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: DEVELOPMENT AND FEASIBILITY STUDY OF AN APP (LADLE) FOR WEIGHT LOSS AND BEHAVIOUR CHANGE
Review round: 2
Reviewer: 2

Output:
Basic reporting: The article is clearly written using professional English throughout. The following amendments would resolve ambiguities:

it is stated that the completion definition was set to reflect existing research. Please cite the research that supports this definition.

typo on line 471 - should be 'it' rather than 'in'.

line 494 - "completion is higher than a few of the intensive interventions". But not if you use the same definition of completion, please clarify and cite the comparison studies you are referring to.

line 499 - clarify that this is 52% of completers

provide ref for lines 501-503.

Table 2 needs a Note defining the abbreviation ITT. Also, the term "finishers" should probably be removed from the column headings, as this is not correct for all data in that column, and those that do use completers data should be delineated similar to those using ITT.

The terms completer and finisher are used interchangeably. While they clearly mean the same thing, consistency would aid the reader. (personally I prefer completer, but perhaps it is just my misspent youth playing computer games that considers "Finisher" to have aggressive connotations)
Experimental design: I do wonder if the comparison information should be in the discussion rather than the results. The studies chosen for comparison was not systematic and there is no formal analysis. Perhaps having claims of "lower" or "higher" in the results section adds too much weight to these comparisons, particularly when other characteristics are so unbalanced. Having the comparisons in the discussion, perhaps in more broad terms such as "comparable" would be more appropriate. The results could then simply be a clear description of the intervention development and a descriptive evaluation of the intervention. However, I do not have strong feelings on this latter point, and I do think that table 2 is helpful.

The role of the "Expert support" should be clarified (p279): "There was also space for participants to ask questions and comment on any aspect of the course. Responses from a Ladle trained professional were given within 24 hours." Apologies that I missed this the first time round. Is this human support element an integral part of the app, and is it included in the estimated costs?
Validity of the findings: The authors have added some clarification to the results section on comparative effectiveness, but they are still not consistently explicit about the differences in definitions of completion, timing of follow up, and analysis method (ITT vs intervention completers). This is absolutely essential for ALL of the comparisons made. Comparisons of mean weight loss are also only described where they reflect favourably on Ladle.

An example is L534:  "superior to that achieved by other apps with no human coach."  I think you mean "some other apps" as you did not do a systematic search and comparison, and did not do a statistical comparison.

Other problematic phrases include
L33 - just say comparable
L390-393 - clarify differences between this study and comparators
L399-400 - ditto
L404 - what is meant by "similarly measured"
L484 - is this a vaild comparison?
L490 - ditto
L522-524 - ditto
L525 - There is insufficient evidence to claim this is "as effective as other approaches"
Additional comments: No additional comments


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

Input:
Title: THE EFFECT OF DIFFERENTIAL SPATIOTOPIC INFORMATION ON THE ACQUISITION AND GENERALIZATION OF FEAR OF MOVEMENT-RELATED PAIN
Review round: 1
Reviewer: 1

Output:
Basic reporting: No comment.
Experimental design: No comment.
Validity of the findings: No comment.
Additional comments: The present study investigated to what extent spatiotopic information contributes to the acquisition and generalization of fear besides proprioception. Based on controled joystick movements, conditioned stimulus associated with pain and conditioned stimulus not associated with pain were used to understand differential learning.

It has been shown that pain-related fear might be more disabling than pain itself, specially for chronic mausculoskeletal pain patients. The present study tested pain fear generalization to movements with similar proprioceptive features but different endpoint location. The authors suggest that fear accrued to proprioceptive conditioning stimulus features.

This is an interesting study that can have a positive impact on the understanding of sensory-motor interactions. In my opinion, in general, the manuscript is clear, unambiguous, well-written, and structured according with PeerJ standards. Literature is well referenced and relevant. Raw data is supplied. The investigation has been conducted in conformity with the prevailing ethical standards in the field.

Data is robust, statistically sound, and controlled. The study design required the expansion of the sections 'material and methods' and 'results', making them extensive, although clear and well written. This led to the manuscript having more than 7,500 words. Since there are no restrictions on the part of the Peer journal, on the number of words, I do not see this as a problem.

The subject itself arouses interest in the scientific community and is far from been fully understood. The authors chose a paradigm to test their hypothesis and remain faithful to it. Considering the way the manuscript was organized, the data presented and discussed, I have nothing to add that can change in an impactful way the message brought by the study.

I take this opportunity to congratulate the authors and, as a reviewer of this important scientific journal, manifest approval and agreement so that the manuscript is approved in its current format.

Minor correction:
Line 27. In the ‘abstract’, please put the name in full and then the abbreviation the first time the term 'conditioned stimulus' appears. The same should be done in Line 162, for the term ‘intertrial interval (ITI)’.
Line 533. “...This means that is our study...”. Replace with ...This means that in our 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: THE EFFECT OF DIFFERENTIAL SPATIOTOPIC INFORMATION ON THE ACQUISITION AND GENERALIZATION OF FEAR OF MOVEMENT-RELATED PAIN
Review round: 1
Reviewer: 2

Output:
Basic reporting: Manuscript is well-written, references provide a clear state of art in the research field. Figures and tables are clear and easy to follow. Abstract needs some improvement, as well as a better basis for the hypothesis of the study.
Experimental design: Research question was clearly defined and evokes for additional research. Some methods information needs to be reviewed or strengthened.
Validity of the findings: Manuscript fails to explore the practical implications of the conclusions.
Additional comments: Abstract

Terms and descriptions are superficial for the first contact with the study. It is not possible for the reader to scale and fully understand the experimental protocols and the determinant variables of analysis of the study. I suggest a more detailed description of the methods, within the allowed word limit.

Introduction
Lines 86-88. Why this hypothesis was established? The first hypothesis is clearly linked to Haddad’s study, but not this one. Could the authors emphasize the basis for this hypothesis as well?

Methods
How sample size was defined? Please, provide information about the participants firstly. I suggest to present table 2 before table 1.
How exclusion criteria were checked? Self-reported? Please, add this information.
It is not clear if all participants performed in all groups or if they were allocated in different groups. Please, emphasize that in the methods section.
Line 133 – “half of the participants” – half of 51? Please, identify in Table 1 the sample size of each group.
Lines 186 – 191 – In the second block, joystick position was on the left or on the right. Therefore, before the trial the participant knew the direction to move the joystick. It is unclear why tone stimulus was necessary.
It is unclear if the pain was randomized inside each block. For example: in the location group, for half of the participants, to move right would always result in pain? Why this protocol was used? One can argue that, in daily activities, people know the movements that result in pain resulting in fear or in avoidance to perform it. Did the authors think about in a ‘control group’, that knew previously the direction that would cause pain?

The practical implications of the study could be strengthened. Authors affirm in the introduction that pain-related fear significantly predicts physical performance and functional disability and that such fear is often more disabling than the pain itself. What conclusions about the differential spatiotopic information can be drawn and eventually used for pain/disability treatments?


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

Input:
Title: THE EFFECT OF DIFFERENTIAL SPATIOTOPIC INFORMATION ON THE ACQUISITION AND GENERALIZATION OF FEAR OF MOVEMENT-RELATED PAIN
Review round: 1
Reviewer: 3

Output:
Basic reporting: This manuscript is generally very well written.  The literature is sufficiently informative, as is method and analysis.  The tables and figures are excellent and he raw data is provided to allow open re-analysis. The rationale is clear and the hypotheses relate clearly to this and to the conclusions.

Some serious but easily addressed issues in reporting are as follows.

I find Table 1 very confusing.  I seem to understand the procedure from the text - but the table does not reflect the text on lines 140-147.  Why does the table not show  fear conditioning and probes for generalisation for the movement group?   Surely the far right column, for example, should list CS presentations for the movement group as these were used as GCs probe stimuli for that group?  It is also not clear from the table that startle probes were ALSO part of the fear conditioning phase.

What I understand to be the precise procedure for taking expectancy ratings (inline) is contradicted by Figure 1 suggesting that the text is not sufficiently clear and is not directly related to Figure 1.

In line 209, allusion is made to in-line stimulus ratings but not enough procedural detail is given to allow reader to ascertain how these may have interfered with conditioning trials - this may be critical.  It is provided a little later but a flag should be provided earlier  for this.

In discussing generalisation, reference is made for the first time to a first and second generalisation test, but there is no mention of this up to this point.   I guess it is simply the arbitrary division of the probe trials into two halves - referred to previously as blocks - , and is so likely inconsequential but it would help the reader greatly to not worry that there is some other procedural aspect they have missed.

Line 65 - break up sentence.

Lines 76-78 also have missing words and are too cumbersome.

