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: UPRIGHT WALKING HAS DRIVEN UNIQUE VASCULAR SPECIALIZATION OF THE HOMININ ILIUM
Review round: 1
Reviewer: 1

Output:
Basic reporting: Excellent.  Superbly well-written and well-researched (and see Additional Comments)
Experimental design: Excellent in all ways (and see Additional Comments)
Validity of the findings: Clearly stated in the text.  All aspects are excellent.
Additional comments: Few authorities in the human/hominin osteology (including me) have likely made much note of the presence of an enlarged, anteriorly-positioned foramen on the lateral face of the human ilium, let alone in early fossil hominins.   In fact, as I began reading this manuscript, I looked at human and nonhuman primate ilia in our collection and had the same “ah-hah” moment that Zirkle and collaborators must have had when making the discovery.  In this well-written and hypothesis-driven manuscript, Zirkle and co-authors lead the reader through the study of the vascular specialization and interpretation of the morphology of the foramen.  They convincingly argue that the specialization is a central element of the enlarged central vascular canal system that is unique to hominins, including in the earliest hominins (Au. afarensis and Au. ramidus).   They make the clear case that the largest external vascular structure (AIF) appears first in the hominin fossil record as a part of the unique morphology associated with bipedality.
	I am impressed with the detail of the science behind the study, including a compelling comparison based on the record of dissection, radiography, and computed tomography of human adults produced by earlier researchers.  Zirkle and collaborators thorough study provides a comparison of the foramen’s morphology in archaeological materials, CT scans, casts of early hominins, photographs, dissections, and descriptions from the literature.  In addition to measurements of the feature, images from CT scans of two Ardipithecus ilia and correction for body size differences firmly establishes the uniqueness of the foramen exclusive to extant and extinct hominins.  The investigators suggest that the enlarged AIF and morphology is associated with increase in blood supply during development.
	In summary, this is an extraordinarily well written article, test of a compelling hypothesis, and documentation of a clear association between morphology and behavioral adaptations surrounding bipedality.  I strongly recommend publication.  In my view, aside from some minor editorial revisions (e.g., upper case for the journal title for the Hammond et al. reference, italics for the journals Science, Skeletal Radiology, and Clinical Orthopaedics, italics for Ardipithecus ramidus, and other minor revisions, the manuscript is ready for publication.


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

Input:
Title: UPRIGHT WALKING HAS DRIVEN UNIQUE VASCULAR SPECIALIZATION OF THE HOMININ ILIUM
Review round: 1
Reviewer: 2

Output:
Basic reporting: The paper is clear, concise, and easy to follow. There are a few places where additional citations and word changes are important.

It would be delightful if some specimens prior to Ardipithecus were discussed--Oreopithecus?? What might be happening during the Miocene that helps us discern the likelihood that an arboreal biped and/or flexible scrambler already had an additional vascular network in the ilium? I understand leaving the discussion of other, later (South African) material to be tested by others, but I think earlier material does need to be included here, at least in discussion.

Line 243, please cite something other than the Zirkle & Lovejoy paper here. The idea of using the glutes during stance phase is hugely important in biomechanics and having an actual biomechanics study (EMG?) cited here will help broaden the interest and background for the vascularization argument put forth by these specific authors.

Most importantly, I am not sure why the authors refer to hominin bipedalism as cursorial (in the final paragraph). While I understand that cursorial can simply mean continuous, or endurance, it will typically be used to imply running; the inference that all forms of our lineage allowed for running does not stem from this study or any data of which I am aware. To this end, another word here is quite important.
Experimental design: Appropriate design for the study. Clear, relevant.
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: MARINE AMPHIPODS (PARHYALE HAWAIENSIS) AS AN ALTERNATIVE FEED FOR THE LINED SEAHORSE (HIPPOCAMPUS ERECTUS, PERRI 1810): NUTRITIONAL VALUE AND FEEDING TRIAL
Review round: 1
Reviewer: 1

Output:
Basic reporting: no comments
Experimental design: The Materials & Methods section needs the seawater temperature of the experiment.
Validity of the findings: no comments
Additional comments: The present MS reported the nutritional value of the marine Amphipoda Paryhale hawaiensis in the wild captured and cultured one, and provide these amphipods to juveniles of seahorse compared with Artemia. This study presented well that the usage of Paryhale hawaiensis is one of the promising feeding items to seahorse aquaculture. While the present form of MS needs several minor revisions, the results would be worthy of publishing in one of the international scientific journals.

