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: HIGH TEMPO MUSIC PROLONGS HIGH INTENSITY EXERCISE
Review round: 1
Reviewer: 2

Output:
Basic reporting: Language is scientific but authors are advised to use specific terms as mentioned in comments. Sufficient Literature references and  field background/context provided by authors.
Corrections advised in Fig 1 A as it is incomplete.
Otherwise complete.
Experimental design: What make you decide music tempo 120 bpm was high for subjects ? Have you followed any classification? Kindly cite reference.
Please provide basic anthropometric characteristics of the study subjects if noted any.

Line 45- use more specific term for "as an external distracting stimulus during exercise" as Dissociation.

Line 55-56: The third explanation postulates that during continual submaximal activity, anindividual is predisposed to respond to rhythmical elements (music being one of many rhythmical patterns); cite reference.

Line 60-65:  The available evidence on this topic is congruent and demonstrates that music can and does   have a consistent and measurable effect on attention, the ability to trigger a range of emotions,  affect mood, increase work output, and encourage rhythmic movement (Atkinson et al. 2004;  Karageorghis 2008; Scherer 2004; Terry and Karageorghis 2011; Yamashita et al. 2006). The  ‘psychophysical’ effects primarily examine the perception of effort (Pandolf 1978), which in  almost all cases involves the Borg’s Ratings of Perceived Exertion scale (RPE).  Emphasis effect of music as mentioned  above—does it correlates with high intensity exercise as well or is just for mild / moderate intensity exercise only.

 Methodolgy:
Line92 Kindly state what do you mean by recreationally active individual ? State whether study subjects are trained / untrained/ ameteurs. Kindly give how much amount of physical activity per day/per week/per month  and how long were engaged before undergoing testing.

Line 97 – Any reference  given in observation section  regarding Physical Activity Readiness Questionnaire   from the Canadian Society for Exercise Physiology to determine physical activity level and to  screen for a history of cardiovascular, pulmonary, metabolic and orthopedic conditions.

Line 100 – informed and written consent was sought from study subjects.
Methodology section haven’t elaborated  the method for estimation of VO2max .
Similarly recording of basic parameters like HR, BP  at resting / baseline level were recorded in which position of body whether supine or sitting position.

LINE 108:
The test was terminated when the participants reached one of the following criteria: 1) volitional exhaustion, 2) RPE value ≥ 19, or 3) RPM ≤ 60. What was the maximumcadence value reached during testing session in both males and female?
Reason for having pretesting session for the study subjects. Kindly justify.

LINE 116 :
Upon exercise termination, four of the five pre-test measurements (minus blood pressure) were  repeated (See description below).  Why BP wasn’t taken into account post exercise.Do all subject undergo test session otn the very same day? How much was the study duration?What were the ambient conditions during high intensity exercise testing?

LINE 143:
Please elaborate –Purpose of visual feedback of pedalling rate (RPM)

LINE 144:
Does playlist implies same song been played for all participants or different song from the playlist ?

LINE 154 :
 During warm up parameters were noted or not?

LINE 158-159:
Were subject informed and explained about reporting of RPE score before initiating testing?

LINE 161:
Kindly explain “Active recovery” Why 2 miunutes time duration wass expected for active recovery with high intensity workout? Explain.
Exercise testing under both that is test and control condition for the same subjects were done on the same day or different days ? If done on same day how much interval was there in between two sessions.

LINE 211 :
What may be possible explaination for raised BL levelsin music condition than without music condition? Baseline/resting HR, BL levels weren’t compared with post exercise levels.

OBSERVATION:

Changes in BP levels wasn’t noted- kindly explain.
LINE 252: Does CNS regulation impacts exercise performance?  What could be putative factor which increase TTF in  high intensity exercise setup?

FIG 1 A No music condition in TTF graph isn’t represented correctly.

 It would  have been better if anthropometric data was also if  represented in graphical form .
Validity of the findings: No comment
Additional comments: Some corrections suggested with relevant explanation as asked in experimental designed from the authors .


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: HIGH TEMPO MUSIC PROLONGS HIGH INTENSITY EXERCISE
Review round: 1
Reviewer: 3

Output:
Basic reporting: Writing quality is mostly very good.
Literature references generally seem very appropriate, though perhaps more recent references can be added if available.
Structure of the paper seems appropriate.
Experimental design: Design is good.
I am unsure about the way in which percentage of time to completion was handled (see comments below).
Validity of the findings: Findings seem valid.
Additional comments: Comments

I very much like the idea behind the study and feel as though the content is timely and appropriate for the journal.

The manuscript is written well and is easy to follow.

The authors are to be commended for writing quality.

Abstract

Some mention of the sample seems appropriate.

Lines 34-36: Perhaps note the point in time when these differences were observed. Likely at or near the end of exercise but this should be stated more explicitly.

Line 38: Not stated is whether music continued during the recovery. This is important.

Introduction

Line 52: Using the word ‘somehow’ seems to beg for an explanation. Provide the explanation perhaps or edit the wording.

Line 65: A citation for the Borg scale should be provided.

Line 66: It seems odd to refer to the effect of music as a trend in the literature. Consider editing to create greater precision.

Line 76: The studies noted as more recent are not in fact more recent than the manuscript suggests. In the next statement you seem to suggest that more recent studies (06 and 09) now contradict conclusions made in the 2011 paper. My request here is that you more carefully consider the timing of publications and related statements so as to most accurately reflect the literature. And the following statement related to the 2011 publication suggests

Method

Line 106: The specifics of the cycle ergometer are described later in the manuscript but the mention here of self-selected cadence immediately left me wondering whether the bike was braked electronically or mechanically. If an adjustment here can be reasonably made please consider it.

Line 107: The language of this statement seems to suggest that the workload changes of the test are somehow specifically linked to VO2max determination. I of course know what you mean but more exacting language may be beneficial.

Line 109: It seems odd that the test would perhaps automatically terminate at an RPE of 19 when an RPE of 20 may have been achievable with additional time on the test. Also, it seems appropriate to provide justification and perhaps a citation for the stated termination criteria.

Line 110: The description of the experimental sessions leaves me somewhat unclear. The abstract notes the trials are ‘time to task failure’ but the methods describe a 4-minute test. This seems contradictory, or at least requires greater clarification.

General Note: The many uses of ‘4-min’ and ‘5-min’ seem inconsistent with traditional grammar/writing rules.

Line 126: The language ‘quotient of VCO2 on VO2’ seems awkward.

Line 129: Check spacing around gas percentages.

Line 138: The braking mechanism for the ergometer should be specified.

Line 141: Perhaps the procedure should be cited.

Line 160: Some justification of the work and recovery intensities and durations should be provided.

Line 161: The cycling protocol is described more clearly here than before, but edits to prior mentions of the protocol remain appropriate.

Line 161: Designation of the trial as being linked to ‘failure’ seems inappropriate if in fact the trial can be terminated by RPE values only, especially if a value of 19 out of 20 would be a reason to terminate the test. This seems like an important matter. I am curious how many trials ended because of RPE. If relatively few then perhaps those trials could be deleted from analyses.

Line 162: If I am understanding the design properly a participant who stops perhaps 15 seconds before the conclusion of one of the 4-min intense segments in comparison to a participant who makes it through those final 15 seconds and the recovery that follows and likely the first 60 plus seconds of the subsequent 4-minute interval would have dramatically different ‘time to failure’ but not necessarily very different capacities. Really, their capacities would likely be far more similar than the time difference would indicate. Some justification for the 4-min hard and 2-min easy approach to the protocol seems useful.

Lines 182 & 185: It is not clear to me how the 4 data collection points for the time variable would necessarily equate to specific percentages of completion because time to failure would not be known in advance of starting the trial.

Note: A justification of sample size should be provided.

Results

Line 199: This disclaimer should maybe be provided earlier in the manuscript because I was not thinking this was the case when considering the time figures in the previous sentence.

Line 205: Again, how would the time points be known in advance? That is, the 75% time mark would be different for a trial that lasted 8 mins in comparison to a trial that lasted 11 mins.

Line 215: The p-value and ES value seem rather incongruent. Some of the difference is likely linked to the small sample size limiting the likelihood of statistical significance but a .5 ES would seem likely to produce a more favorable p-value. Please consider a careful inspection of the data to seek a better understanding of why this may have occurred.

Line 217: Reporting actual HR values more in this section would be helpful.

Line 221: The reported HR difference in recovery are really large and almost unexplainable. Please consider working towards developing a defensible position on this difference.

General Note: I don’t think the literature review and introduction of the research idea provide a compelling case for why the Ve, TV, and Bf are included in the analyses. Reporting Ve seems more reasonable and appropriate as a marker of overall ventilator work but inclusion of TV and Bf in particular seem unnecessary without greater context and justification.

Discussion

Line 253: The lack of a significant difference in RPE is perhaps expected because music likely created a distraction that impacted ratings in a predictable manner.

Line 268: I am not an expert on cardiovascular control but it seems that the manuscript is suggesting that TV and Bf are under separate control mechanisms. If this is the case it seems more coverage of this topic is warranted in the manuscript than is currently provided, both to address these findings and more generally to help the reader better understand the context of the research design.

Line 277: The post-exercise HR values are very surprising, especially as it relates to the magnitude of the difference and because the music was removed immediately upon completion of exercise. My lack of expertise on neurophysiology of the heart limits my ability to properly evaluate this particular aspect of the design and related findings but I am drawn to the possibility that this finding should be explored a bit more.