Line 339 has a crucial word or two missing.
Experimental design: This study has a very elegant and simple design.   The mastery of the stimulating and recording equipment is satisfactory and reported well.  The study is vert well conducted with no confounds that I can see, but there are compromising procedures.  Specifically, in the current inline ratings procedure, if I understand it correctly, you may expect to see a blocking or overshadowing effect whereby the rating Q and response acquire conditioned fear functions that may overshadow the effects of the subsequent CS+.  It does not take from the differential effects across conditions and stimuli, but it is not good practice if we can avoid it and it is compromising conditioning to a greater extent, I would argue, than the starting point position of the joystick, which the authors discuss in the Discussion section. This could be acknowledged in the Discussion.
Validity of the findings: Generally the conclusions are solid and relate to the hypotheses. So it passes in that regard. I was surprised, however,  not to see more speculation on how these results may relate to our understanding of real world clinical issues around plain management and dysfunction.

One bigger issue is that the question is not as novel as it sounds,  but it is sufficiently novel for publication if the authors are clear about the difference between discovering a new effect and discovering a new process.  The former is not as exciting as the latter. Specifically, the generalisation shown here, and the effect of overlapping features,  is of the same type shown  in several previous studies, but is novel (not an essential feature of the MS I know), to the extent that it also involves testing the effect of overlapping features on that acquisition and generalisation.  So the results are not telling us much new in terms of conditioning and generalisation processes but they ARE interesting form the point of view of understanding responses to pain. This is not very clear unless the reader is quite expert in the fear conditioning literate and the outcome may come across as over stated.  The exciting outcome, is the use of well understood effects, to interpret real life issues relating to movement reduction etc in clinical pain populations.  I felt the manuscript would benefit form leaning more in that direction and ensuring that the reader does not mistake a advance in application of principles for an conceptual advance at the process level.
Additional comments: Overall this is an excellent study that is very well reported and deserving of publication.


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

Input:
Title: THE EFFECT OF DIFFERENTIAL SPATIOTOPIC INFORMATION ON THE ACQUISITION AND GENERALIZATION OF FEAR OF MOVEMENT-RELATED PAIN
Review round: 2
Reviewer: 1

Output:
Basic reporting: I see no need to re-review this manuscript. In my opinion the manuscript is ready for publication in its current version.
Experimental design: I see no need to re-review this manuscript. In my opinion the manuscript is ready for publication in its current version.
Validity of the findings: I see no need to re-review this manuscript. In my opinion the manuscript is ready for publication in its current version.
Additional comments: I see no need to re-review this manuscript. In my opinion the manuscript is ready for publication in its current version.


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

Input:
Title: THE EFFECT OF DIFFERENTIAL SPATIOTOPIC INFORMATION ON THE ACQUISITION AND GENERALIZATION OF FEAR OF MOVEMENT-RELATED PAIN
Review round: 2
Reviewer: 2

Output:
Basic reporting: I would like to thank the authors for the relevant responses and the revisions submitted.
The new version of the manuscript attends all the questions I raised previously.
Manuscript is well-written, references provide a clear state of art in the research field. Figures and tables are clear and easy to follow.
Experimental design: Research question was clearly defined and evokes for additional research. The additional information provided in the methods section after review allowed to better understand all the protocols designed.
Validity of the findings: No comment.
Additional comments: No additional comments


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

Input:
Title: ELLIPSE PACKING IN TWO-DIMENSIONAL CELL TESSELLATION: A THEORETICAL EXPLANATION FOR LEWIS’S LAW AND ABOAV-WEAIRE’S LAW
Review round: 1
Reviewer: 1

Output:
Basic reporting: The work is clear and well-written. The background is well presented and the structure is good. It is self-contained.
Experimental design: The question posed is interesting. The approach original. The work is of high standard and of interest to a wide range of problems.
Validity of the findings: The findings are robust and interesting.
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: ELLIPSE PACKING IN TWO-DIMENSIONAL CELL TESSELLATION: A THEORETICAL EXPLANATION FOR LEWIS’S LAW AND ABOAV-WEAIRE’S LAW
Review round: 1
Reviewer: 2

Output:
Basic reporting: .
Experimental design: .
Validity of the findings: .
Additional comments: This paper investigate cells structures as ellipse packings and thereby provide insight into a set of observed laws for such structures.
The investigation seems sound and the results reasonable. What remains unclear, however, is in what way these results are useful for understanding cellular structures.
It is mentioned in the text that the results can be used for simulating cell topology during growth. How this should be done remain unclear, however. In general, it needs to be demonstrated,
or, at least, argued for in what way the results are useful in this respect before the paper can be considered for publication.


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

Input:
Title: ELLIPSE PACKING IN TWO-DIMENSIONAL CELL TESSELLATION: A THEORETICAL EXPLANATION FOR LEWIS’S LAW AND ABOAV-WEAIRE’S LAW
Review round: 1
Reviewer: 3

Output:
Basic reporting: .
Experimental design: .
Validity of the findings: .
Additional comments: This paper  presents a range of statistical data on 2d foams.  It adopts a novel method of analysing 2d cellular patterns, involving the fitting of ellipses to individual cell outlines. I was not persuaded of the necessity for this and indeed some of the conclusions, but I do recommend publication. The work is substantial, and well placed in relation to the literature. Moreover it takes data from a range of biological materials. This will make it useful to many people, particularly as the question of the relation of biological cells to analogous physical systems is topical at this time.


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 MUSIC INTERVENTIONS ON ANXIETY DURING LABOR: A SYSTEMATIC REVIEW AND META-ANALYSIS OF RANDOMIZED CONTROLLED TRIALS
Review round: 1
Reviewer: 1

Output:
Basic reporting: In general, this manuscript is well written. But I found two grammatical errors in line 201 and line 305. “Total 1,310 participants” and “Aldridge et al declared that” should change it to “A total of 1,310 participants” and “Aldridge et al. declared that”, respectively.
Experimental design: The greatest problem in this paper is that the time frame of data retrieval is from April 25, 2018 to the end of December, 2018.  For a meta-analysis, this is not a comprehensive search for relevant literature.
Validity of the findings: No comment
Additional comments: This is a meaningful, interesting study in which the authors confirmed that music interventions may decrease the anxiety levels measured using the VAS-A and physiological indexes related to anxiety (HR, SBP, and DBP). However, some issues should be solved. The specific points need to be carefully corrected as follows:

Abstract
-- Authors need to report specific the name of databases and time frame in this part.

Introduction
-- It is necessary to introduce some basic information about music intervention in this part.

Method
-- All abbreviated name should be corrected. For example, “H.-H.L.” change it to “HHL”.
-- I suggest that the authors should write the inclusion/exclusion criteria based on the “PICOs” approach.
-- Line 113-114.  “without deafness (unless corrected with a hearing aid) or severe psychiatric disorder” This is an exclusion criterion rather than an inclusion criterion.
-- Please cite the references for related heterogeneity criteria.

Result
--Authors should describe basic feature of the included studies in brief. The best way is to perfect table 1 rather than a detailed and lengthy description.
-- The references of the included studies should only be listed once.
-- For subgroup analyses, results from comparisons across groups can provide more useful information.


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 MUSIC INTERVENTIONS ON ANXIETY DURING LABOR: A SYSTEMATIC REVIEW AND META-ANALYSIS OF RANDOMIZED CONTROLLED TRIALS
Review round: 1
Reviewer: 2

Output:
Basic reporting: no comment
Experimental design: I did not have access to the additional tables and figures, which was lacking in the review process. If they have not been placed in the submission process, it is essential that they be sent.
The term music therapy is used several times and appears in the titles of some figures, but the authors do not make clear the difference between music therapy (performed by a professional Music Therapist) and musical interventions. This differentiation is of fundamental importance considering that the term Music therapy appears at different points in time. Questioned? Was a study differentiated with music therapy and music in medicine? Was Music Therapy considered as a musical intervention? How many and which studies were with Music Therapy? I consider it important to clarify this aspect as well as information on how the data were analyzed considering that there is a difference between these practices.
Validity of the findings: The validity of the data should be better analyzed after clarification on experimental design
Additional comments: The article was well written and there is only one aspect of doubt pointed out in the design of the study. This area lacks studies of this nature, which allows this to be an essential publication.
I did not have access to the additional tables and figures, which was lacking in the review process. If they have not been placed in the submission process, it is essential that they be sent.