    My comments for improving the MS are,

1) The introduction needs more detail, especially a clear explanation on what is the progress of the authors' previous studies;

Vargas-Abundez et al. (2018) Feeding the lined seahorse Hippocampus erectus with frozen amphipods. Aquaculture 491, 82–85.
Vargas-Abundez et al. (2021) Marine amphipods as a new live prey for ornamental aquaculture: exploring the potential of Parhyale hawaiensis and Elasmopus pectenicrus. PeerJ 9, e10840.

2) The introduction needs a more detailed explanation of the characteristics of the life history and ecology of Paryhale hawaiensis based on numerical data.

3) What is a plastic label fastened to the neck of the seahorse (Line 213)?  If the authors could prepare photos on the label, readers could understand the rearing method.

4)  Figure 3 was based on the estimated regression. I think that Figure on the average and S.D. would help readers to understand the validity of estimation.

5）The discussion would need a more detailed explanation of what the purpose of seahorse aquaculture is and why the usage of amphipod Paryhale hawaiensis is suitable for this aquaculture purpose.


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: MARINE AMPHIPODS (PARHYALE HAWAIENSIS) AS AN ALTERNATIVE FEED FOR THE LINED SEAHORSE (HIPPOCAMPUS ERECTUS, PERRI 1810): NUTRITIONAL VALUE AND FEEDING TRIAL
Review round: 1
Reviewer: 2

Output:
Basic reporting: I found the research topic interesting.  The idea of culturing a food item in a biofloc system is attractive.  It is scalable and would allow for the commercial production of valuable feed for marine fish.  This could go well beyond seahorses.
Experimental design: The study design has some serious issues and I question a few of the key aspects of the design which limits the study publication potential.    Your diet study and your biofloc culture appear to be disconnected but joined into this manuscript.

Feel free to correct me, unfortunately I feel the following issues warrants rejecting the manuscript:

The diet study design:

1. Where is the biofloc, pellet grown feed in the diet study?
LINES 212-217   i) amphipod diet: 100% captured amphipods; ii) Artemia diet: 100% enriched Artemia; iii) mixed diet: a 1:1 mix of the amphipod and the Artemia diets.

It is possible that the seahorses do well on 'captured' amphipods but do poorly on cultured amphipods? If so that would invalidate the premise of growing the amphipods for food. Their fatty acid profiles are very different and this would seem important.

2. Your starting weights for the seahorses are not reported correctly.  You report a mean and S.D. for your starting weights, but what you should report is a range.  Your range was 0.72 --- 2.48 with a mean of 1.3.  This starting weight invalidates your study design; unless you can demonstrate that starting weight had no effect on weight gain or SGR. Given that your starting weight is so vastly different and it is likely that your seahorses were at different development states. Clearly, your artemia feed treatment does not provide the nutrition needed as the fish didn't gain weight.  Weight gain and SGR are tied to development and age.

3. Your fatty acid raw data shows some issues.  It appears that your duplicate samples (mentioned on LINES 251-252) indicate some method issues. It is to be commended to take duplicate samples in a diet study, the purpose is to check analytical precision.  In chemistry, it is expected that two subsamples from the same sample would yield nearly identical results.  If results are not identical or close to identical it indicates an error in the process.   Samples 1-a and 1-b, provide a good example.   These do not look like the same starting materials.

                  1-a           1-b
C22:6n3	  8.71	   2.37
Σ SFA	42.83	44.39
Σ MUFA	26.00	41.47
Σ PUFA	31.17	14.14

These differences cause this reviewer to believe that you have a process error in your fatty acid prep or sampling.
Validity of the findings: I'm concerned about the above issues confounding your findings.
Additional comments: Great topic, writing needs some work, the manuscript is too long, needs some focus.  The issues with experimental design should result in a rejection or a major rewrite and resubmission. I would argue for a rejection.


