on the NCBI Sequence Read Archive. However 93 samples are listed under the designated BioProject PRJNA506875. The manuscript states that 92 tick samples were sequence (no controls sequenced), please include the BioSamples that were used in the present study (e.g. BioSample SAMNXXXXXXXX – SAMNXXXXXXXX). The authors have made additional data available on FigShare repository.
Experimental design: The authors make use of ecological modelling methods in the context of tick-associated bacteria, including pathogens. The authors are to be commended on the detailed explanation that is made available in the supplementary information. In particular references regarding their classification of bacteria as endosymbiont or pathogen. This is a challenging and contentious aspect of tick microbiology that many authors do not justify. The authors have also given extensive detail on modelling aspects used in the manuscript.
Validity of the findings: Absence of sequences from negative controls in 16S NGS studies is concerning and raises questions as to the use of appropriate controls. Evidence of both (i) bacteria present in extraction kits known as the and (ii) unavoidable cross-contamination during sample processing and DNA extraction of ticks, are well reported tick microbiome and general microbiome/eDNA studies. (E.g. Couper et al. 2019, DOI: 10.1002/ece3.5361; Gofton et al. 2015, DOI: 10.1186/s13071-015-0958-3). Can the authors please confirm if controls were included in sequencing libraries?

Taxonomy of OTUs – while the authors have provided detailed additional information in the supplementary information there is little details on the taxonomic assignment of OTUs other than the following sentence “We used 97% similarity to determine OTUs and classified them with the wang method (Wang et al. 2007) using SILVA taxonomy.”. As the authors have assigned species level taxonomy to OTUs (table 2) can they please expand on how they assigned this level of taxonomy (e.g. BLAST confirmation and results?). In the case of Rickettsia spp. in particular the accurate taxonomic assignment through a short 250 bp amplicon of the 16S rRNA gene is extremely difficult (Greay et al. 2018 10.1186/s13071-017-2550-5; Raoult and Roux 1997 Clin Microbiol Rev 10(4). If the authors used additional taxonomic assignment analysis or confirmed by other molecular tools can they please expand.
Additional comments: [1] Line 54-55 – The number of OTUs is highly ambiguous, and influenced by numerous factors including target amplicon, bioinformatics and library preparation (Youssef et al. 2009, DOI: 10.1128/AEM.00592-09; Chen et al 2013, DOI: 10.1371/journal.pone.0070837). As such reference reporting on a designated number of OTUs in a specific tick species is misleading. Unless the authors wish to expand on comments about the number of OTUs in ticks including biases involved, this should be removed.

[2] Line 160– The use of the word “quantified” is inaccurate as the authors did not quantify bacterial load (via qPCR or otherwise). The number of 16S NGS reads is not directly relative to bacterial quantification in a community, unless attempts to account for such biases were made (e.g. Kembel et al. 2012, DOI: journal.pcbi.1002743, Rosselli et al. 2015, DOI: 10.1038/srep32165). Unless these methods were implemented it is recommended wording be changed to “characterisation of bacterial community” or “profiling of the tick bacterial community” or similar.

[3] Line 160 – Reference to the V3-4 region of the 16S gene is incorrect if primers 515/806 were used as is stated in the manuscript. This primer pair amplifies only the V4 region (see http://www.earthmicrobiome.org/protocols-and-standards/16s/).

[4] Lines 177 – throughout manuscript - The authors use the words amplicons and sequences interchangeably. If they are referring to different aspects can they please clarify this clearly, otherwise the authors should ensure wording is consistent throughout the manuscript.

[5] Line 180-181 – A threshold of >5 amplicons is extremely low, can the authors please justify this? Without any sequence data from controls, the level of contamination or cross-talk is difficult to assess.

[6] Line 236-237 – See above for comments regarding no amplification from controls.
References – some references are lacking italics for species names e.g. lines 472-474, 480.


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: BACTERIAL MICROBIOTA COMPOSITION OF IXODES RICINUS TICKS: THE ROLE OF ENVIRONMENTAL VARIATION, TICK CHARACTERISTICS AND MICROBIAL INTERACTIONS
Review round: 2
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: Considering that the authors have complied with the feedback from the reviewers and they have modified it accordingly, I am happy to recommend acceptance 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: BACTERIAL MICROBIOTA COMPOSITION OF IXODES RICINUS TICKS: THE ROLE OF ENVIRONMENTAL VARIATION, TICK CHARACTERISTICS AND MICROBIAL INTERACTIONS
Review round: 2
Reviewer: 2

Output:
Basic reporting: The manuscript was improved.

The authors considered the suggestions and responded convincingly to my comments, and in my opinion, to the comments of the other reviewers as well.
Experimental design: The description of the materials and methods was improved.
Validity of the findings: The authors achieved a correct presentation of their results, with adequate contextualization of the strengths and weaknesses of the study, and their contribution to the research area.
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: BACTERIAL MICROBIOTA COMPOSITION OF IXODES RICINUS TICKS: THE ROLE OF ENVIRONMENTAL VARIATION, TICK CHARACTERISTICS AND MICROBIAL INTERACTIONS
Review round: 2
Reviewer: 3

Output:
Basic reporting: The manuscript is well written and would be of interest to Peerj readers and those in the field of tick research. Sequence data is provided on the NCBI Sequence Read Archive and supplementary information made available.
Experimental design: The authors have provided extensive details on methodology, made available in the supplementary information. I am happy that they have addressed reviewer comments regarding experimental design.
Validity of the findings: The results are presented in a consistent manner. Any limitations in the data have been discussed and they have incorporated reviewer comments.
Additional comments: The authors have thoroughly addressed all reviewer comments, and I am pleased to recommend the manuscript be accepted.