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 MUSIC INTERVENTIONS ON ANXIETY DURING LABOR: A SYSTEMATIC REVIEW AND META-ANALYSIS OF RANDOMIZED CONTROLLED TRIALS
Review round: 1
Reviewer: 3

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


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

Input:
Title: EFFECT OF MUSIC INTERVENTIONS ON ANXIETY DURING LABOR: A SYSTEMATIC REVIEW AND META-ANALYSIS OF RANDOMIZED CONTROLLED TRIALS
Review round: 1
Reviewer: 4

Output:
Basic reporting: English language editing is necessary - especially in chapters "Introduction" (e.g. l. 62; l. 72-73, l. 79) and "Discussion"
References: The references given in the introduction section are not quite fitting. Nearly all references in the introduction are questionable, because the topics do not fit or the quoted studies are irrelevant for the current context. A number of studies is out of date (published before 2000 but not of outstanding scientific impact). The current references refer to completely different fields, such as ICU, chronic pain or cancer.
I would strongly recommend to stick to the current topic (labor pain and anxiety), you might back up the introduction with some general information on music + analgesia and music + anxiety (there exists a number of high quality reviews and meta-analysis on this topic).
In the discussion section, a rather comprehensive overview, that fits to the current topic is included - Maybe you could restructure the paper and use several paragraphs from the discussion rather in the introduction section!!

Background: Please, give a precise definition of "music therapy". The term is used in a very unspecific manner, therefore it is very important to make clear, what you mean by "music therapy". There exist good definitions by the American Music Therapy Association (AMTA) https://www.musictherapy.org/
or the World Federation of Music Therapy (WFMT)  https://www.wfmt.info/

Article structure, figures, tables:
- The structure of the article is conform to an acceptable format of ‘standard sections’
- All appropriate raw data have been made available in accordance with the Data Sharing policy.
- Figures are relevant to the content of the article; It would be helpful to add a label to the figures in the supplement, in order to make sure, the figures are comprehensible in a stand alone version.
Experimental design: Search strategy:
- did you search for a specific period? Please state the space of time, studies would be eligible.
Data extraction:
- why did you choose the outcome variables you chose? This should be explained in more detail.
- what do you mean by "visual analoge scale"? Please give more details on the operationalization (e.g. " It is a scale that is comprised of a horizontal or vertical line and anchored at both ends by words indicative of extremes of magnitude" - if the studied did not measure their data according to this definition but askes participants to rate their mood on a scale 0-100% or allocate it to a number 0-10, this would not be a VAS but rather a NRS (numeric rating scale) - please be precise here!
Did all scales refer to a range of 0-10? Did you convert any different measures to this scale? Please be more precise on this topic!
-  the what about the psychometric more sophisticated questionnaires? Why did you not follow up the data of these questionnaires? A strengh of a meta-analysis can be to pool outcomes from different sources!
- l. 127: "The secondary outcomes were physical signs, such as BP and HR." ==> please, be precise in naming the secondary Outcomes!
- Control Group --> inconsistent Information. L. 114: "2) inclusion of a control Group" vs. L. 117/118: "4) studies with an effective intervention as control arm rather than a Placebo" - please be precise here. Why did you exclude studies with an effective intervention as control arm rather than a Placebo?
- l. 139-141: "A p value < 0.05 was considered significant. Statistical heterogeneity was assessed using I2 and Cochran’s Q tests. A p value < 0.10 for the χ2 test of the Q statistic or an I2 > 50% was considered indicative of statistically significant heterogeneity." ==> why did you use different alpha-Levels?= please explain or adujst!
Validity of the findings: - It is unclear why the results restrict to 14 studies overall and to 7 (or 6?? l. 217) studies in the meta-Analysis. Please be again more precise in describing you Methods.
- Did you weigh the studies? If yes, please explain your algorithm!
- l. 172/1733: "Self-selected music under recommendation was used in most studies (at least 9 studies" --> please give exact numbers!
- l.155-159: why did you stress the origin of the studies? Is is rather unusual to have so many Asian studies??

l. 213-217: "Ultimately, 7 studies ... were included in our meta-analysis... Primary Outcome: in 6 selected studies" --> what happended to study #7?

l. 222: "t value = 1.18, df = 4, p = 0.304" --> unusual report, rather 't-test: t(4) = 1.18, p = .304'

l. 230-231: "difference in means −= −0.28" unclear ??

- the trials included in the review are very, very heterogeneous. It therefore is doubtful to conduct a meta-Analysis on the sparse data.
I would suggest to emphasize the narrative review rather than extracting data of feeble informative value.

- l. 260-263: why did you emphasize age? Other variables might have been
number of birth (firstborn, later-born)

ethnic/cultural Background (there exists evedence concerning differences in pain and anxiety perception in different cultures)
Family Status (married/single etc., big Family Background,single-child-family…)
Additional comments: - the paper Reports interesting Facts concerning the use of prerecoded Music on anxiety Levels of Women during Labor.

- make sure to report new findings and to distinguish your results from previous, often quite similar papers.

Major concerns are
1) the choice of papers for the review/meta-analysis and the chosen outcome criteria --> please be more precise in the methods section. Currently you put quite an effort into the attempt to avoid any bias but you do not manage to outline your search and selection process.
2) the background section is completely inconclusive, since nearly all papers quoted do not refer to the topic "labor+anxiety and music" --> a complete revision of the introduction section is inevitable!
3) the results of the meta-analysis are very flabby, due to the heterogenity and the very small number of the raw Studie --> rather emphasize the narrative review instead of statistical fireworks without clinical relevance


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 MUSIC INTERVENTIONS ON ANXIETY DURING LABOR: A SYSTEMATIC REVIEW AND META-ANALYSIS OF RANDOMIZED CONTROLLED TRIALS
Review round: 2
Reviewer: 1

Output:
Basic reporting: No
Experimental design: NO
Validity of the findings: NO
Additional comments: I am satisfied with the changes the authors made.


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 MUSIC INTERVENTIONS ON ANXIETY DURING LABOR: A SYSTEMATIC REVIEW AND META-ANALYSIS OF RANDOMIZED CONTROLLED TRIALS
Review round: 2
Reviewer: 2

Output:
Basic reporting: The authors met the requests of the reviewers; therefore I consider the article suitable for publication. They updated some references, and made the table title corrections.
Experimental design: The authors responded to the requests of the reviewers.
Validity of the findings: The authors responded to the requests of the reviewers.
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: EFFECT OF MUSIC INTERVENTIONS ON ANXIETY DURING LABOR: A SYSTEMATIC REVIEW AND META-ANALYSIS OF RANDOMIZED CONTROLLED TRIALS
Review round: 2
Reviewer: 3

Output:
Basic reporting: Clear and unambiguous, professional English used throughout.

Literature references, sufficient field background/context provided.

Professional article structure, figures, tables. Raw data shared.

Figures should be relevant to the content of the article, of sufficient resolution, and appropriately described and labeled.

Self-contained with relevant results to hypotheses.
Experimental design: Design improved, no further comments
Validity of the findings: Validity of findings greatly improved
Additional comments: Many thanks for your effort in implementing the suggestions. The article improved greatly and in my opinion does not further re-editing.
One minor comment: in the section "data extraction" you mention secondary outcomes, i.e. physical signs. Please spell out the abbreviations since they appear for the first time in the running text (l. 145 in the tracked changes document)


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: CLARIFYING A TROPHIC BLACK BOX: STABLE ISOTOPE ANALYSIS REVEALS UNEXPECTED DIETARY VARIATION IN THE PERUVIAN ANCHOVY ENGRAULIS RINGENS
Review round: 1
Reviewer: 1

Output:
Basic reporting: Present study determined TP in Peruvian anchovy applying SIA method associated statistical approach. Large range of nitrogen isotope ratio within anchovies was resulted in the large variation of TP within species. I agree this interesting result and reasonable approach (e.g., mixing model and TP estimation) is acceptable, but need more concern for TP estimation and discussion for different diet preference within anchovy groups.
Experimental design: Present study applied “tROPHIC POSITION” for TP estimation, which is a new statistical approach. This approach looks nice to demonstrate TP of consumers by range scale. However, I think author need to explain more details about this new method. For example, nitrogen isotope ratio of phytoplankton was assumed as a baseline in this ecosystem. But nitrogen isotope value of phytoplankton has large range in this study. Thus I am wondering if SD of nitrogen isotope ratio of phytoplankton also included in the “tROPHIC POSITION” or only average value is used for baseline.
In addition, this study mentions that nitrogen isotope ratio of zooplankton is difficult because of uncertainty of TP. This assumption is reasonable, but need to concern relatively short turnover rate of phytoplankton than fish. In particular, study area is strongly affeceted by upwelling and hence variable nitrogen isotope baseline.
Validity of the findings: 1. Author mentioned spatial difference in the carbon and nitrogen isotope ratio due to the upwelling intensity with previous studies, indicating large variability of isotopic baseline.
Similar isotope ratio of carbon among anchovies is suggested as an evidence for identical nitrogen isotopic baseline, due to the similar pattern of spatial variation between carbon and nitrogen isotopes reported in the previous studies. However, this evidence are not enough, and also unclear. Present study collected samples for diet resources in 16 sites with large range of isotope ratios. This can be demonstrated spatial variation of carbon and nitrogen. This is maybe more helpful to the readers.
Also, author mentioned that sampling area is recognized as a spawning and nursery area. If anchovy migrate from other habitat, their isotope might be recorded information of the former habitat, meaning different nitrogen/carbon isotopic baseline.
Therefore, more information of the migration scale for anchovies is required.