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: MARINE AMPHIPODS (PARHYALE HAWAIENSIS) AS AN ALTERNATIVE FEED FOR THE LINED SEAHORSE (HIPPOCAMPUS ERECTUS, PERRI 1810): NUTRITIONAL VALUE AND FEEDING TRIAL
Review round: 1
Reviewer: 3

Output:
Basic reporting: No comment, except the abstract and introduction length. In my personal opinion abstract is too long (250 words is enough). Concerning the 'introduction': It is well written, clear and concise, focusing from general to precise aim of the paper. however some paragraphs should be summarized (e.g. L83-100): Sometimes there are many references (L79/L84).
Experimental design: The second objective should be better defined and seahorse species should be included in the paper objectives. Ideally some hypothesis linked to objectives would be desired as well.
Methods are well described and statistical analysis is well described. However It would be nice if authors can explain better Fatty Acids determination on seahorses: "Fatty acid determinations were performed on seahorses once at the end of the trial. Duplicate subsamples from each of six individuals fed each of the three diets were randomly taken for this purpose". Authors should clarified if animals were scarified or not, how, if the entire animal was used for analysis or just some tissues. It is unclear for me as well, from the 48 seahorses (16 for each treatment), how many and the selection criteria employed to sample tissues.
Validity of the findings: The study presents the use amphipods as an alternative live prey for seahorse (Hippocampus erectus) aquaculture. The literature justify the research presented as some bottlenecks, not only in seahorse, in general in ornamental aquaculture of marine species  exist since many years compromising their own sustainability. Authors described also the lipid composition of targeted amphipod species enlarging the knowledge about nutritional quality of these preys.
Results: Lipids values are expressed in %, but authors should specify in this % is in dry or wet mass. Other authors should express lipids as g/100 g dry weight. It would be easy to compare with values obtained in other seahorse preys such as mysids (Herrera et al. 2010).

I have some concerns on the discussion, which sometimes showed some speculative sentences that I described later on General comments section.
Finally for conclusions,  It seems more a summary of ideas. Conclusions should be reformulated with the main demonstrated facts extracted from the paper.
L578-582: This two sentences should be summarized and merged on only one.
L582. Considerations about biofloc technology can be inside the discussion but not as paper conclusion.
The improvements in FA profiles could be linked to statistical differences, which there are not specifically tested on the paper, therefore we can not assume them as a conclusion.
Additional comments: In general the paper is well written and should be published. However I have main thought regarding the study: In general it presents the utility of amphipods as alternative prey for seahorses promoting their growth and showing also their lipids and FA profile. Ideally some reflection about protein content should be included in the discussion, as this information is missed in the paper. High quality lipids (specially HUFA) are important in the early phases of development of any organism, but for growth, proteins are at least very important too.

Specifically there is some comments described as follows:
L211. Seahorse Feeding trial. Seahorses had 10 months when the experiment starts. I am just wondering if during the two additional experimental months any reproduction behaviour was registered. One year in other seahorse species implies mature specimens that when they are fed with high quality food (in this experiment animals were even overfed) involve at least courtship behaviours. In addition there is no data about sex ratio (males:females) and their length measurements (only weight). Normally females can growth faster as normally they are less involved in reproduction (compared to males). It would be nice if authors can add some comments about it.
L438.  Authors observed that " ...amphipod introduced into the biofloc system changed markedly in color compared to pellet-fed amphipods." as a result "...microalgae and biofloc ingestion". This statement is merely speculative as they said "amphipod gut content was not analyzed". It would be ideal if authors can add a figure showing amphipod color changes.
L451 and L462. These two paragraphs state the benefits of biofloc technology. Viewing the length of the discussion, I suggest that this information was summarized in few lines always trying to find the relationship with the paper outcomes.
L470. 4.2. Effect of amphipods on the seahorse performance and fatty acid profile. I suggest to change "performance" by "growth" as authors did not assess other parameter except animals growth.
L499. Authors state that  seahorses "... likely ingested a higher amphipod biomass compared to that of Artemia, thereby partially explaining the increased growth in the two diets that contained the former." It should be nice to add some results regarding ingestion acceptability. Did the authors observe any difference between preys ingestion?
L557. Statements about digestibility are based on some references that mostly focused on studies carried out during seahorse early developmental stages (Blanco et al., 2015; Novelli et al., 2016; Ofelio et al., 2018) , which it is not the case of experiments developed on the manuscript (seahorse are almost 1 year old). Therefore this issue should be clarified or references deleted. Moreover the rest of  paragraph state different hypothesis to explain digestibility issues which there are not tested under this manuscript. Therefore even if it is important to pointed out, text should be summarized.
L555. This paragraph can be deleted or merged summarized somewhere before in the discussion.
L561. I am partially agreed with authors statement about "... monodiets inconvenience to provide essential nutrients". It always depends on the nutritional quality of preys: Wild-capture mysids has been used successfully employed in the reproduction of seahorses (H. reidi) as monodiets (Otero-Ferrer et al. 2016, 2020). On the other hand feeding regimes can be adapted to animal life-stage or culture objective: e.g. broodstock feeding regimes should target high-quality lipid content, instead juveniles and subadults should search high-quality protein content.  In this context amphipods seems to be a prey in which its nutritional quality can be easily modulated.