Line 292: The word ‘effort’ may not be appropriate given that RPE is a dependent variable within the study. Maybe ‘physiological performance’ would be better.

Conclusions

Line 306: Perhaps also mention other dependent variables that did not differ, not just RPE.

References

Seem good and appropriate.

Figures

Figure 1: Check spelling of ‘measures’ at the top of the figure.

Figure 1: The notation of variable should include the ‘minus’ of the SD not just the plus.


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: HIGH TEMPO MUSIC PROLONGS HIGH INTENSITY EXERCISE
Review round: 2
Reviewer: 1

Output:
Basic reporting: no comment.
Experimental design: This is not an original study.
Validity of the findings: no comment.
Additional comments: Thank you for your revision.
I am still not convinced about why you compared ventilation, tidal volume, and RER, and not other respiratory parameters. Please try to justify them in your discussion.


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: HIGH TEMPO MUSIC PROLONGS HIGH INTENSITY EXERCISE
Review round: 2
Reviewer: 2

Output:
Basic reporting: All reference as suggested earlier are cited.
Explanations asked were submitted and found to be rational.
Earlier comment regarding Fig 1 A  withdrawn. Fig I1 A is appropriate.
Experimental design: Yes the article is within scope of the journal.
Research questions were defined properly.
Methods described are appropriate.
Validity of the findings: No comments
Additional comments: Research article can be accepted as the corrections and changes have been incorporated by the author with explainations.


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: HIGH TEMPO MUSIC PROLONGS HIGH INTENSITY EXERCISE
Review round: 2
Reviewer: 3

Output:
Basic reporting: Appropriate.
Experimental design: Reasonable.
Validity of the findings: Good.
Additional comments: The authors are commended for their excellent work in attending to the many comments provided by the various reviewers. Any remaining concerns on my part are not particularly substantive.


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: HDAC4 MEDIATES PERICHONDRAL OSSIFICATION AND PHARYNGEAL SKELETON DEVELOPMENT IN THE ZEBRAFISH
Review round: 1
Reviewer: 1

Output:
Basic reporting: In this manuscript, DeLaurier demonstrated that disruption of hdac4 caused increase of ossification in the Ch cartilages, with enhanced expression of runx2a and sp7. Also, maternal-zygotic mutants showed enhanced ossification than the zygotic only mutants. This study revealed a role of hdac4 in cartilage ossification. The manuscript is well written, but still needed further improvement.

Major issues:

1. In Fig3, The expression of hdac4 at 72hpf is very different from the previous reported paper (DeLaurier et al., 2012).  The in situ seems to be in bad quality, it is not convincing about the colocalization of sox9a.

hdac4 and sox9a are highly co-expressed in the bb and cb3-5. Are there any defects in these skeletal elements?

2. Is hdac4 highly expressed in the first arch? The MZ mutants show severe defects of 1st arch cartilages.

3. Are the zygotic hdac4 mutants showing any defects in the neurocranium?

4. In Fig 4, authors claimed that runx2a and sp7 were increased in the mutants. Authors need to quantify the changes and reveal the ratio of embryos showing the defects.

5. In Fig 5, MZ hdac4 mutants show more severe defects than the zygotic mutants. Authors should detect the bone marker runx2a, runx2b and sp7 expression in the MZ mutants.
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: HDAC4 MEDIATES PERICHONDRAL OSSIFICATION AND PHARYNGEAL SKELETON DEVELOPMENT IN THE ZEBRAFISH
Review round: 1
Reviewer: 2

Output:
Basic reporting: The manuscript is well written, follows an appropriate structure for reporting of scientific information and the figures are of extremely high quality to convey the information being presented. The information provided in the background and discussion is thorough and provides adequate context to interpret the results presented.

However, in general, the manuscript could benefit from some relatively minor modifications to improve the readability for a more general audience (outside the zebrafish/developmental/craniofacial/skeletal fields). Most notably, this includes clarifying methodologies used in each of the figures and double checking gene/protein nomenclature, specifically for cited mouse work. These suggestions are indicated with an asterisk (*) below.

Specific suggestions (in order of appearance) are as follows:
BR1. Line 145: Add “The” at start of the sentence so it reads, “The plasmid...”.

BR2. Line 204: would prefer use of hyosymplectic over hyomandibula (if they can be used interchangeably at this developmental stage) for consistency with main body of the text and figures. There are no other appearances of hyomandibula in the main body of the text.

BR3. Line 236 to 238: Reword the fish have “no discernible abnormalities” for clarity.  The fish appear normal to the eye, but a phenotype can certainly be discerned at this stage as demonstrated in Figure 2. The allele for which this statement is being made is also not specified (aik3, based on the Figure, but this should be mentioned in the main text).

*BR4. Line 244: briefly reference the method used to detect cartilage and ossifying elements. One sentence is sufficient. (For example: add “At 7dpf, flat mounted sections of fixed/fresh (?) tissue stained with... were analyzed for bone defects” etc.) At a minimum, include some description of staining and mounting or mention of the methodology used in the figure legend.

*BR5. Line 279: Again, a mention of the method used (in situ) to assay gene expression would be preferred in the main body, but at a minimum in the legend for Figure 3. This is especially important because zebrafish are still transparent at this stage (72hpf) and transgenic fish are often used to detect gene expression/localization. One phrase/sentence would be sufficient.

*BR6. Line 291 to 292: Double check nomenclature (specifically for mouse) for all genes/proteins to help clarify when talking about genes/transcripts or proteins for different species. Except in rare cases, mouse proteins should be in all caps. (Please check entire manuscript, however, other instances to check include lines 374, 459 and 461.

BR7. Line 299 to 312: Panels in Figure 4 are referred to out of order in the text. Suggestions:
- a general reference to Fig. 4 in line 299, or “(Fig. 4 A-H)”
- line 303, “(Fig. 4 A-D, indicated by arrows)”
- line 306, remove reference to figure
- line 307, “(Fig. 4 E-H, indicated by arrows)
- line 310, add “(Fig. 4 I-L, indicated by arrows)
- line 311, remove reference to figure
- add sentence at the end of the paragraph (~line 312) describing Fig. 4 M and N; for example: “A summary of the changes seen in runx2a, runx2b and sp7 are depicted in Fig. 4 M and N.” This also provides an opportunity to restate the findings of Fig. 4, if desired.

BR8. Line 321: would prefer something denoting the “enlarged quadrate” in Fig. 5B (arrow, asterisk, etc.) instead of, or in addition to, a reference to Fig. 2.

BR9. Line 474: check instance of mef2c-/-;mef2c+/-, missing a/b designations

BR10. Figure 2 legend: E’ there is only one arrow in the figure even though two are mentioned in the legend. Either omit reference to second “lower arrow” (not mentioned in the main text) or add arrow to the relevant important feature in figure. If adding a second arrow, would prefer use of “arrowhead” in place of “lower arrow” (or the other arrow, depending on relative location) to distinguish between the two shapes when/if arrow is added to figure so the reader can easily distinguish which element is being referenced as an “irregular border” vs. “spread of ossification to ventral aspect...”.

BR11. Figure 3 legend: In “A”, remove text, “red indicates bone”, as there is no red/bone in this schematic. Move this text to “I”. In “E” figure refers to ventral view, but legend says lateral. Also, see note BR5 about mentioning methodology used.
Experimental design: In general, the goal of the study is quite evident and the experiments used to assess the role of Hdac4 in zebrafish early larval perichondral ossification are appropriate and well-designed. There are a few major considerations, however, that I feel must be addressed by the authors prior to publication. These are as follows (in order of importance):

ED1. While the authors do a sufficient job convincing the reader that early ossification occurs in the Hdac4 mutant lines, it is difficult to assess the degree to which this is true. For example, in line 365 to 373, the comparison of mutants to more developed zebrafish is an important one. This point is well-made and the impact would be even greater if the authors could provide an image of a wild type, 12dpf (or similarly staged), fish prepared using their staining and tissue preparation method.
Again, in the discussion (see lines 406 to 408 and lines 410 to 412), it is difficult to assess the degree of precocious ossification alluded to in these statements without reference images using a similar method on older larvae. The craniofacial atlas referenced in Eames et al. 2013 shows some ossification of the ceratobranchial (cb) as early as 4dpf. If this statement is specific to a certain ceratobranchial bone (e.g. cb1/2 vs cb5) that distinction should be acknowledged. Similarly, the atlas denotes an anguloarticular bone (aa) is detectable as early as 6-8dpf (“Other bones visible here between 6 dpf and 8 dpf include the anguloarticular (aa) and quadrate (q) bones...”). Otherwise, the differences may be due to methodological or staging in these fish, which would be easily resolved by presenting comparison images of older larvae.

ED2. Somewhat related to ED1, the results presented in Fig. 2 and Fig. 5 (particularly Fig. 2B/C and Fig. 5G) would be easier to interpret and more impactful if data relating to developmental staging of these fish were available (e.g. swim bladder or eye size, see Parichy et al. Dev Dyn [2009] "Normal table of postembryonic zebrafish development: staging by externally visible anatomy of the living fish"). This would demonstrate that potential growth differences in these fish were considered (at a minimum) and/or controlled for (ideally), which seems relevant and especially important given that the phenotype in zygotic mutants is later described (lines 365 and 372 in the discussion) as appearing in a normal pattern as opposed to being ectopic.