2. Based on TP, author identified two groups of anchovies, indicating different diet preference. However, in my idea, assuming large acceptability of diet in anchovies, TP of anchovy may be distributed continuously large range rather than grouping. Author should discuss the possible reason for this grouping in anchovies.

3. In many part of this MS, author emphasizes higher TP than 2 in anchovy (previous knowledge) as new finding in this study. However, in my feeling, this is not interesting and weak for main discussion. This result clearly showed different feeding strategy between anchovy and jack mackerel, which may imply importance role of these fish in ecological view. I recommend more deep discussion (or reason) for how and why anchovy have large range of TP.
Additional comments: Because I am not a native in English, I can judge English in this MS. However, I found several mistakes and strange English in the MS, which should be revised before published. For example,
- Author use both trophic level and trophic position. I recommend identical term.

- Line 400 Docmac et al, 2016: cannot find in the reference lists (Docmac et al. 2017 ?)

- For carbon isotope value author did not use minus sign “−”

- Line 239 npermutations = 9 999 : need to correct
- Line 341 Espinoza et al’s : when?


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: CLARIFYING A TROPHIC BLACK BOX: STABLE ISOTOPE ANALYSIS REVEALS UNEXPECTED DIETARY VARIATION IN THE PERUVIAN ANCHOVY ENGRAULIS RINGENS
Review round: 1
Reviewer: 2

Output:
Basic reporting: Overall, this was a very well-written stable isotope study focused on the trophic ecology of the Peruvian anchovy. Aside from a few grammatical errors, the language was quite clear with excellent English. The Intro, while probably a bit longer than necessary was thorough and appropriate references were included. The Figures were clear and well-constructed. I might suggest stacking panels A & B of figure 3 so that a single x-axis can be used for both panels but that is a minor comment and more stylistic than anything else. The use of horizontal lines throughout Table 1 is a little jarring and not typical of most journal formats so I point it out but leave it to the copy editors to determine if that is an issue. The data were provided in zip format with the ms.
Experimental design: The authors’ findings that stable isotope-based estimates of trophic position of the fish were markedly higher than estimates from stomach contents (in agreement with several other recent studies) were based on sound, well-established methods (although I note one consideration that should be addressed below under General comments). The study falls within the scope of PeerJ and focuses on an ecologically important issue. In several instances, noted below, the methods were not entirely clear. Assuming the authors provide clarification, description of the methods is/will be quite clear and replicable.
Validity of the findings: The data appear to support the authors’ contentions, the conclusions are reasonable and the Discussion is appropriate.
Additional comments: General comments
Overall, I think this is a very nice study that I enjoyed reviewing. I have only one major comment (follows) and a number of specific comments (below).

The only concern I have is the potential for seasonality in the phyto/zooplankton d15N values to bias estimates of fish trophic position due to seasonal changes in the strength of upwelling. The authors state that fish and putative prey were collected during the austral winter but that upwelling in the region is typically strongest during the austral spring-summer (Line 172-173). Upwelling brings more isotopically heavy, deep water NO3 to the surface and it’s quite possible that could result in heavier phytoplankton and zooplankton d15N values during the spring-summer (e.g., as discussed in Hannides et al., 2009. Quantification of zooplankton trophic position in the North Pacific Subtropical Gyre using stable nitrogen isotopes). Also, spring-summer seasons in many areas are associated with higher somatic growth rates of fish and therefore can have a stronger influence on the stable isotope values of fish muscle tissue (e.g., Perga and Gerdeaux, 2005. ‘Are fish what they eat' all year round?). If the anchovies collected during the winter have tissues that are more reflective of summer conditions when prey had heavier d15N values, then the net result would be an overestimate of trophic position when using lighter winter d15N values as a baseline. Since the higher than expected TP values of anchovies estimated here are a key take-home message from this paper, I’d like the authors to address this point. I don’t think this potential issue invalidates the current study, but I think it is a consideration that needs to be stated for readers, discussed and noted as deserving future research. If, on the other hand, there is previous literature from the study region that demonstrates no seasonality in stable isotope values of phyto/zooplankton linked to changes in the strength of upwelling, then the authors should definitely make this known to readers and cite the appropriate literature.

Specific comments
Line 96 – upwelling systems are generally considered very productive for pelagic fisheries (as noted by the authors) and the HCS is no exception, therefore I would recommend deleting ‘anomalous’ from this sentence
Line 107 – add ‘in’ after ‘role’
Line 109-110 – presumably the HCS anchovy stats skew the global mean TL of catch downward….
Line 118 – do you mean ‘HCS’?
Line 117-121 – I know what the authors are arguing here, but ‘changes in our understanding’ of diet doesn’t actually impact ‘the movement of energy and nutrients through the food web’. Please adjust the wording of this sentence to reflect how our knowledge can influence our understanding of food web structure rather than the actual structure itself.
Line 145 – delete ‘is’
Lines 156-166 – These paragraphs can be integrated
Line 203-205 – lipid removal is considered standard practice for high-lipid fish tissues but it is not considered necessary for low-lipid tissues. If available, please provide a measure of lipid content of tissue or accepted proxy (e.g., C:N ratio) from pre-extracted tissue so readers can evaluate the need for lipid-removal
Line 204 – oven dried or freeze-dried? If oven, please note temperature
Line 213-216 – this section is a little unclear. Where phytoplankton really ‘placed’ on filters or were they filtered onto the GFFs? For zooplankton, it sounds like they were dried on GFFs, acidified, then scraped off the GFFs, homogenized, then analyzed…is this correct? How were samples acidified? It sounds like they were fumigated (if so, please make this explicit in the text), but if acid was directly applied, then please note that fact.
Line 216 – change ‘sampled’ to ‘samples’
Line 249-250 – Presumably, literature diet studies were used to decide which groups of plankton to use as potential prey from the range of taxa identified (Lines 208-211). Please provide 1 or more suitable references
Line 268 – change ‘mas’ to ‘mass’
Line 268 – delete extra set of parentheses around the SD of mass
Line 274 – here and elsewhere, a single notice that values following the +/- sign are SDs is sufficient. It shouldn’t be necessary to repeat this notice for each usage
Line 274 – this is a HUGE range in dN for one species!
Lines 299-306 – I’m confused as to why the authors elected to use PERMANOVA for these two univariate statistical models. Why not just use ANOVA or GLMs (if you want to include covariates) to look at group-based differences in dN or dC? Perhaps I’m missing something?
Line 348 – a word is missing in this sentence
Line 349 – Can you add ‘naturally’ before ‘labelled’ to emphasize that these observations come from naturally occurring heavy isotope values in deep ocean water rather than experimental labelling with heavy dN?
Line 353-356 – could spatial differences in phyto assemblage composition also contribute to the observed variability?
Lines 406-409 – It is also important (more so?) to consider body length relative to TP when looking for ontogenetic shifts b/c body length is more likely to positively covary with individual gape size, which is a very important factor influencing the types and sizes of potential prey as fish grow. The overall length range of fish analyzed here was quite narrow and presumably this is why the authors focused on mass which had a wider total range.
Line 417 – I think the authors meant to use ‘synoptic’ rather than ‘symptomatic’
Line 418 – please translate square NM to square km to remain consistent with use of metric in the ms


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: CLARIFYING A TROPHIC BLACK BOX: STABLE ISOTOPE ANALYSIS REVEALS UNEXPECTED DIETARY VARIATION IN THE PERUVIAN ANCHOVY ENGRAULIS RINGENS
Review round: 1
Reviewer: 3

Output:
Basic reporting: No comment
Experimental design: In the section Material & methods:

First observation:
Sample collection:
On the second paragraph, the authors do not give details of hour collection.
This factor is important to know, taking into account some reports about light influence on fractionation at inverse relationship.
One of these reports is:
Needoba, JAN, Waser, NA, Harrison, PJ, Calvert, SE. (2003). Nitrogen isotope fractionation in 12 species of marine phytoplankton during growth on nitrate. Marine Ecology Progress Series 255, 81-91.

Second observation:
Data analysis:
On the first paragraph, is not so clear why use Pearson correlation index and Welch t-test?.
There are not information on previous analysis such as normality and homocedasticity test.