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: MARINE AMPHIPODS (PARHYALE HAWAIENSIS) AS AN ALTERNATIVE FEED FOR THE LINED SEAHORSE (HIPPOCAMPUS ERECTUS, PERRI 1810): NUTRITIONAL VALUE AND FEEDING TRIAL
Review round: 2
Reviewer: 1

Output:
Basic reporting: The present study assessed the nutritional value of a marine gammarid Paryhale hawaiensis and evaluated through a feeding trial its potential use as a natural prey for the lined seahorse Hippocampus erectus. The authors indicated this species of gammarid possess high levels of valuable lipids and polyunsaturated fatty acids, including the long-chain PUFAs (LC-PUFAs) arachidonic acid (ARA) and so on. The authors’ experiment indicates that gammarid Paryhale hawaiensis is a more suitable prey item for the lined seahorse than Artemia. I understood that the authors responded to all comments and improved the revised MS. I think that the revised MS is suitable for publication.
Experimental design: no comment
Validity of the findings: no comment
Additional comments: No additional comments


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

Input:
Title: MARINE AMPHIPODS (PARHYALE HAWAIENSIS) AS AN ALTERNATIVE FEED FOR THE LINED SEAHORSE (HIPPOCAMPUS ERECTUS, PERRI 1810): NUTRITIONAL VALUE AND FEEDING TRIAL
Review round: 2
Reviewer: 2

Output:
Basic reporting: The basic reporting of this revision remains mostly unchanged from the original submission.   I found the revisions to help the flow of the manuscript.

A few areas that need to be greatly improved if this moves forward.

The graphs:

The PCoA is not interpreted as well as it should be.  The vectors and eigenvalues should be reported.  The results of the PCoA are not presented with enough information to determine the validity of your data analysis.  For example, there are clear outliers in your data set from the raw data (see Experimental Design discussion), because your number of replicates and number of factors are so similar, PCoA might be a better choice here than a PCA, but outliers have large effects on PCoA with small samples. In fact, the use of PCA and PCoA to find outliers is a very useful method of analysis for machine learning.  If you do a literature search for PCA/PCoA and fatty acids (FAMEs), you will see that many authors reduce the dataset down to the most important fatty acids. While there is no hard and fast rule on sample size > factors, outliers are real problems with small data sets.  Furthermore, from your vectors, you can see some clustering of vectors into groups, it is possible for some values to strongly affect others and therefore masking your data.

You should at a minimum provide some interruption of your data that allows the reader to understand these impacts.  I would suggest you reduce the number of factors down greatly, and discuss the vectors (FAMEs).   Most marine FAMES papers do not focus on FAMES less than 16.

I'm not exactly sure why you chose to use two different multivariate methods (one on individual FAME values, others on value groupings/ratios).  You clearly have a reason for this, perhaps you can improve the literature around multivariate methods by providing some discussion on why PCoA or nMDS was chosen.    Why not a PCA?  Most applications are using PCAs for FAMEs.

Can you provide some incite here?

I'm not sure how informative your nMDS is compared to the PCoA.  These reduction methods are really great data exploration methods but sometimes abstract representations of the data.  You should use the most informative information and present that in the body of the manuscript and provide supporting data in your supplement information.

I would suggest you cluster the groupings onto your PCoA graph and show these in one figure.

Explore the R vegan package or Python skbio for better utilities.

Your growth graph should be fit to the model data and not connect the dots.  Show the positive and negative errors around your data.

Graph quality could be greatly improved using GraphPad for all your graphs or using R

The Tables:

Add tables to support your multivariate work.  Can be in the supplemental.

Your tables 1 and 4 do not say if you are reporting S.E. or S.D.  Recall that S.E. is used for reporting data where the sample size changes.   If your samples size is the same throughout then report S.D.
Experimental design: The authors deal with the size difference in the rebuttal. However, I still would caution that growth is very impacted by the life stage. Diet studies normally want to see an increase in body mass or size, for example, we use 300% growth on fingerling trout for a diet study.  We need to see 300% growth from the start weight prior to the termination of the study. This provides time for small influences to make real differences.

This said it is clear that Artemia is a poor diet and that amphipods/mixed diets support growth.    I'm not sure how your fatty acid data is impacted by sizes for seahorses.
Validity of the findings: I still remain troubled by the fatty acid data quality.

I still question this, I do not find the author's rebuttal to be substantial.