ED3. The presentation of two CRISPR-generated alleles is a strength of the paper. However, it is unclear why some figures include data for both alleles, some for only aik2, and some for only aik3. A rationale of why certain allele(s) were chosen for each experiment (including the generation of maternal-zygotic mutants using the aik3 allele) would be appreciated.

ED4. In Figure 2, a more thorough explanation of criteria for classifying “mild” and “moderate” ossification patterns is needed. It is hard to distinguish the difference between the normal and “mild” phenotype shown in Fig. 1 D’ and Fig. 1 E’ (there also appears to be a small bone collar formation at the upper right area of ossification in D’ “the normal phenotype”, similarly sized to that designated by an asterisk in E’). Was the observer blinded during scoring? Were objective criteria used to classify patterns or subjective measures? Did scoring consider ossification of only the ceratohyal, the ceratohyal and the hyosymplectic, or both, or the entire tissue preparation?

Specific, minor considerations:
ED5. Line 190 to 192: A brief expansion of the histological methodology used would be welcome, as full-text to the reference cited is not widely accessible.

ED6. Line 258: Justification for normalizing to cartilage area should be included. If this is due to staging concerns, would another measure (independent of skeletal formation) be more reliable?

ED7. Line 316: A sentence describing the rationale for moving to maternal-zygotic mutants should be included.
Validity of the findings: Overall, the conclusions the authors make are supported by the data in the manuscript. A few changes relating to the rigor of statistical testing and the validity of statements made are below (in order of importance):

V1. Line 258 to 271: Justification for using a different statistical test on similar analyses should be given. If there is no justification, data should be re-analyzed using only ANOVA -or- ANCOVA.

V2. Line 355 to 359: Authors may need to adjust interpretation depending on revised statistical analysis.

V3. Line 344, and line 350 to 354: The prediction that the gene product for aik2/3 is a truncated protein is unfounded. The presence of amplified cDNA is not sufficient to suppose that a protein product is generated, or that the RNA is not subjected to NMD. Quantitative real-time PCR would be a much more reasonable experiment to assess relative cDNA expression in WT, hets and mutants for each allele. Of course, Western blots and/or immunostaining against an N-terminal portion of Hdac4 would be required to conclusively make this statement/prediction although antibodies for Hdac4 may not be readily available for zebrafish. It would be preferable to support this prediction with additional experiments or to remove these statements from the manuscript.

V4. Line 269: typo in the p-value, cannot be greater than 1.0
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: HDAC4 MEDIATES PERICHONDRAL OSSIFICATION AND PHARYNGEAL SKELETON DEVELOPMENT IN THE ZEBRAFISH
Review round: 1
Reviewer: 3

Output:
Basic reporting: The micrographs are of high quality and overall the imaging and figures are very nice.

The manuscript is clearly written for the most part. However, there are numerous examples of incorrect gene nomenclature usage. For example, on line 368 the authors refer to the mouse Hdac4 gene and it is italicized, on line 374 the authors discuss Hdac4 and Runx2 mouse genes and neither are italicized. There are numerous such incorrect and inconsistent nomenclature usages like on line 461 the zebrafish gene mef2ca is capitalized and it should be all lower case.

There are numerous statements that require references. For example the sentences ending on lines 56, 61, 64 and others need references.

The Mef2c gene is “myocyte enhancer factor 2c” NOT “myocyte enhancing factor 2c”

In my opinion, “intercross” should be used rather than “in-cross”.

Stylistically, the authors could improve the readability by avoiding the passive voice throughout.
Experimental design: The manuscript is within the aims and scope of the journal. Examining the functional role of hdac4 in zebrafish development with a genetic mutant is meaningful especially since the only loss of function phenotype reported thus far is a morpholino-based study which often do not phenocopy germline mutations. The methods are sufficiently described.
Validity of the findings: To strengthen the manuscript to "pass" the following points need to be addressed.

In order of importance:

1. The ossification phenotype in the zygotic mutants (Fig 2D-F) is very subtle. Moreover, the range of ossification reported to be due to hdac4 mutation can be observed in wild type animals at this stage (7dpf). In light of these facts, extra careful quantification is required. Specifically, the authors need to quantify bone blind to hdac4 genotype.  Perhaps using the intrinsic fluorescence of Alizarin red in whole mount larvae from a heterozygous intercross prior to genotyping will speed analyses.  Quantifying ossification BEFORE genotyping then determining if the ossification phenotype segregates with hdac4 genotype will lead to a much more convincing demonstration that hdac4 mediates perichondral ossification as the authors propose.

2. An additional concern with the data in Figure 2 is that the “moderate” phenotype in Fig 2F looks like an overall developmentally advanced individual based on a more developed branchiostegal ray set, a more well-developed interopercle and larger cartilage elements (eg palatoquadrate) than those shown in the control or “mild” in D and E respectively. These observations imply that the ‘moderate’ phenotype is an overall advanced individual rather than a phenotype specific to a certain bony element. This sort of stage variation is common even in wild-type larvae. Indeed, the authors report some wild type individuals with the ‘mild’ phenotype in their raw data, weakening the claim that this phenotype is due to hdac4 mutant genotype. Even in the MZ mutants the ossification phenotype appears to be within the normal range of ossification variation that is observed in wild-type animals.

3. Similarly, the subtle differences in runx2a and sp7 expression in figures 4B,D and 4J,L are within the variation seen among wild types. If the authors wish to make the point that these subtle expression differences are meaningful they should be backed up with quantitative analyses like qPCR. At a minimum, the fluorescence in situ signal from many individuals should be quantified BEFORE genotyping to determine if the in situ signal phenotype segregates with hdac4 genotype.

4. In Fig  5, the loss of neurocrania and first arch structures while the second arch appears completely wild type is both fascinating and convincing. This phenotype should be the centerpiece of this study. The low penetrance of such a severe phenotype is also very interesting and worth study. This manuscript would be dramatically strengthened by focusing on understanding the loss of arch structure phenotype, rather than the subtle ossification phenotype. Do the NCCs migrate normally? Is Arch morphology and size normal? Are there arch morphology differences between arch 1 and 2? Are patterning genes only disrupted in arch1 while arch 2 is wild type?

5. Does the MZ phenotype (Fig. 5) match the previously reported MO phenotype? It is difficult to imagine that the previous MO study was blocking maternally deposited transcript because splice-blocking MO were used (DeLaurier 2012). The similarities and differences between the MO and MZ mutant should be clearly fleshed out. An interesting experiment to better understand this phenotype an how it contrasts with the previously reported MO phenotype would be to inject the MO into MZ mutants.

6. It is certainly important to consider how hdac4 may be functioning during craniofacial development, i.e. through what target genes and interactions. However, this manuscript contains no evidence whatsoever that hdac4 functions via mef2c. Without any data, the mef2c/hdac4 interaction in this system is purely speculative and therefore any reference to an hdac/mef2 interaction should be relegated to the discussion and clearly stated as speculative.  ie, removed from the 'results section' and the abstract. For example, the authors state their hypothesis in the abstract that “mutant larvae have an increase of expression of runx2a and sp7 in the ceratohyal cartilage, which we hypothesize is due to the de-repression of Mef2c through loss of Hdac4.” But then they do not test this hypothesis at all.

7. It is curious that the authors report an ossification phenotype in the hdac4 heterozygotes. Is there any reason to suspect that the alleles are dominant? Dominance needs to be addressed. If the authors suspect haploinsufficiency then this should be stated.

8. The authors argue that maternally deposited hdac4 functions in skeletal development. They could formally demonstrate that hdac4 mRNA is maternally deposited if they were to examine the amplified cDNA sequences in zygotic mutants obtained from heterozygous intercrosses. If any wild type sequences were found in cDNA generated from homozygous mutants they would unambiguously demonstrate that wild type hdac4 mRNA is maternally contributed in zygotic hdac4 mutatns. The authors already have the required PCR products to test this hypothesis (1E,F)
Additional comments: Clean mutagenesis, including multiple alleles, leading to an interesting craniofacial phenotype  (5 J-M) make this project an important contribution to our understanding of craniofacial development. I strongly suggest shifting the focus of this work to the most robust (and unusual) phenotype rather than the subtle increase in ossification. At a minimum the ossification phenotype needs to be much more carefully quantified as described in the ‘validity of the findings’ section above.

Please find a marked up PDF with some candid comments that may help the authors edit this manuscript. Many of the comments are redundant with those in the review but some additional frank (non proofread) comments are provided which I hope will be helpful.


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: HDAC4 MEDIATES PERICHONDRAL OSSIFICATION AND PHARYNGEAL SKELETON DEVELOPMENT IN THE ZEBRAFISH
Review round: 2
Reviewer: 1

Output:
Basic reporting: Authors have well addressed my questions. I have no more concerns.
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: HDAC4 MEDIATES PERICHONDRAL OSSIFICATION AND PHARYNGEAL SKELETON DEVELOPMENT IN THE ZEBRAFISH
Review round: 2
Reviewer: 2

Output:
Basic reporting: All changes made by the authors are satisfactory.
Experimental design: The authors have made changes that significantly improve the experimental design criteria of this study. The addition of a 12dpf wild-type larvae in Fig. 5B adds a critical point of reference and the authors comments in the rebuttal letter about imaging methodologies used appropriately address the discrepancy between their study/results and those of Eames et al. In addition, the new scoring criteria in Fig. 2 are an improvement from the previous submission.
Validity of the findings: 1. Justification for use of appropriate statistical tests for data in Fig. 2 has been adequately described.