I advice follow the routine:
- test the normality of the data (may be with Shapiro-Wilk), if the data is normally distributed, follow the next step.
- test of homocedasticity (Levene test), if the variance are not equal follow the next step.
- Welch t-test to examine differences in the data when compare two variables.
- Finally, apply the Pearson correlation test, which is parametric.

The other way is use the non-parametric tests, and Spearman correlation test is adviced.
Validity of the findings: No comment
Additional comments: No additional comments


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

Input:
Title: PEST SEQUENCES FROM A CACTUS DEHYDRIN REGULATE ITS PROTEOLYTIC DEGRADATION
Review round: 1
Reviewer: 1

Output:
Basic reporting: The manuscript is clearly and well written overall. However, the writing of “Materials and Methods” session need improve due to the use of non-standard English. For example, “scattered”, “a proportional vermiculite and soil mix”, “after they were…’’ in the paragraph (line 103-105), and “shifted into” in multiple places, were not properly worded.

Other comments related to writing:
Line 54, “suggest” should be “suggests”
Line 56, what is the superscript of “segment6”?
Line 56 and 77, “motives” should be “motifs”
Line 62, “present” may be changed to “contain”?
Line 355 , “represent” should be “represents”
Line 361, remove “,” after et al.
Line 450,  rephrase “ transient tobacco leaves”
Line 457, “protein fusion” should be “fusion protein”
Line 458, “its” should be “they”
Line 636, “plant cell” should be “ plant cells”

Table 1, orthologous should be “orthologs”. The title may be changed to “ Distribution of potential PEST sequences in subgroups of DHN orthologs”

Legend of Figure 5, “in A. thaliana the pMDC32-GUS…..” should be “ in the A. thaliana pMDC-GUS…..”
Experimental design: Meet standards. No other comments.
Validity of the findings: no comment
Additional comments: Figure 6D and 6E, in staining (6D), the “pMDC-GUS::PEST-mut L1 and L2 seem to have high GUS activities than the control pMDC32-GUS L1; however, in the fluorometric assay (6E), the result indicates the opposite, please discuss in 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: PEST SEQUENCES FROM A CACTUS DEHYDRIN REGULATE ITS PROTEOLYTIC DEGRADATION
Review round: 1
Reviewer: 2

Output:
Basic reporting: Pass.
Experimental design: Pass.
Validity of the findings: Pass.
Additional comments: The methods are very clearly explained, with credible results shown in the figures and supplemental information..The findings significantly add to the body of knowledge on dehydrins, as noted by the authors addressing an aspect of dehydrins - degradation during stress-recovery - that has received little attention so far. So, PEST sequences seem to be part of the picture. It leaves the reader wondering if/why it is not always part of the picture for every dehydrin, or at least always part of the picture for some specific subset of dehydrins. Perhaps this could be addressed somewhat more in the Discussion.

As a routine practice, each DNA construct should be checked by sequencing to make sure that the intended junctions have been made. For some constructs this validation was stated. For most it was not. The authors should insert statements to confirm that all constructs were validated by sequencing.


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: P5CR1 PROTEIN EXPRESSION AND THE EFFECT OF GENE-SILENCING ON LUNG ADENOCARCINOMA
Review round: 1
Reviewer: 1

Output:
Basic reporting: In the work of Cai et al., they have shown that “Overexpression of PYCR1 in lung cancers was further supported by data analysis from the Oncomine platform, which revealed higher PYCR1 mRNA expression levels in lung adenocarcinoma tissues.” It would be good to include this statement in the introduction and provide more rationale of studying PYCR1
Experimental design: *It would be better to mention a little more about the rationale to investigate the role of P5CR1 protein in lung adenocarcinoma.

*It didn't look like there was a significant difference in the results of wound healing experiment. (by independent sample t-test? it would be inappropriate if there are more than two groups) What post-hoc test was used? Please do not show any symbol if the p value is not < 0.05. Also, is there any difference in Fig. 3A? Which statistical methods was used?

*Please state how the authors conducted the statistical analysis of the difference for the CCK-8 assay.
Validity of the findings: *It is suggested to relate the results with other previous results, including different types of cancers so the readers could gain a general idea of the role of PYCR1.

*Please explain that why silencing of the PYCR1 gene did not affect the migration of lung adenocarcinoma cells.
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: P5CR1 PROTEIN EXPRESSION AND THE EFFECT OF GENE-SILENCING ON LUNG ADENOCARCINOMA
Review round: 1
Reviewer: 2

Output:
Basic reporting: In this manuscript Yang She and collaborators present data to demonstrate that PYCR1 gene affects the biological behavior of lung adenocarcinoma suggesting a potential therapeutic target for lung adenocarcinoma with this approach. Overall, this is an interesting study but as indicated below, several results need to be backed up.
1. The major concern is the need to share the information regarding to if PYCR1 protein levels are affected by PYCR1-silencing recombinant lentivirus in lung adenocarcinoma cells. In addition, shRNAs can have off-target effects.In general, multiple shRNA should be tested for consistent and reproducible evidence.
2. Figure 1A, 1B, and Table1, there is no indication the scoring system for immunohistochemical staining
3. In introduction provide a rationale between NSCLC and lung adenocarcinoma.
4. Please check the histology of NCI-H1299 cells (adenocarcinoma or large cell carcinoma).
5. To point out the reproducibility of the data and their statistical significance, this information should be integrated with the number of experiments with similar or overlapping results. Otherwise the means (+/-SD) of all the experiments performed should be reported along with the appropriate statistical analysis.
6. The paper requires careful editing by a native English speaking scientist.
Experimental design: no comment
Validity of the findings: no comment
Additional comments: No additional comments


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

Input:
Title: P5CR1 PROTEIN EXPRESSION AND THE EFFECT OF GENE-SILENCING ON LUNG ADENOCARCINOMA
Review round: 2
Reviewer: 1

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


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

Input:
Title: P5CR1 PROTEIN EXPRESSION AND THE EFFECT OF GENE-SILENCING ON LUNG ADENOCARCINOMA
Review round: 2
Reviewer: 2

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


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

Input:
Title: ASSOCIATION BETWEEN ISCR-BASED PHYLOGENY, SEROVARS AND POTENTIAL VIRULENCE MARKERS OF HAEMOPHILUS PARASUIS
Review round: 1
Reviewer: 1

Output:
Basic reporting: There is some speculation on horizontal gene transfer in the study. There is not an analysis, such as linkage disequilibrium or other, to measure or quantify it.

Some sentences are very difficult to interpret (Lines 189-190, lines 236-238))

The objective of section ‘Higher diversity of PVMs profiles were found in isolates from multi-strain infected farms’ is not clear. It seems to deal with strain diversity, but it is not clear by the wording used.

Discussion is too long and somewhat repetitive

Verb tense and plural/singulars forms need to be checked through the text.
Experimental design: One important aspect that the authors do not take into account when analyzing and discussing their data is the absence of non-virulent isolates in their collection. The collection is composed of only isolates from clinical samples. This is a major drawback of this study. Other studies that they use for comparison and discussion include non-clinical non-virulent isolates. This aspect need to be considered. In fact, the authors state in the introduction that H. parasuis can be commensal or pathogenic and it is important to differentiate between both status, but this is impossible if no commensal H. parasuis is included in the analysis. This absence of commensals could explain also the results with vtaA and OmpP2. This need to be taken into consideration in the discussion section, too. An extra information that could be included in the analysis is to investigate the clinical origin of lung isolates as coming from pneumonia cases or Glässer’s diseases cases. Sometimes lungs are used for diagnosis when the clinical picture is not barely a pneumonia, but systemic Glässer’s disease and this could be indicative of the invasive potential of the isolates.
Validity of the findings: Besides the comments about the isolate collection used in the study, some analysis on specific cases can be added to improve the discussion of the results:

Is the 16% of multi-strain infection associated with respiratory disease/isolation? This would be an interesting information, since it is considered that systemic disease is caused by a single strain.

It would be also very interesting to know if there are other pathogens involved in the clinical cases, especially in the cases when systemic isolates were found not to have putative virulence markers.
Additional comments: The title does not represent the content of the manuscript. The study shows similar results than previous studies on population structure of H. parasuis, and does not give new insights on clinical virulence.
This study reports the population structure obtained with IscR and the correlation of the clades with strain serovar and the presence of putative virulence markers. These data are interesting, but there are some important considerations that need to be addressed by the authors. (see above comments)

Section ‘Separation of clinical isolates into two deeply divergent groups by iscR-based phylogenetic  analysis’: the results presented here in relation to the amino acid substitutions are difficult to follow since in the beginning (Fig 2A) the comparison are made against the sequence from Nagasaki strain, and therefore the interpretation is completely different to what is written. The comparison to other bacteria (Fig 2B), which defines the substitutions, should be presented first.