For example:

Captured amphipods

C16:0	29.84, 23.67, 11.21, 25.24
C16:1	0.74, 0.53,13.67, 7.90

16:0 and 16:1 are very diagnostic ratios of fatty acids.  within a single treatment "Captured Amphipods" the authors have vastly different ratios. It is hard to believe that animals that are captured from a similar source would have such different ratios.   It is more likely that there are some method problems in their fatty acid workup.

The challenge here is that you draw conclusions as to the effects of the FAMES on the PCoA.  But your data is highly variable in ways that suggest methods problems.  Do you still have your raw data from the GC?  Perhaps some separation issues?   Again, I really like the idea of the study, but these are serious issues.

If this publication is to proceed, these issues need to be specifically highlighted to the reader.
Additional comments: I still like the idea of the study.  There are some real issues with the fatty acid data which are not possible to be addressed in a revision.  I'm happy to see if the authors can address these but again argue that the data is seriously challenged.


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: MARINE AMPHIPODS (PARHYALE HAWAIENSIS) AS AN ALTERNATIVE FEED FOR THE LINED SEAHORSE (HIPPOCAMPUS ERECTUS, PERRI 1810): NUTRITIONAL VALUE AND FEEDING TRIAL
Review round: 2
Reviewer: 3

Output:
Basic reporting: No special comments, previous issues related to abstract and introduction have been solved by the authors. Just some additional references to be added will be further mentioned in the additional comments section.
Experimental design: Previous issues solved. Perhaps, instead "captured amphipods", a most appropriated term would be "wild" or "wild-captured". Please replace one of the two options along the manuscript.
Validity of the findings: No special comments, previous issued resolved.
Additional comments: Lines referred to attached pdf:
L211. Please clarified: If wet weight was calculated individually but SGR was calculated for each experimental group following the formula presented.
L290. "The Total lipid content was high amongst amphipods from all three  sources, with no significant
 differences (F = 4.56; p = 0.07) among groups." This sentence should be clarified  and rewritten in a more clear way. The lipid content was high compared to "what"? Artemia? please clarify.
L330. The indistinguishable growth rates observed in Seahorses fed in excess with Artemia after almost 2-months experiment results quite curious. Apparently no courtship, gender or pregnancy effect has biased this result, nor prey acceptability as well, makes me think about the protein content of these preys, probably very low, as a potential cue behind this result. Something should be added in the discussion. Other than lipids aminogram or energetic content of each diet could clarify these results.
L447. To add a couple one or two references.


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: IDENTIFICATION AND EVALUATION OF THE NOVEL GENES FOR TRANSCRIPT NORMALIZATION DURING FEMALE GAMETOPHYTE DEVELOPMENT IN SUGARCANE
Review round: 1
Reviewer: 1

Output:
Basic reporting: Yan et al. manuscript identified candidate genes for normalization of qRT-PCR analysis during sugarcane female gametophyte development. Authors performed a genome wide approach by using available RNAseq data to identify suitable candidates based on coefficient of variation of average RPKM values and level of expression (highest RPKM values).
Twelve selected candidates were further analyzed, together with 6 well known, widely used normalization genes, with proper tools (geNorm, BestKeeper and NormFinder).
Selected reference genes (FAB2 and MOR1) were finally validated through the expression analysis of 2 other genes, comparing qRT-PCR and RNAseq data.
Experimental design: The experimental design is valid, and proper techniques are used.
Validity of the findings: The identification and validation of reference genes to be used for qRT-PCR normalization in specific developmental processes constitutes a very valuable tool that guarantee for accurate gene expression analysis in future studies concerning female gametophyte development in sugar cane.
Additional comments: In general, the manuscript is OK, it provides novel and useful data. However, English language and supplemental data should be improved to be accepted for publication.

The English language should be improved. Some examples where the language could be improved include lines 53, 72, 92, 100, 134, 166, 222, 251, 301– the current phrasing makes comprehension difficult.
Figures are clear and relevant, however, supplemental files need more description:  in Table S1, what are the values and units? Expression levels, FKPM? In TableS2, what are the values, Cts? There is no SD shown, please, add to the table. In Table S3, add SD values to FKPM and Cts.

Minor corrections
In line 59, Saccharum spontaneum should be italicized.