2. The only change requested that has not been addressed by the authors surrounds the interpretation of RT-PCR data in the context of nonsense-mediated decay (NMD).

The authors provided a description of the efforts to detect Hdac4 in their tissue samples using available antibodies. While these efforts are appreciated, it is unclear why the authors did not pursue quantitative real-time pcr of cDNA samples, which may have been more fruitful and less work than attempting the antibody approach. Similar band intensities resultant from RT-PCR amplification to saturation is not adequate to make an implication about the presence or absence of NMD. (Even if it were, the data in Fig. 1 is from adult fin clips, which may or may not reflect NMD occurring in larval skeletal tissues.) In my experience, RNAs subjected to up to 90% NMD will still amplify relatively "normally" during RT-PCR, especially if transcripts are abundant in the tissues processed, and the degree to which a transcript may be subjected to NMD may also be tissue dependent.

For these reasons, in addition to the lack of protein data or quantitative RNA analysis, removal of the suggestion that the aik2 and aik3 alleles are not subjected to NMD (or genetic compensation) in the Discussion secton (Page 21, L824-828 of tracked-changes word doc) is warranted. Further, the authors seemingly contradict this implication later in the discussion (Page 21, L842-846 of tracked-changes word doc) by referring to the "haploinsufficient" phenotype (which often results from NMD). The mutant allele may be non-functional, hypomorphic, or even produce a dominant-negative effect manifested by aberrant protein products. The authors have no data to distinguish between these outcomes, and this should be acknowledged more openly in the discussion. This is particularly true given the slightly different phenotypes manifested by the aik2 and aik3 alleles.
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: HDAC4 MEDIATES PERICHONDRAL OSSIFICATION AND PHARYNGEAL SKELETON DEVELOPMENT IN THE ZEBRAFISH
Review round: 2
Reviewer: 3

Output:
Basic reporting: While the manuscript is well written overall, I am still not certain that appropriate, consistent gene/gene product nomenclature is used throughout. I defer to the editor on this matter but, for example, line 101 “… cytoplasm, MEF2C becomes unbound to Hdac4, and can activate…”  I’m not clear why MEF2C is all caps while Hdac4 only has the first letter capitalized when talking about these two gene products in the same context. I strongly suggest further careful editing to ensure consistent capitalization, and italics are used according to the nomenclature guidelines in zfin/MGI etc.

Similarly, when referring to the zebrafish hdac4 alleles the authors should only say the allele designation once (hdac4^aik3), i.e. reserve the hdac4^aik3/aik3 designation for cases where you are specifically referring to homozygous mutants. This confusing usage is throughout the manuscript. Lastly, use the complete allele name when referring to an allele hdac4^aik3 not just aik3.

The authors should carefully edit to find all cases with the above mis usages and strive for consistency and adherence to convention.

Lines 561-4 “the proportion of approximately one quarter of maternal-zygotic mutants with defects suggests there may effects of other unknown alleles influencing the phenotypic outcome of loss or reductions of hdac4 on neural crest development.” It is confusing to use the word alleles here. To me this implies that there are hdac4 alleles in the background that modify the phenotype. A better word might be unknown loci, or modifiers, or genes.

The text edits in this revision significantly help clarity and readability.
Experimental design: The authors have taken appropriate measures to clearly describe how the different phenotypes are scored. Importantly, given the subtlety of the phenotypes, the blind scoring strategy as described is a crucial improvement.
Validity of the findings: The precocious ossification phenotype is subtle, making it difficult to measure. That said, the improved, blind, scoring strategy and statistics help in this regard and the increased ossification of the ch appears to be valid by these measurements.

The inclusion of Fig. 5B a wild type at 12 dpf is included to make the claim that the MZ mutants resemble the 12 dpf wild types. This image begs the question, what do the MZ or zygotic mutants look like at 12 dpf? Is the precocious ossification still detectable? Quantifying the phenotype at the 12 dpf stage would strengthen the overall claim that ossification is sped up in hdac4 mutants.

The in situ results (Figure 4 A-H, increased runx2 expression) remain particularly subtle and would be greatly strengthened by qPCR. That said, the authors have gleaned all they can from these in situ findings and according to their scoring approach reach valid conclusions.
Additional comments: It is interesting that the authors do not recover homozygous hdac4^aik2 adults. The two alleles are quite similar so I’m not sure why one would be lethal and the other viable. Assuming the backgrounds are identical, this is curious and if the authors have any speculation as to differences between these alleles it might be useful to include in the discussion.

This nice manuscript is an important contribution, especially as an addendum to the MO phenotype that might mislead researchers as to the role of hdac4 in craniofacial development. I look forward to a careful characterization of the severe loss of craniofacial structure phenotype in future studies. It is striking and I agree that the similarities to the fscn1a MZ phenotype are considerable, perhaps offering a lead-in to some novel genetic interactions controlling craniofacial development.


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: HARDLY HABITUAL: CHIMPANZEES AND GORILLAS SHOW FLEXIBILITY IN THEIR MOTOR RESPONSES WHEN PRESENTED WITH A CAUSALLY-CLEAR TASK
Review round: 1
Reviewer: 1

Output:
Basic reporting: The manuscript is very clearly structured and the logic of the arguments is easy to follow. The language is clear, unambiguous, and professional throughout. I commend the authors for sharing an example video file and for producing very clear and helpful figures. The authors refer to the relevant prior literature appropriately.

Background information:

1. The two introductory paragraphs introducing current evidence on behavioural flexibility and conservatism in chimpanzees from both experimental and field studies are very well written, they are detailed and refer to the relevant literature, but are still concise. A minor point here: Currently these first two paragraphs are somewhat unbalanced given that several examples for behavioural flexibility are given in paragraph 1, but no examples for conservatism in paragraph 2. I suggest to divide the sentence from lines 56-60 into two (cut after “Harrison & Whiten, 2018)”) and to insert a sentence or two between these sentences to provide examples (ideally one from captivity and one from the wild).

2a. I have a more important point regarding the third paragraph (lines 69-88). While I find the authors’ suggestion of causal information and habit formation as possible drivers of chimpanzees’ conservatism highly interesting, I would like to encourage them to expand on their theory a bit more, especially given that the theory seems to be novel: The authors say that chimpanzees’ “ability to adopt novel strategies may be inhibited when they receive limited/no causal information about a task or its potential solutions” (lines 72/73). It is somewhat unclear what “novel strategies” means here. Do the authors mean (as I suspect) the more efficient solutions to an “old” task for which one has already learned a solution (thus describing the switch from an old to a more efficient solution) or do they mean any novel solution to a task (i.e., both more efficient solutions to old tasks for which a solution already exists as well as solutions to tasks that have not been encountered before, see the ill-structured tasks as described in Chappell et al., 2013)? This should be clarified, possibly by changing “novel strategies” to “more efficient strategies”.

2b. Second, the authors speculate that “[p]erhaps when chimpanzees cannot discern the causal influences of their actions, they perpetuate them, even when task demands change.” (lines 74/75). Could the authors provide an example from the literature where they think this could have been the case? If they have provided some examples of chimpanzee conservatism in the introduction (see comment above), they could simply refer to one of the examples here.
Furthermore, does this explanation also hold for studies in which chimpanzees are shown a more efficient strategy by a demonstrator? That is, couldn’t one argue that a demonstration by a conspecific already provides the individual with the necessary causal information to solve the task and that it is something else than a lack of causal information that makes chimpanzees fail to adopt a more efficient strategy? A brief statement on the authors’ stance here would help clarify the scope of the theory.

2c. The authors then introduce the concept of habitual responses; again, while the suggestion of a role of habit formation for chimpanzees’ conservatism is intriguing, more information in this paragraph is needed to clarify the assumed theoretical links between habit formation, causal understanding, and conservatism. First, the authors quote Köhler in saying that “chimpanzees’ failure to solve novel tasks” can be due to “crude stupidities arising from habit” (lines 76/77). What I find a bit confusing here (see comment above) is what exactly is meant by “novel tasks”. As far as I understand Köhler, he was describing cases in which chimpanzees bring to a novel task a solution that has worked well repeatedly in a different context, whereas most current experimental studies on chimpanzees’ conservatism seem to examine these animals’ ability to invent a novel, more efficient solution to an old task. In how far are these cases comparable? Is one more the case of previous experience influencing the innovation of a task solution whereas the other describes the possibility of habits that were formed on a task to perpetuate despite changes in this task’s demands?

2d. More importantly, however, the authors need to make clearer how they see habitual responses, causal understanding, and conservatism relate to each other. Are the formation of habits and the lack of causal information two partially independent factors affecting conservatism (with more causal information reducing the likelihood of habit formation), are they mutually exclusive or orthogonal concepts? While I don’t expect the authors to come up with a fully fledged theory, providing some more information would nevertheless be very helpful, as it is unclear throughout the manuscript how exactly these concepts are assumed to interrelate.