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

Input:
Title: ASSOCIATION BETWEEN ISCR-BASED PHYLOGENY, SEROVARS AND POTENTIAL VIRULENCE MARKERS OF HAEMOPHILUS PARASUIS
Review round: 1
Reviewer: 2

Output:
Basic reporting: No comment
Experimental design: No comment
Validity of the findings: No comment
Additional comments: The manuscript Peer J 33889 describes an interesting study about the phylogeny and potential virulence markers of of Haemophilus parasuis, etiological agent of Glässer’s disease. Before being accepted some subjects must be revised:
•	The English language should be improved (especially verbs) to ensure that an international audience can clearly understand the text. Some examples where the language could be improved include lines 30, 33, 34, 35, 72, 88, 92, 110, 129, 180, 187, 192, 211, 245, 260, 277, 292, 313, 314, 336, 349, 370, 381 among others

•	Introduction section is too long and it must be shortened about one paragraph

•	References in the text must be ordered chronologically, not alphabetically

•	Line 101: “etc” cannot be used in this context. All sites from which samples were recovered must be cited

•	Line 102: The author must indicate CO2 conditions of incubation

•	Line 107: “Serovar” instead of “serova”

•	Line 221: “clade” instead of “calde”

•	Line 242: “respectively” must be deleted in this context

•	Line 269: “recently” cannot be used when one of the references is dated on 2010

•	Discussion section is too long and it must be shortened about one page

•	Line 369: What is the meaning of serovars 4, 5 et al.?

•	Line 378: “It shows that” must be deleted in this context


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

Input:
Title: ASSOCIATION BETWEEN ISCR-BASED PHYLOGENY, SEROVARS AND POTENTIAL VIRULENCE MARKERS OF HAEMOPHILUS PARASUIS
Review round: 2
Reviewer: 1

Output:
Basic reporting: Some improvement of the English would be appreciated

In some sections Vta gene is used, while the correct name of these genes is vtaA, and as for all gene names should be written in italics and lowercase. The same applies to other gene names.
Experimental design: The use of nasal cavity samples for diagnosis is not commonly used. Only samples from lesions should be considered clinical samples for Glässer’s disease. In fact figure 3 does not include this isolation site
Validity of the findings: The results are interesting and the conclusions and comparison to data from the literature are adequate
Additional comments: The manuscript has been 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: ASSOCIATION BETWEEN ISCR-BASED PHYLOGENY, SEROVARS AND POTENTIAL VIRULENCE MARKERS OF HAEMOPHILUS PARASUIS
Review round: 2
Reviewer: 2

Output:
Basic reporting: The authors have now corrected almost all the recommendations done in the first review of manuscript and, consequently, it should be accepted after these minor subjects are modified:
 Lines 53 and 54: References “Lawerence et al.” and “Brockmeier et al.” are not in an orthographic order
 Line 93: “And” must be added between “effusion” and “seroperitoneum”
 Line 175: “Data” (datum is singular, data is plural) instead of “datas”
 Line 182: “genes” instead of “gen”
 Line 256: “Our study was also consistent” instead of “Our study also consistent”
Experimental design: No comment
Validity of the findings: No comment
Additional comments: The authors have now corrected almost all the recommendations done in the first review of manuscript and, consequently, it should be accepted after these minor subjects are modified:
 Lines 53 and 54: References “Lawerence et al.” and “Brockmeier et al.” are not in an orthographic order
 Line 93: “And” must be added between “effusion” and “seroperitoneum”
 Line 175: “Data” (datum is singular, data is plural) instead of “datas”
 Line 182: “genes” instead of “gen”
 Line 256: “Our study was also consistent” instead of “Our study also consistent”


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

Input:
Title: THE HIGHLY TOXIC AND CRYPTOGENIC CLINGING JELLYFISH GONIONEMUS SP. (HYDROZOA, LIMNOMEDUSAE) ON THE SWEDISH WEST COAST
Review round: 1
Reviewer: 1

Output:
Basic reporting: Some parts are not enough citations.
Experimental design: Sampling data is not enough for estimate growth rate of Gonionemus sp.. If possible, the authors should add more data during all months when medusae appear.

And please add more detail of culturing methods (sizes of tanks, feeding frequency).
Validity of the findings: The authors should discuss why Gonionemus sp. appeared in Swedish waters. Water temperature is one of important trigger for medusa budding. See Kakinuma (1971).
Additional comments: The authors examined medusa growth rate and identified using morphology and molecular method with beautiful photos. I think the paper will be acceptable after minor revision but please consider growth rate and condition of medusa budding.


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

Input:
Title: THE HIGHLY TOXIC AND CRYPTOGENIC CLINGING JELLYFISH GONIONEMUS SP. (HYDROZOA, LIMNOMEDUSAE) ON THE SWEDISH WEST COAST
Review round: 1
Reviewer: 2

Output:
Basic reporting: This is an important contribution to the taxonomy, biogeography and the knowledge of sting-associated records of hydromedusae in general, and the genus Gonionemus in particular. The text is well written, concise and unambiguous, and the English is clear. The question is clear and well contextualized, especially considering the previous paper by Govindarajan et al. (2017), which have discussed the possible origins and scenarios of invasions of lineages within Gonionemus. The authors present detailed descriptions with relevant information on morphology and cnidome, which is accompanied by high quality, exceptionally clear figures. Methods are well-described and sufficient to address the question proposed. Raw data on COI haplotype sequences was supplied.
Experimental design: My only concern here is related to the absence of Table 1, which was not supplied with the submission. I am not sure if it was my mistake (I couldn’t find it), but a Table is cited in line 140. I believe it is very important that the authors include a Table describing the provenance of the material used in the analysis, identifying the ones used for morphological description, and the ones from which COI sequences were obtain. Additionally, I believe it is very important that the authors identify the haplotypes in this table, to make it easier for the reader to understand Figure 6 and the discussion that is related to it. Without a reference of a table with the description of the material, their locality and haplotype designation, it is very difficult to follow the discussion provided by the authors. I also suggest that the authors include the same information about the sequences from GenBank and from Govindarajan et al. (2017).
Validity of the findings: No comment
Additional comments: Another suggestion I have is related to the identification of the species Gonionemus sp. The authors provide such a detailed morphological description, with sequences from several localities, that I was wondering if it wouldn’t be possible to improve their contribution to the taxonomy and systematics of this group. Is it still not possible to refer to this species as G. murbachii? Or at least provide a more solid delimitation for this lineage? In my opinion, a discussion concerning the morphological diagnostic characters of the species and their relationship with other closely related species would be relevant.

Additional minor comments:

- Please correct the year of the paper Govindarajan et al. in the references section.
- Table 1 was cited in line 140 but was not provided with the submission.
- Please remove the additional tentacle in line 3 of the legend of Figure 3.
- Please include a comma after ric and before ring canal in the legend of Figure 3.
- In the legend of figure 3 the radial canal is referred as rac but in the figure it is referred as ra.
- Line 149: I suggest the authors replace the word the for in
- Lines 180-181: the authors describe the male gonad indicating Figures 3B and 3D, but in these figures, the gonads are identified as developing gonads (gd), and not as male gonads. I believe this needs more clarity.
- Legend of Supplementary Figure 6: remove the word in before A,B in line 4.
- Please exclude the word with in line 262.
- Line 291: this sentence is not entirely clear and needs modification.


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: TOWARDS AN IN-DEPTH CHARACTERIZATION OF SYMBIODINIACEAE IN TROPICAL GIANT CLAMS VIA METABARCODING OF POOLED MULTI-GENE AMPLICONS
Review round: 1
Reviewer: 1

Output:
Basic reporting: This article is very well written and the methods are explained in excellent detail. The structure is clearly defined and the key findings and developments are easily accessible. Raw data is publicly available.
Experimental design: This is predominantly a methods paper, that presents a technique for highly multiplexed NGS diversity analysis for Symbiodiniaceae targeting multiple genetic markers. I believe this is a good step in the right direction, since previous studies have tended to target only a single marker due to the prohibitive costs involved. The authors demonstrate that three markers with varying taxonomic resolution can be targeted for a similar cost by pooling samples before attaching Nextera tags. This will encourage labs with limited resources to expand their methods to include to multi-gene analysis, which will ultimately help us to better-understand the diversity and evolutionary history of this group.
Validity of the findings: Experimental controls were appropriate and the laboratory analysis appears to be thorough. The new LSU primers developed in this study will be of broad utility, since the amplicon shows good specificity and taxonomic resolution. This study took the conservative approach of only assigning taxonomy in cases where a 100% similarity match was found with a previously deposited sequence. Therefore the assignments can be considered robust.
Additional comments: This is a good contribution to the field, and I look forward to adopting these methods into my own research. Thank you for the opportunity to review.