Minor issues:

1. Introduction, lines 84-88: You mention the Horner & Whiten (2005) study as support for your suggestion that habit formation might result in conservatism. I struggle to see where the chimpanzees in this study showed perseveration in solving an opaque box or formed a habit in the first place. The authors report that “When subjects […] transferred from the clear box to the opaque box, although there was a slight increase in the reproduction of irrelevant actions, this was not significant” (p. 170); therefore, in my understanding, chimpanzees did not seem to perseverate on the irrelevant actions. In addition, chimps who were presented with the opaque box first did reproduce more of the irrelevant actions, but dropped most of them once they were presented with the clear box. To me, chimpanzees in this condition did not perseverate on the irrelevant actions either. I suggest that you either elaborate on which result from that study you interpret as habit formation and/or perseveration or that you choose a different study that supports better your suggestion that habit formation might result in conservatism.

2. In the abstract, it is written that it was “investigated if this conservatism is due to the perseveration with a specific solution or if it could be attributed to the development of habitual motor responses”. It does not become clear what is meant with perseveration with a specific solution – how is this different from conservatism? What seems to be missing here is perseveration due to what (possibly a more cognitive explanation?).

3. Very minor grammar point: line 276: remove hyphen in “commonly-used”?
Experimental design: The research is in conformity with the ethical standards in the field; the investigation and analysis was rigorous and was conducted to a high ethical standard. Methods are described in detail and allow replication of the study by an independent researcher. However, I have three points here:

1. Coming back to the paper’s slight vagueness of how habitual motor responses, causal understanding, and conservatism interrelate, this vagueness seems to continue in the formulation of the research question. In the abstract, the authors state that they investigated if chimpanzees’ conservatism “could be attributed to the development of habitual motor responses when repeatedly solving a task”. This might suggest to the reader that the authors manipulated the formation of habitual motor responses in chimpanzees to see whether habit formation had an effect on conservatism. However, in the introduction (lines 90-95), the question is formulated differently, namely whether chimpanzees would form habitual motor patterns in the first place. This is a different (albeit not less interesting) research question. I urge the authors to change the respective part in the abstract and any other places in the manuscript to keep the formulation of this research questions consistent throughout. This is especially important because the validity of the task can only be assessed in the light of the question (i.e., if the aim had been to manipulate the formation of a habitual motor response, the experimental manipulation would have failed as the subjects showed a variety of strategies rather than habits).

2. The authors also mention that prior to their study, the formation of habitual motor responses had not been studied in chimpanzees nor gorillas. Given that knowledge about habit formation in chimpanzees is so limited, the authors could provide more information in the introduction as to why they have come to consider habitual motor patterns as a possible reason for chimpanzees’ conservatism. While some background information is given from lines 75-83, the introduction mainly focuses on the causal transparency/opacity aspect of a task; a couple of more sentences on what we (not yet) know about habit formation in chimpanzees would be useful, in order to be better able to locate the paper in the current literature and to clearly identify the current knowledge gaps. The authors could also consider moving the sentence from lines 102/103 to the previous paragraph.

3. A second research question is given in the introduction which is missing in the abstract, namely whether causal information can prevent conservatism. Again, this second question should be added to the abstract, as it seems to be a crucial question.
It does not become clear how the two research questions (or better: the concepts described in them) relate to each other. As suggested above, the authors should provide more information about the relationship between causal understanding, habits, and conservatism in the introduction.
Validity of the findings: The data have been provided. The data seem robust and all analyses to be statistically sound.

1a. I commend the authors for mentioning a possible limitation of their study, namely that the apes might not have gotten long enough exposure in order to form habits (lines 424-426). To follow up on this, might it not be possible that the Ape-plunk task is particularly unsuited for studying habit formation as it allows for such a variety of task solutions? Might the task actually be especially discouraging by design with regard to the formation of habits, and thus not only be less suited to study the research question as to whether apes form habits but also lack comparability to previously used tasks studying conservatism in apes? I suggest that the authors discuss the specific nature of the task they used.

1b. Somewhat related, the authors set out to investigate whether causal understanding of a task would prevent apes from becoming conservative; and while the data quite convincingly show that the authors succeeded in creating a task that was causally transparent for the apes, the Ape-plunk task also differs from previously used tasks in a second way, namely that it allows for a variety of strategies to be successful (24 in phase 1, 12 in phase 2). Causal transparency thus seems to be conflated with the number of strategies to be employed in phase 1. Therefore, can the authors really draw the conclusion that what prevented the apes from becoming conservative was the causal understanding of the task rather than the fact that they could use different solutions throughout the test and so were not “forced” by the task design to habitually use one or a more limited number of solutions? This question should also be addressed.

2. A point related to comparability of this study with previous ones: maybe a brief note should be made in the sentence from lines 427-428 (which states that in the Davis et al. (2016) study conservatism was found only after 20 trials) that the Davis et al. study did not allow for such a great variety of solutions.

3. Line 358-360: The authors suggest that the lack of habit formation might be explained by the apes’ causal understanding of the task. What about the possibility that there was a significant memory component involved, i.e., that apes simply forgot which specific strategy they had used in the previous trial? This memory demand becomes more pronounced when one considers the fact that the test was administered over several sessions. I would like to hear the authors’ opinion about this possibility.

Minor point: Discussion, line 322. The authors state “This suggests that there was no relationship between consistency of action sequences (habit formation) used in the first phase and ability to employ efficient action sequences when contingencies changed (flexibility).” I doubt whether the use of the term habit formation is warranted here as no habit formation was observed in phase 1. So wile it is valid to say that no relationship between consistency of action sequences in phase 1 and phase 2 was found, it is probably not valid to conclude that there is no relationship between habit formation and consistency of actions in phase 2 – as habit formation was not induced in this experiment.
Additional comments: This  is  a  well-written, clear, and concise paper aiming to investigate the roles of causal understanding and habitual motor responses for chimpanzees’ conservatism in response to changing task demands. The authors contribute to the current debate by theorizing that chimpanzees’ conservatism found in several previous studies could result from the apes’ lack of causal understanding of the tasks. However, if the causal links between actions and the effects on the task are understood, the subject might be better able to evaluate the efficiencies of different actions and to switch to a more efficient strategy when task demands change. In order to investigate whether causal understanding of a task will prevent conservatism, the authors use a clever, new task, designed to be as causally transparent as possible. Indeed, the data suggest that the Ape-plunk task seems to be sufficiently causally transparent to the apes, with a large majority of the chimpanzees and gorillas solving the task efficiently from the first trial. The study also nicely demonstrates the apes’ flexibility in response to changing task demands. While there are no “uncorrectable issues” in the manuscript, some additional work should be done to elaborate on the suggested theory, especially how habit formation is thought to interplay with causal understanding and conservatism, as well as to clarify the research question regarding habit formation (i.e., is the question whether the apes form habitual motor responses or whether habitual motor responses affect flexibility?). Related to this, the authors should also discuss whether their task – whilst being a nice and clear problem-solving task which will likely be used in many future studies – is suited to address the question of the role of habit formation, as the variety of possible efficient solutions might heavily counteract the formation of habits. Some more work is also suggested on the discussion section, regarding the conclusions one can draw from the study. It remains unclear whether the apes’ lack of conservatism is due to the task’s causal transparency or the variety of possible solutions – these two aspects seem to be somewhat conflated in the task design. As the Ape-plunk task differs from previous studies not only in its causal transparency but also in the number of solutions possible, the authors should also discuss a bit more how their study relates to others.


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: HARDLY HABITUAL: CHIMPANZEES AND GORILLAS SHOW FLEXIBILITY IN THEIR MOTOR RESPONSES WHEN PRESENTED WITH A CAUSALLY-CLEAR TASK
Review round: 1
Reviewer: 2

Output:
Basic reporting: Literature references are sufficient.
Structure is correct.
Figures are relevant. Raw data are made available.
The work is self-contained with relevant results for hypotheses.
Experimental design: Research question is well defined. The gap in knowledge being investigated is clear.
Work was conducted rigorously up to a high technical and ethical standard.
Experimental design was well defined and sufficient for replication.
Validity of the findings: Data are robust, statistics are sound, but see below under general comments.
Conclusions are well stated and address the original research question. Conclusions are supported by results.
Additional comments: The manuscript is well written and reports some interesting results. Chimpanzees and gorillas quickly learned the task in phase 1 and used flexible responding when switched to the modified task in phase 2. The unexplainable top-down/bottom up species difference in phase 2 between chimpanzees and gorillas is intriguing. I have a few comments, mostly about the statistics

The authors do not state what the error bars represent in Figure 5. If they are SE, then it looks like there is a significant difference in Phase 1 because the chimpanzee and gorilla bars do not overlap. However, this rule of thumb only applies to a parametric test. Using a nonparametric test (Wilcoxon rank-sum test) the result did not reach statistical significance (p = 0.07; Lines 271-273). Since the authors used a nonparametric test, which compares medians, they should report the results using medians, not averages and SE, perhaps with boxplots. This is also true of any other tests in which averages are compared and reported using nonparametric tests.

If the authors really wish to report means (averages) as they do throughout results, they might check for assumption violations and use a parametric test (independent and paired t). If the authors are going to report averages they should also include standard deviations or SE each time (e.g., Line 272).

The authors state that the gorillas used the bottom up sequence (1, 2, 3, 4) in 26.6% of trials in phase one and that this was not significantly different than the chimpanzees, however they do not state the chimpanzees’ percentage (lines 281 – 284).

The authors report a p = 0.05 result as “did not differ” (Lines 326-327). Most hypothesis testing uses p ≤ 0.05 as the cutoff, so perhaps that should be reported as difference in run length between the phases?