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: TOWARDS AN IN-DEPTH CHARACTERIZATION OF SYMBIODINIACEAE IN TROPICAL GIANT CLAMS VIA METABARCODING OF POOLED MULTI-GENE AMPLICONS
Review round: 1
Reviewer: 2

Output:
Basic reporting: This manuscript reports a nice little case study exploring a very promising way to explore biodiversity in Symbiodiniaceae. It is clear, and a t least from a non native English speaker point of view the English is fine enough. The literature references and structure are good.
I did not check the Raw data, I only check that the authors mention the pathway to access the raw data in the manuscript and I trust the authors did not invent this information. I believe there is no need of specialist to check access to raw data, therefore the editor can do it well enough, and if PeerJ expects reviewers to check in depth raw data, they should consider seriously paying the reviewers for the time it will take (and not give 10 days to do it).

The paper is self-contained and completely relevant to its purpose.

For more details see the general comments to the authors.
Experimental design: This research is totally suitable for PeerJ (I would even think it can go to a better journal), its question is well defined and certainly will provide useful critical information for researchers not only studying giant clams but for all the coral reef research community and potentially beyond this field too (Symbiosis, eDNA fields etc...).
The authors clearly followed high standards in Symbiodiniaceae research, although some little glitches (domains outside of Symbiodiniaceae) appear here and there but nothing that cannot be modified/discussed easily.
Methods are described fine enough.

For more details see the general comments to the authors.
Validity of the findings: The findings are totally valid, the data robust and the conclusion relevant. I pointed at a number of minor issues in the general comments to the authors, but I believe none of these issues affect the fundamental findings of this research.
Additional comments: I really enjoyed your manuscript and I am looking forward both to see the next steps of this research (multiplexing, more complex assemblages) and to potentially apply this approach to some of our questions. And of course I look forward to see the final version of this manuscript published.

Good luck for the revisions!

L. 36 rationale (typo)

L. 51-52 I could not find Mercier and Hamel 1996, but I could also not find any other scientific report on how giant clams act as a shelter for a large number of orgnanisms. From my experience, they do indeed host a few organisms and their shell can be colonized by a variety of organisms, but I don’t think they are more of a shelter than any rubble (if we remove giant clams for the reef, will many organisms lose their shelter? Personally I think not and I would recommend to delete this statement, but if yes (I think some of the co-authors are more specialists of giant clams) then I would recommend to add some more accessible reference maybe.

L. 55 I am a bit surprised to see a reference to an article from a German aquarist magazine here. Did you really read this reference and are you sure it is appropriate? (Same comment overall for the first part of the introduction). In this specific case, I would rather recommend referring to one or few of the following: Romance and Grossman 1989 Palaios, Watanabe and Oba 1999 Journal of Geophysical Research: Oceans, Watanabe et al. Palaeogeography 2004 or other relevant papers as there are quite a lot of research done in this field.

L. 56-58 Maybe split this sentence in two? I am not sure how the “as a highly prized resource” relates to “the habitat degradation”. Actually the simple way may be to remove the “as” in the beginning of the sentence. Or split it, and in this case you could define a bit better the value of giant clams (i.e. as food and recently as jewelry in China for example). Actually, my personal feeling is that the economic value of the giant clam is much more important than their ecological role in the reef, maybe this part could be first in the paragraph (again I am no giant clam specialist).

L. 74 It is up to the authors but personally I am not sure Shinzato et al 2018 needs absolutely to be referred to. Nowadays any research can be published without meaning it is good research and Shinzato et al 2018 is a perfect example of combined poor methodology and overstatements (the title with “zoothantellae”already illustrates the attention given by authors, reviewers and editors to the manuscript, but aside from this typo there are I think enough approximations in this paper to encourage me to classify it in the category “papers to forget”). That being said, this is my personal opinion and I let you free to decide.

l.80 to 105: Personally I would be very interested to know why you selected this approach rather than the the Stat et al. 2017 method, is it impossible/difficult to adapt the fusion primer approach to multiple markers. I can imagine troubles at the creation of the first demultiplexing on the illumina machine, but if you thought about it already, it might be nice to explain it a bit so readers like me will save some time thinking how to use fusion approach with multiple markers. At the moment I did not find a solution, so I can easily imagine you have good reasons to use the Nextera approach…

L. 111 Archipelago? (Without “s”)

L. 164 this is again more a personal style comment, but I would remove the “unprecedented” in this sentence as it sounds a bit like you are a “carpet merchant” try to sell some products. “… provides speed and accuracy (…) without the need for time-consuming alignment steps.” Is a good enough reason to use this software and sounds more objective.

L. 181 Please indicate the BLASTn  parameters (at least e-value threshold) as it is important to assess the reliability of the BLAST assignment.

L. 208 According to your text, this is the raw read output. To me this seems a very low output for a 500-cycle MiSeq Reagent Kit v2.

L. 227 I am not sure to understand why your Symbiodiniaceae reference was so incomplete? Why were those 27 genotypes no in your database? Are they unreliable? I guess there is a good reason as it would be very easy to append your reference database, but I cannot understand it clearly based on the text. Maybe it is related to the next sentence, but please clarify (how many of the 11 retained sequences were in your database?). Similarly I am confused why, based on Fig.2 it seems your reference data base did not include C1 as this one appears to have been identified through NCBI…

L. 248 This is the only “red flag” I see in the manuscript. Both plants in the example (two North American/Canadian plants have nothing to do with Polynesian coral reefs and are obvious contaminations, and possibly misidentifications. While this does not absolutely affect the general findings of the manuscript, these findings indicates that the limit of reliability of the approach has been reached and probably this percentage of reads is just trash. And this should be considered and discussed. Even for an eDNA study I would be very suspicious to find  DNA of these plants in Polynesian waters, so the fact that it is present in Tridacna biopsies... Please, although it seems an increasing trend, don’t produce one of these metabarcoding paper where authors don’t know what are the organisms sequenced and blindly trust bioinformatics, especially as your manuscript is a very nice little study making its point quite well I think.

L. 269 related to the previous comment, please don’t tell me you are suggesting that terrestrial north American plants are host-associated organisms. I think it is just a misformulation or a misunderstanding from my part as the generation of scientists that cannot use its brain to interpret bioinformatic results is coming rapidly enough without our generation needing to show the example.

L. 277-278, I would remove the first part of the sentence as the money values are constantly changing and very country dependent (with the same methods, it is certainly not the same price per sample in Australia, USA, Japan or Indonesia). I would just state “The costs depend on the number…”

L. 283-288 While you are at the future perspectives, I would also suggest to list that this approach succeeded its first test in a low diversity setting (mainly A., with a bit of C. and a tiny bit of G.), the next step would be to test if higher diversity and more equal proportions in the samples will introduce more bias.


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: TOWARDS AN IN-DEPTH CHARACTERIZATION OF SYMBIODINIACEAE IN TROPICAL GIANT CLAMS VIA METABARCODING OF POOLED MULTI-GENE AMPLICONS
Review round: 1
Reviewer: 3

Output:
Basic reporting: Overall I thought this was a high quality paper. The introduction provided a clear justification for the research and I was able to follow all sections with ease. In particular, I found their descriptions of dual-index sequencing, and the novel contribution their study makes, very lucid.  The standard of writing was high, with very few errors (see General Comments). I was able to access all raw sequence files and it was simple to relate them back to the sample sites described in the paper.

However, I do think Figure 2 could be improved. I had to look at this figure for a long time before I understood all the information it was providing. I think the main problem is that the lines separating different sub-genera on the outer circle are very faint, which makes the clade designation labels and accession number arrows a lot more confusing than they need to be. For example, in the ITS2 circle, the line separating A3 and A6 on the outer ring is nearly impossible to see, and I cannot tell at all where C1 ends and the NCBI cluster (HG942434-AB294624) begins. Could these lines be made black or clearer in some other fashion in order to make the proportions of different sequences recovered more understandable?

Also, as an aside, I’m curious as to why the nearly all the Cladocopium sequences for the ITS2 region, and every Cladocopium sequence for the LSU, was identified with the NCBI database and not the in-house reference databases. Is this because the in-house databases were not particularly comprehensive? I think a comment from the authors to explain this would be useful here.

In addition, this is not necessary but I wonder if the authors would consider making Table S4 a primary table in the manuscript. I personally found it very compelling evidence that the pooled samples recovered very similar sequence proportions to the controls and went a long way to convincing me of the efficacy of this approach.
Experimental design: The work carried out was clearly justified, and suitable for beginning to answer the question they posed. However, given the novelty of this approach, I would note the following points:

I would strongly encourage/expect the authors to provide the full code they used in their bioinformatic pathway, as this would improve reproducibility and encourage other authors to utilise this approach.