Some of the gorillas seemed to be using a predominantly 1, 2, 3, 4 sequence in phase 1. If that was the case, the result might influence Discussion. The authors already state that the chimpanzees might have been using a simple associative rule “pull out the straw on which the reward is resting” to solve the phase 2 task without necessarily using cognitive flexibility (Lines 380 – 385). If a gorilla was using a bottom up strategy in phase 1, they would not need to make any cognitive flexibility adjustments moving from phase 1 to phase 2 to be successful. Perhaps this could be mentioned somewhere in Discussion.

Minor suggestions
Line 71: Add “cues” after causally-relevant.
Line 351: ‘a lot” is a bit colloquial; ‘a wide range of variation”?


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: OCEAN ACIDIFICATION ALTERS MORPHOLOGY OF ALL OTOLITH TYPES IN CLARK’S ANEMONEFISH (AMPHIPRION CLARKII)
Review round: 1
Reviewer: 1

Output:
Basic reporting: This paper reports the effect of ocean acidification on otolith development in larval clownfish. This study includes SEM analysis in addition to otolith parameters usual measured which makes it one on the most in depth studies looking at ocean acidification effects on otolith development.

The MS is very well written and crafted. Its contains all the appropriate references. The raw data has been shared but in the form of mean tank values. Raw data should be provided for each individual fish. Figures and table are clear.

I don't understand why you present the survival, settlement and growth data first in the results and discussion. The focus of this paper is otoliths so lead with this.
Experimental design: The experimental design and methods appear good, however, there is some vital information missing with regards to the brood-stock which make it difficult to fully assess the strength of the experimental design.

1.	You need to describe the history of the brood-stock pairs, were they wild caught, or lab bred (if lab bred what generation)?
2.	Need information on how many pairs and clutches were used in the experiment and how the clutches were split between tanks, so the reader has an idea of how much genetic diversity was in the experiment. Its also difficult to assess the strength of the experimental design without this info.
3.	When reading the experimental design section it sounds like you had 12 tanks and ran the experiment only once but looking at your supplementary data file I can see you ran it 7 times? This needs to be made clear. Where the same pairs used each time? Addressing point 2 should clear this up.
Validity of the findings: I have an issue with the way the data has been analysed. I can understand taking the mean of tank as the individual fish aren’t independent replicates. However, to take the mean of a tank across the 7 times (taking the mean of a mean) you ran the experiment is inappropriate as you are masking a lot of natural variation and thus forcing an R2 that is unlikely to be representative of what is actually going on. The mean for each tank each time the experiment was performed should be included in the analysis, so you should have 21 points per treatment not 3. Then tank should be included in the model as a random factor. Parental pair or clutch (these should be uniquely labelled) should also really be included as random factors in the model nested within tank. It would also be interesting to see how the relationship looks if you plot the data for each individual fish, this could go into supplementary materials. I would also be interested to see how the relationships looked if the 3000uatm treatment was excluded as it appears some of the significant trends are being driven by this treatment which is unrealistic for coral reef fish. Just out of curiosity did you try analyzing the data looking at comparisons between treatments and were they any significant differences, especially between the 390-1600 treatments which are more relevant levels for coral reef fish in the future.

Additionally, you don’t specify/it isn’t clear why you choose to do a regression analysis over a standard treatment comparison approach. This relates to the question you are asking. Perhaps re-defining/refining your aims could clear this up.

The discussion in well written. However, care needs to be taken when comparing your results to some past studies as the data was analysed differently and it does seem that some of your significant trends are being driven by the non-ecologically relevant 3000uatm treatment.
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: OCEAN ACIDIFICATION ALTERS MORPHOLOGY OF ALL OTOLITH TYPES IN CLARK’S ANEMONEFISH (AMPHIPRION CLARKII)
Review round: 1
Reviewer: 2

Output:
Basic reporting: There is an extensive use of tables while it would be better to use charts as they are easier to be visually understood. There is a specific comment in my revision.
Experimental design: No comment
Validity of the findings: I found too many speculations in the discussion part, that is unnecessarily long. I think that authors should keep speculations at minium and always keeping in mind the central topic of the work (pretty much what is stated in the title).
Additional comments: In their paper, Holmberg and colleagues investigate the potential effects of future sea acidification on the morphology of otoliths and other biological characters in the Clark’s anemonefish. They used a lab experiment testing the impacts of different water pH, eventually similar to those foreseen for the future. Most of their results are in line with the ones from other studies on the same topic, even though in their experiment they considered a set of non-ordinary morphometric characters. Overall, I found the experiment designed and conducted in a solid way, even though, considering the rationale and the final objective of the study, it would have been better to increase the number of aquaria for each level of treatment. The micro-structure analysis of otoliths would have also helped to shed some light on different issues on which authors are not particularly sure (e.g. the assessment of settlement). I do not have any major concerns about otolith analyses while I found some problems concerning the estimation of mortality, settlement timing and somatic growth that I explain below.

MAIN COMMENTS
As pointed out by authors, it is not surprising that the extreme value of pH investigated did not result in a higher mortality rate in larvae. The high natural mortality that occurs during settlement has probably blurred the effect of acidification on this variable. As in many others studies, in my opinion assessing and comparing mortality in fish larvae is a not easy task and the outcomes should be discussed with caution, also considering that it is an experiment conducted in controlled conditions.

One concern regards the assessment of acidification effects on the number of settled individuals. Authors found that SL was reduced by increased pH. This could be an indication that settlement was going to be delayed by the experimental conditions. But in order to claim that there was a delay you have to know which is the average age at settlement in control conditions, that authors do not have as they stop the experiment even before the end of the larval phase in controlled conditions. From this perspective the outcome and the relative conclusions made by the authors are very doubtful in my opinion. In this sense, which was the rationale for choosing just 10 days for rearing larvae and not waiting, at least, till the end of the potential larval phase (that in the species should be around 15 days)? I also wonder if eggs hatched at the same time. If not, this would add further uncertainty to the assessment of settlement delay. I found the conclusions and the hypothesis about this part to be improper and over-speculative. In my opinion this part of the study should be removed.

It is interesting that SL decreases with increased acidification, something on which there are controversial results coming from the few studies carried out till now. Concerning this result I have a concern about the potential influence of size at hatching on the estimation of the final standard length. Size range at hatch for the species considered can be 0.3 mm, so it is not that different from the range found by authors on the final standard length between aquaria averages (that seems to be <0.5 mm from the regression). Considering these comparable ranges, how can authors be sure that the final difference in terms of standard length was not due to an initial difference of size in recently hatched larvae (even considering a random distribution of larvae in the tanks)?

Finally, even though I found the discussion interesting and well written, in my opinion it is too long and not very focused on the results of the study. Even though the journal accepts certain speculations I think that they should be kept at minimum and avoiding to go out of context. In this sense, in my opinion it is not proper to speculate about potential impacts of ocean acidification on behavior or sensory reception as authors did not have any data on these topics at all. For this reason I suggest to shorten the discussion.

MINOR COMMENTS
Abstract
line 27: no need to specify what ocean acidification is in the abstract, in my opinion. The concept is self-explanatory.

Introduction
Table 1. I would directly specify in the table the effect like ‘increased S area’ or ‘decreased S roundness’. It would be clearer. Also because for some metrics like ‘S shape’ an increase is difficult to understand.

Line 134: it is not very clear what ‘lateral face’ is, even looking at the supplementary material. Is it the dorsal part of the otolith?

Material and Methods
line 233-236: why are these variables visually scored by operators? Especially for lateral development that is a measure of convexity, was there no way to ‘measure’ it and then assign it to a score?

line 243: how many otoliths (and % on the total) were discarded? Also considering the unsettled individuals.

Results
There is an extensive use of tables. In many cases I would use graphical representations like bar-charts to show results. Tables, especially when they are large, are very difficult to read.

Results are very wordy and probably too long. I would reduce them a bit keeping the main output of the analyses. I do not find useful to report all the detail for all the otoliths analyzed.

You should explain what RC1 and RC2 are in the text, for a non expert reader.

Why did you show a PCA in a table?

Figure 1. Please, do not extrapolate regression lines and confidence intervals.

Discussion
line 402-403: how is this possible?


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: OCEAN ACIDIFICATION ALTERS MORPHOLOGY OF ALL OTOLITH TYPES IN CLARK’S ANEMONEFISH (AMPHIPRION CLARKII)
Review round: 1
Reviewer: 3

Output:
Basic reporting: The manuscript is clear and unambiguous, with professional English used throughout

Literature references are "over-sufficient", the authors cannot be blame for that and I guess the peerJ format allows that, but it is true that the long lists of references (3-4 lines of only references sometimes) is sometimes disturbing. But this shows the good knowledge of the authors and this provide a powerful contextual frame.

Article structure is correct, but authors should consider to reduce its length significantly.
Figures are of good quality, while being not very "graphically" attractive. This is purely esthetism, but I cannot say that more attractive figures would be a great value here.
Tables are fine, so are the raw data shared.

This is an interesting, substantial, and coherent body of work, but as written in my detailed review, the authors should consider re-organizing or re-wording some of their paragraphs and ideas before this manuscript gets published.
Experimental design: This is original primary research within aims and scope of the journal

The research question is well defined, relevant and meaningful

The investigation is super rigourous, well backgrounded, and performed to high technical & ethical standard

Some methods may be detailed more (despite my general comment to, most of the time, there are too many details and the overall manuscript is too long and has to be shortened), see my list of comments for that.
Validity of the findings: Rational & benefif to literature is clearly stated, even if this could be improved in the discussion as detailed in my list of comments.