An additional supplementary file regarding the design of the new primers (Line 119) would be useful, particularly how they were designed to avoid cladal bias, given the highly variable proportions of different genera recovered using the different markers for S147, S150 and S152 in Figure S2.
Validity of the findings: In general, their conclusions are justified based on the data they produced, and I am satisfied that they have provided proof-of-principle for this technique. However, I have two concerns that should be addressed further:

I think it is important for the authors to add a caveat regarding the decrease in sequencing depth with their approach. It is clear there are generally far fewer sequences recovered per pooled gene than in the control samples (Table 1), something that is not mentioned at all. While this doesn’t matter for their own study, it may have more of an impact for future authors who want to have a complete overview of fine-scale diversity, or study potential low-frequency intragenomic variants etc.

Lines 275-288: It may be a slightly sensitive subject, but given the emphasis that has been placed on “considerable cost saving”, beginning in the abstract and throughout the manuscript, I expected a more detailed and tangible demonstration of this (e.g. “using traditional dual-index HTS methods the sequencing we achieved would have cost $XX, but with the methods utilised in this study it cost $YY.”) Even if the authors do not want to mention specific costs, some relative measure would be useful for readers to assess the strongly promoted significant cost savings, and weigh it against potential negatives such as decreased sequencing depth (e.g. was this method twice as cheap? 1.4 times cheaper?). This seems to be one of the major benefits of this technique that the authors want to push, so I think it needs to be fleshed out slightly to justify the emphasis placed on it.
Additional comments: I have a few small comments that I believe would enhance the quality of the manuscript.

Line 36: ‘rational’ should be ‘rationale’

Lines 56-58: I know what the authors mean, but this sentence is a bit ambiguous, as it could imply that there are endosymbionts inside giant clams that are endangered (rather than the clams themselves). Could be improved with e.g. “… range, the family Tridacnidae also contains…”

Lines 84-91: This is a useful and concise summary – excellent.

Line 130: How was the size of amplicons assessed?

Line 164: I’m not sure about the use of the word ‘unprecedented’ as the only description of Kallisto. I don’t disagree with the authors, I just think an extra line on how it is unprecedented would aid readers who may be less familiar with the latest bioinformatic techniques/literature.

Lines 173-175: I am not very familiar with this approach, so it may be just a lack of knowledge on my behalf, but this sentence is not clear to me. Is it possible to expand slightly to better explain this analytical step?

Line 180: spelling mistake in Symbiodiniaceae

Lines 263-275: Could this discussion be added to regarding the results of Figure S2, which show markedly different proportions of symbiont genera recovered between the 3 gene regions for samples S147, S150 and S152? What are the implications for interpretation when this is the case?

Line 307: missing full stop

Line 310: missing italicization

Line 320-322: This sentence could be rewritten, particularly regarding the awkward use of ‘noteworthy’ at the start, and the double emphasis on those genotypes having not been previously reported (only one of “yet” and “before” is needed).

In the first sheet of Table S3, it is not clear what the column ‘EE’ is – can this be clarified?

It may be just the program I used to open it, but Figure S1 appeared to be cut off and I could only view a small portion of it (Figure S2 was fine though)

Regarding Table S1, I don’t think the presentation of date is self-evident; could signpost better in caption: “…date collected (Year-Month), …”

Regarding Table S2, a very tiny thing, but the ITSD_illu primer name is missing the underscore.


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: TOWARDS AN IN-DEPTH CHARACTERIZATION OF SYMBIODINIACEAE IN TROPICAL GIANT CLAMS VIA METABARCODING OF POOLED MULTI-GENE AMPLICONS
Review round: 2
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: Thank you for addressing all my comments and concerns. I think the manuscript is well improved and ready to go!

Cheers for the good work!


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

Input:
Title: TOWARDS AN IN-DEPTH CHARACTERIZATION OF SYMBIODINIACEAE IN TROPICAL GIANT CLAMS VIA METABARCODING OF POOLED MULTI-GENE AMPLICONS
Review round: 2
Reviewer: 2

Output:
Basic reporting: I am impressed with the efforts the authors have taken with their revisions of the manuscript; all my (reasonably minor) concerns have been addressed. This was already a high-quality paper and it is now especially so. It is unfortunate that the bioinformatic pathway could not be provided at this time, but I am happy with the suggestion the authors make regarding it in their rebuttal document. I am pleased to be able to recommend this paper for publication.
Experimental design: No additional comments
Validity of the findings: No additional comments
Additional comments: No additional comments


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

Input:
Title: THE INTERACTIVE EFFECT OF HERBIVORY, NUTRIENT ENRICHMENT AND MUCILAGE ON SHALLOW ROCKY MACROALGAL COMMUNITIES
Review round: 1
Reviewer: 1

Output:
Basic reporting: Your introduction needs more detail. I suggest a more in-depth look and representation of the literature on the specific effects of the factors you are testing in this study, especially mucilage. Some citations span ecosystems other than shallow rocky shores, which was good, but did not include some of the more relevant papers such as Smith et al. 2001, 2010 etc. Certain concepts were not explained in the introduction, including: the relationship between mucilage abundance and “blooms”.

I thank you for providing the raw data, however your supplemental files need more descriptive metadata identifiers to be useful to future readers, specifically an explanation of the Time, Unit, and Treatment codes is not included.

There is an unlabeled table on page 32. Is this a supplemental table? It needs a caption.

There seem to be no standard error bars on the inorganic P bars on Figure 1. Please add them, if they are not there.

The caption for Figure 2 indicates that the MDS plot should have black or white symbols with shapes around them but the actual figure only has labels.

Figure 3 does not have a legend for the bars’ color patterns, which should be associated with mucilage (M+ and M-).

In line 47 the (Lewis & Bryan 1941) citation is not necessary, as this is the abstract.

In lines 52-57, the responses are unclear. This section should explicitly discuss the results of the study.

Include a reference to the appropriate table or figure when discussing results (in the discussion section).

Line 106, please explain what you mean by “interact detrimentally”.

Line 112, the correct citation is (Lewis & Bryan 1941) within the text. Please correct the Silva citation on line 188 and the Thivy citation on line 190.

Line 131, please define C zone.

The sentence that spans line 157-159 belongs in the results section.

Please add the year for Huang & Boney study in line 250.

Lines 302-305 should be re-written for clarity.

According to the author guidelines, the acknowledgement section should not be used to acknowledge funders. This information will appear in a separate Funding Statement on the published paper.
Experimental design: Please include a map of the MPA to make visualization of the study area easier. In lines 134-137, you describe the experimental design. It would be helpful to include a panel in the map figure that shows a conceptual representation of the experimental design as well.

In line 157, the authors wrote that the “the concentrations of inorganic N and P (ammonia, nitrate, nitrite and phosphate) were estimated”. Please describe the chemical analyses, including the instrument(s) used for these analyses. The precision of the instrument’s measurements should also be included.

Were the plots surrounded by cages in order to exclude real herbivory, considering one of your treatments was artificial grazing? How did you control for herbivory after the artificial grazing treatment was imposed?
Validity of the findings: There is no evidence (in the form of a figure or table) for the results presented in lines 192-199. There is an unlabeled table on page 32, which may contain this information, but without a caption, it is impossible to tell what the information in the table is conveying.

It would be useful to have a table with the SIMPER results to show the percentages of the differences in assemblage associated with the dark filamentous algae, encrusting coralline algae, and macroalgae (lines 200-204).

The discussion in lines 214-217 on the long-term macroalgal response to disturbance needs more explanation. It is unclear that Table 2 has to do with the second sampling effort at T2 (line 217).

The discussion, with some grammatical and English language clean-up, does a good job of explaining your results in the context of some of the literature. If you could expand on the discussion of the importance or implications of the findings as it pertains to MPAs, the field of study, or management in the conclusion section, this would strengthen this section and make it relevant to a broader audience.

It is important to include a discussion of the limitations of the study in the discussion section (eg. small sample size, seasonal variance, etc.).

In the text of the results please discuss the quantitative enrichment of N and P in the nutrient treatments. N was enriched about 2 times the concentration of the water around the non-enriched plots, while P was enriched much more. Discuss how this may have affected your results, if at all.
Additional comments: I suggest the authors get editing help from someone with full professional proficiency in English.

A more in-depth discussion of the results is required with accompanying evidence in the form of relevant figures and tables.

I enjoyed reading your article. I learned a lot about mucilage and its interactive effects with nutrients and herbivory ☺.