Data is robyus, statistically sound & controlled

Conclusion are well stated but can still be improved, especially after considering the real meaning of the "settlement" term used by the authors. I recommend the authors to change this wording.

The authors should consider re-writting part of their Results and Discussion to meet with what is expected from such sections. Results section should presents... results only, and Discussion section is not a review section. Overall, the authors have all the sufficient data and knowledge to perform these improvements, so there should be no problem with that. I highly recommend the authors to read my details comments in regard to that.

Speculation is appropriate.
Additional comments: General comment
This is a great manuscript relating a substantial amount of work on a clownfish otolith metrics in the context of acidification pressure. This manuscript is very professional and globally well written. The changes that have to be performed are not related to the quality of the writing, but to the manuscript organization between Introduction, Mat&Met, results and discussion. Some wordings are inappropriate and require modifications. I highly encourage the authors and read carefully all the detailed comments provided below.
I believe that this study can be published if these comments are taken into account.

lines 41-43
I’d rather summarize here the main idea and hypothesis of your discussion on your results, ocean acidification and otolith’s functions, instead of saying that “you reviewed the literature”, as this does not provide any important information. Be more specific perhaps.

line 44
missing a word I guess “these impacts may have consequences on teleost fitness in the near-future ocean”?

line 54
“serving to abate”: awkward wording, please change to something that does not imply that the absorption of CO2 by the ocean intentionally “serves” to decrease the CO2 greenhouse effect. This is an inappropriate “personification”.

lines 55-56
You should simplify this sentence   “impacts seawater chemistry by decreasing pH and carbonate (CO32-) concentration (Doney et al., 2009).

lines 56-57
I would be more direct: “both can impact” instead of “both are expected to impact”. Please also refer to more, including more recent, literature to justify this point.

lines 72-84
I would delete the first sentence, as it introduces well you research interests, but does not serve this introduction. Then I would organize this paragraph a bit differently, by introducing that Teleostii dominate most aquatic habitats and are internal calcifiers (which would make the connection with your previous paragraph). And then I would write that in addition to the already known impacts of acidification on other of these fishes’ biological functions, their calcified structures might also be vulnerable in acidified contexts.

lines 85-98
This a very well written paragraph, but as the entire manuscript actually, this is too long. You could summarize that very briefly by saying that otoliths are calcified structures of the inner ear that function as sensory organs. The pH gradient in the endolymph is also a good point in the context of your study, but apart from that I advise the authors to be more concise in their writing.

line 103
I would add “therefore” or a synonym in “… elevated seawater pCO2 therefore stimulates…” to precise that you refer here to the demonstration you made with Table 1.

lines 110-111
I don’t understand what the authors actually mean here.

lines 133-139
I suggest the authors to use (i), (ii), … etc. to list the different metrics that they investigated (as this is again very long).

line 139-144
While references are over-abundant in the previous parts of the introduction, I would like the authors to here justify their arguments with more literature.

line 145
Authors should precise here that the otolith morphology they will look at corresponds to the metrics they described in the previous paragraph. As previously, the authors should consider to make the transition clearer between their paragraphs, and this could be done most of the time by a simple word (e.g. here: “we investigate ocean acidification impacts on these otolith metrics in A. clarkii” or something similar).

lines 150-154
Authors should consider to simplify their point here and move the most part of these information in the Mat&Met section. I advise the authors to be more direct, and so in the whole manuscript, to shorter it significantly.

lines 166&168
Authors should check if the volume metric required by peerJ guidelines is liter of if gal is also accepted.

lines 176-181
If possible, I recommend the authors to use (i) (ii), etc. instead of “1.”, “2.” etc… as this will be clearer given that lists start with numbers.

line 185
As authors use liter here for volume metric, they should consider homogenizing their manuscript by using always the same metric type all along the manuscript (in reference to my previous comment on gal).

lines 210-216
I do not agree with the terms “settlement” and “settle” used by the authors. I recommend the authors to use “settlement-stage” instead, as ‘settlement’ refers to an ecological transition that does not occur here in aquaria, especially as you euthanized individuals at that time. For instance: “Due to the natural variability in developmental time required for individuals to reach their settlement-stage” would be more appropriate wording than what the authors have used. I recommend the authors to consider re-wording all the sentences where they clumsily refer to “settlement” and “settle” instead of “settlement-stage”.

lines 216-217
Unnecessary sentence as it does not provide any clear information.

Mat&Met – general comment
The use of “we” at the beginning of every sentence is probably one of the reason why this section is, similarly to the introduction, too long. I recommend the authors to be more synthetic, and to not hesitate to use the passive form instead of “We + verb”.

lines 279-284
I would add this paragraph in Mat&Met to confirm the proper functioning of the pH treatments and conditions, but not here in the results section. Or, in the case the authors comment these results, then OK for the results section, but here the authors only cite the Table 2, without any particular interest of that being in the results section.

lines 288-289, 305/308, 322-324
I recommend the authors to switch directly to the results. There is no point of presenting what they will then present. Authors should just present directly their results. This sentence is typical of a figure or table caption, but should not stand in a results section. The manuscript is well written, but really not synthetic, and these particular cases are symptomatic of what should be improved by the authors.

Figure 1A-B
I do not feel convinced by these regressions with n=3. In addition the variability is important, and the differences are also not convincing, as evidenced in Table 3 with the means and standard deviations. I would therefore rather limit these results to comparisons between the pH conditions, not taking risks with the estimations made by the authors with the “each 100 µatm increase in pCO2”.

lines 292-296
Again, I do not think that authors measured settlement by putting fish in a habitat context and observing the number of fish settling in an anemone and the number of fish staying in the water column. What their measure is the acquisition of certain developmental features, related to the settlement stage in situ. I recommend the authors again to change their wording appropriately, as this result actually represents the proportion of fish reaching certain anatomical and developmental criteria, but not an ecological step.

lines 308-314
I do not understand the point of this text. Perhaps in the Mat&Met, but still, this does not provide any result here.

line 318
Authors should cite their Table 4 here.

line 332-333
This is the interesting part. The previous sentences are unreadable or at least too long. In this result section, the authors should consider justifying their “biological” results with statistics. If they want to detail more the statistical significance of all that, authors should consider to write extra files or extra Mat&Met for the reader to have access to such details, but here, in the result section, the messages have to remain clear.

lines 334-340, 347-359, 367-382
Same comment as above

lines 341-346, 360-366
Very interesting! But maybe more a discussion than a result? Anyway, this is great and way more interesting than the long list of statistical details… The authors should consider rewriting their results section taking this comment into account.

lines 405-418
Again, the authors should avoid to use the term “settlement” in this context, and talk more about “settlement-stage” pigmentation or morphology.
Also, the author should consider to add some literature references here, the work by Victor 1986 is great, but there are several other publications relating that: authors should have a look in particular at McCormick 1999 (delayed metamorphosis in A. triostegus) and Holzer et al., 2017 (A triostegus metamorphosis).
I would be more careful with the conclusions given here (but maybe this is because of the inappropriate “settlement” wording): mortality and somatic growth are not impacted, but competency (developmental ability to settle) is impacted. This is very interesting and the authors should give more emphasis on that in my opinion.

line 440
What do the authors conclude from these 24 studies reviewed? It would be a great value to put in parallel the great point made by the authors (importance of investigating each otolith independently) with their review (as done in this article), but also to dress some sort of a conclusion or perspectives taking into account both these things.

lines 441-455
This is great and interesting, however I wonder if it is essential to mention the “number” of paper consistent with the authors’ findings, or in the case it would also be appropriate to mention the number of papers inconsistent with these findings? I would remain simple here and simply mention that this is consistent, with some specificity that the authors mention very well, with most studies, and I would also provide small insights on findings that are new and/or inconsistent.

lines 456-474
This is too long and too much a bibliographical work rather than a discussion of the results.

line 487
I cannot agree more. This is all very interesting and warrants review, but this is not the topic of this manuscript, as I previously evoked many times.

lines 476-565
This is a great paragraph, but this has, in my opinion, nothing to do in this particular research article. Authors should consider writing a separate review, as they barely refer to the findings of their study here.

lines 569-607
This is finally some real discussion challenging the authors’ findings with existing literature and hypotheses. I recommend the authors to organize their entire discussion like this paragraph if they review their manuscript.

Discussion comment
Could the author discuss more their results on asymmetrical responses in the context of lateralized processes in coral-reef fishes? I recommend the authors to have a look at studies relating lateralized behavioral, anatomical and developmental processes in coral reef fishes at the settlement stage, in particular when facing external perturbations such as acidification (see Ferrari and Besson papers). Putting the results of this study into such a context would be a great value for this manuscript.

Conclusion comment
I would like the authors to justify or discuss more the reliability of their curves given the small number of replicates used for these curves (n=3).


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: OCEAN ACIDIFICATION ALTERS MORPHOLOGY OF ALL OTOLITH TYPES IN CLARK’S ANEMONEFISH (AMPHIPRION CLARKII)
Review round: 2
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: Firstly, I would like to apologise for misinterpreting that data table you provided. You have done an excellent job in addressing the concerns that I raised. There is only one issue I have left; the fact only one clutch was used. While a lack of  genetic diversity may be great for running statistics care should be taken when drawing conclusions from one genotype as a number of papers have demonstrated family dependent effects of ocean acidification. Seeing as your results are largely in agreement with previous studies that used multiple clutches it doesn't seem to be too big of an issue. However, I strongly feel that this should be mentioned as a caveat in the discussion, just so the reader is aware of this point (as is may get missed in the methods) This is a very well written paper, great 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: OCEAN ACIDIFICATION ALTERS MORPHOLOGY OF ALL OTOLITH TYPES IN CLARK’S ANEMONEFISH (AMPHIPRION CLARKII)
Review round: 2
Reviewer: 2

Output:
Basic reporting: All comments and request of clarifications that I have provided on the first version of the manuscript were substantially  fulfilled. I do not have any further comments on the second version of the manuscript.
Experimental design: No comment on the experimental design as in the first version.
Validity of the findings: All the request were fulfilled.
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: OCEAN ACIDIFICATION ALTERS MORPHOLOGY OF ALL OTOLITH TYPES IN CLARK’S ANEMONEFISH (AMPHIPRION CLARKII)
Review round: 2
Reviewer: 3

Output:
Basic reporting: No comment
Experimental design: No comment
Validity of the findings: No comment
Additional comments: Authors have modified their manuscript taking into account all the comments I made during my first review. Their manuscript is still well written, the research question is well defined and their methods and investigation are rigorous, well described and relevant. Data is robust, statistically sound and controlled.

Improvements made to the manuscript are significant. In particular, authors reduced the length of their manuscript, re-worded properly some ambiguous or erroneous sentences or terms, without reducing the clarity and meaning of their manuscript.

I recommend the article to be accepted as it is.


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

Input:
Title: RELATIONSHIP BETWEEN PHYSICAL AND COGNITIVE PERFORMANCE IN COMMUNITY DWELLING, ETHNICALLY DIVERSE OLDER ADULTS: A CROSS-SECTIONAL STUDY
Review round: 1
Reviewer: 1

Output:
Basic reporting: 1. The authors used a clear English language.
2. Raw data was supplied.
3. Tables have a good quality and are relevant.
4. The background presented in the introduction section needs improvement. A contextualization should be done to justify the use of the covariates in the analyses performed. Additionally, the gap existent in the literature considering the relation between hand-grip strength and cognitive function needs to be presented.
Experimental design: 1. The research question is well defined. However, two points need to be improved.
- The existent gap in the literature that is filled by the study needs to be clarified.
- Some analyses presented in the results section were not the aim of the study. The aim presented was: “to examine the relationship between physical performance measures, such as the exercise intensity and distance walked in the 6MWT, hand-grip strength, and cognitive performance parameters in community dwelling, ethnically diverse, older adults”. However, the relationship between cognitive performance were also investigated with other parameters (e.g. PASE, age, B-blocker use, etc.). Please, clarify why these analyses were done.
2. Some aspects should be improved in the Methods section:
- Before using the acronym RPE, it is necessary to show what it means. It is also important to inform whether subjects were familiarized to the RPE scale use.
- Please describe the instructions given to the participants regarding the 6MWT.
- Considering the hand-grip strength, it is not clear which value was used in the analyses (was it a mean of the peak values for each hand? was it the peak value of both hands, independent of the hand that presented the peak value?).
- Please, explain how is the score of the 3MS test given.
- For the categorical parameters, which statistical test was used to investigate the associations with the cognitive performance?
Validity of the findings: 1. Results for all correlations performed should be presented in the paper. Only the significant correlations were presented in the Results section. Please, present all values.
2. Despite the significant correlations found for some parameters, all of them were only weak or moderate. This fact should be considered in the Discussion section and taken into account in the Discussion and Conclusion.
3. The Conclusion is based mainly in the results of the relationship between cognitive performance and peak exercise intensity during the 6MWT. All physical performance measures should be considered, to answer the research question presented in the aim of the study.
Additional comments: 1. The background presented in lines 1-4 in the Abstract does not contextualize the aim of the study. Please, revise it.
2. Please, change +/- to ± in the Abstract.
3. As commented previously, some results presented in the Abstract were not the aim of the study. Please, clarify why these analyses were done.
4. Review the p value presented in the Abstract for the relation between the peak heart rate during the 6MWT and the 3MS test (p<0.071).
5. Please, clarify the sentence in lines 102 to 105 in the introduction section. It is confuse due to the use of the terms “baseline”, and “three and six years later”.
6. The parameters 6MWT %HRMAX should be described in the Methods section, before using it in the Table 1.
7. In the Table 2, the n for each cognitive test is different (89-90-87-88). Why are they different? In the Results section the authors described that from 95 participants, 5 were excluded due to not meeting the inclusion criteria or missing data. Accordingly, data from 90 participants should be presented.


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

Input:
Title: RELATIONSHIP BETWEEN PHYSICAL AND COGNITIVE PERFORMANCE IN COMMUNITY DWELLING, ETHNICALLY DIVERSE OLDER ADULTS: A CROSS-SECTIONAL STUDY
Review round: 1
Reviewer: 2

Output:
Basic reporting: The article is simple with the use of simple tools but that brings data applicable in practice. One suggestion is to make a theoretical foundation involving more the heart rate as a control of the intensity of the training with the elderly and to bridge the cognitive that is already well documented that must be moderated for both acute and chronic effects, because the works that the authors cite in the introduction they evaluate intensity with other variables.
Experimental design: The inclusion and exclusion criteria should be clearer as this population receives a fall prevention intervention.
The authors can not say that they controlled the intensity of the exercise, but that they controlled the intention of the FC during exercise. To control the intensity of the exercise we have to control other variables.
Validity of the findings: Data is replicable
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: RELATIONSHIP BETWEEN PHYSICAL AND COGNITIVE PERFORMANCE IN COMMUNITY DWELLING, ETHNICALLY DIVERSE OLDER ADULTS: A CROSS-SECTIONAL STUDY
Review round: 2
Reviewer: 1

Output:
Basic reporting: The authors addressed all aspects pointed out in the review.
Experimental design: The authors addressed all aspects pointed out in the review.
Validity of the findings: The authors addressed all aspects pointed out in the review.
Additional comments: The authors addressed all aspects pointed out in the 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: INTESTINAL PARASITIC INFECTION ALTERS BACTERIAL GUT MICROBIOTA IN CHILDREN
Review round: 1
Reviewer: 1

Output:
Basic reporting: There are many awkward phrases, improper grammar and incorrect sentence structure throughout the manuscript.  Some examples are: Line 34 – “children population in developing countries . . .”.  Line 45 – it is not determinant in gut microbiota . . .”. Line 90-91 – educing the second minimal to non-inflammatory . . .
The authors should closely review the text for readability and proper English usage.

The introduction is long and somewhat disjointed.  The authors should review the introduction and look toward streamlining it as well as providing a better focus.
Experimental design: It is unclear why 23 samples were selected for study from the more that 200 samples that were collected.  How were the 23 chosen? Did only a small percentage of the samples contain parasites?  Specifics concerning the selection process are necessary.

How were the controls defined?  Was it only the lack of detectable parasites?
More information about the patient samples would have been useful.  Had any of the individuals been recently treated for a parasitic infection?  They could be negative for parasites, but have significant changes due to a previous infection.

It appears that only single samples from individuals were used for the study.  Would multiple samples taken during and after clearance/treatment of the parasites provide more robust data?
Validity of the findings: It is not clear why figure 1 was included in the manuscript.  It should be removed or its utility clearly demonstrated in the text.

The inclusion of the map in figure 2 would be more useful if there was a discussion of what, if any, significance there was for the location of the child care centers.  Were there differences in socio-economic levels, urban versus periurban or suburban location.  Otherwise it is unclear what the map adds to the manuscript.

The authors discuss the change in enterotypes, but it is not made entirely clear if there is some significance to this switch.  Particularly with regards to the specific individuals in the study.
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: INTESTINAL PARASITIC INFECTION ALTERS BACTERIAL GUT MICROBIOTA IN CHILDREN
Review round: 1
Reviewer: 2

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: In the present study authors sampled the infected stools of 23 children from 4 different children’s care-centers in Medellin, Colombia and identified the eukaryotic parasites by traditional and molecular methodologies. Subsequently, 16S rDNA sequencing was performed to measure the microbial diversity, and later, influence of the different species of parasites on microbial diversity of gut was calculated. This study finds significant influence of the intestinal parasites on the diversity of gut microbiota. Though, study is encouraging, several sections of manuscript need significant improvement as mentioned below:

1.	Dietary habits of individuals have not been reported in the manuscript. Dietary habit contributes to the changes in the composition of gut microbiota, which can have significant role on the findings of this study.
2.	First and last two sentences of the abstract need to be re-written.
3.	Introduction section is too lengthy and unfocused. Several facts have been unnecessarily discussed. For example: Line 89-197 can be reduced drastically.
4.	Last paragraph of the introduction is not systematically written.
5.	Discussion section is also lengthy. Especially, most of facts discussed in lines 353-416 can be removed.
6.	At several places, including the figure legends, microbiota has been referred as microbiome.
7.	Legend of figure 1 should be changed, something like “Schematic of human gut microbiota”.
8.	Legend and description of the figure 6 should be written in more detail.
9.	Legend of Table 1 can be changed to “Description of samples used in this study”.
10.	Legend of tables 2 & 3 is well focused and descriptive.