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: IDENTIFICATION AND EVALUATION OF THE NOVEL GENES FOR TRANSCRIPT NORMALIZATION DURING FEMALE GAMETOPHYTE DEVELOPMENT IN SUGARCANE
Review round: 1
Reviewer: 2

Output:
Basic reporting: It would be nice if all figures have the  same writing font
Experimental design: No comment
Validity of the findings: No comment
Additional comments: The manuscript entitled “Identification and evaluation of the novel genes for transcript normalization during female gametophyte development in sugarcane” submitted to PeerJ describes an attempt to find suitable reference genes to be applied in studies of RT-qPCR during female gametophyte development. It is particularly important determining the suitable reference genes in the exact conditions of the experiment that one would like to determine gene expression variation of a particular gene or set of genes. The manuscript seems to be well-written, although it contains some inconsistencies that will be further indicated. If all the raised points be properly addressed and answered, an edited version of the manuscript can be published at PeerJ.
Comments
1.	A lot of words in the text are hyphenated, especially at introduction. Please correct that.
2.	If you are using a two-step qPCR, the correct name is ‘RT-qPCR’ and not qRT-PCR. qRT-PCR is used for one-step qPCR.
3.	Please, define AC and MMC.
4.	Avoid ‘strong’ words when describing your results or your scientific problem. For example, on line 58 (desperately) and on line 64 (outstanding), etc.
5.	On line 110 put Saccharum spontaneum in italics.
6.	It would be nice a figure showing the morphology of the five different stages of female gametophyte development.
7.	On line 225 the reference is duplicated.
8.	On Figure 4 it would be better if the relative expression were transformed to log, to see the expression values between 0 and 1.
9.	This is a manuscript about finding suitable references genes in a given condition. It is nice that the authors provided as supplemental material all the information about primer efficiency. However, it is important to show the melting curves for each primer. This information would help other researchers to replicate the experiments using the same set of primers.
10.	Also, at table S2 what are those values? Ct values?


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: VOCAL COMMUNICATION IN WILD CHIMPANZEES: A CALL RATE STUDY
Review round: 1
Reviewer: 1

Output:
Basic reporting: The manuscript is concise and is well written for the most part, and the authors make their arguments clearly.  I have just a few minor criticisms of the presentation:

In the Introduction, the authors state that PAM allows conversion of “call density” into “animal density”.  What is “call density”?  Density is a spatial measure, and readers can infer that “animal density” means population density (in this case, the population density of chimpanzees in the Issa region), but I don’t think it make sense to talk about the “density” of pant hoots.

46: Chimpanzees don’t “live in a fission-fusion structure”; they have high fission-fusion dynamics.

52: What does “inform on fitness” mean?  And precisely how could pant hooting rate be associated with fitness, or be a reliably indicator of the caller’s quality as a mate?

96-97: “in function of sex” – this would be better as something like “A sex difference in pant hooting rates exists in adults, with males calling more than females, and high-ranking males call more often than low-ranking males”.

103: “Sexual dimorphism” -- just “sex differences” is better, but if you want to use a technical term, the appropriate one is “sexual diethism”.

113: “A higher visibility”: omit “a”

115: You say “one important function”, but then give two functions.  Re-phrase this.

138: “was comprised of nine…” should be “comprised nine…”

231: This should be “sex differences in dispersal”, not “sexual dispersal”

239: “Male-male competition to attract females is primordial” – “primordial” is a strange term in this context.  “Primary”?  “Most important”?  However, I disagree that male competition in chimpanzees is mostly about “attracting” females.  This implies that it functions mostly to influence female mate choice, but female choice seems to have limited importance in chimpanzees other than its influence on whether male consortship attempts succeed (although the small size of the study community and small number of adult males may provide females there with more potential to exercise choice than is typical).

314: “Given that inter-individual call rate is likely to be greater than within-individual rate” -- ?  Something is missing here.  Do you mean that “variation” in call rate is probably greater among than within individuals?  If this is what you want to say, what do you mean by “within-individual variation” in call rates?
Experimental design: The authors have explained their methods and I have no serious concerns about the statistical methods.  However, I have one question about definitions and another more substantive one about the methods.

First: Did you categorize “arrival” pant hoots (those given on arrival at a food source) as associated with “traveling” or with “feeding”?  How did you determine when a pant hoot was associated with feeding?

Initially, I wondered why you talked about the correlations between calling rates, on the one hand, and the “proportions” of time spent feeding or traveling, on the other, and did not simply ask whether rates were higher while the chimpanzees were feeding and/or traveling than they were when the chimpanzees were engaged in other activities.  Then I realized that you used each hour of a focal follow as a data point, and that in these analyses, the dependent variable was the number of calls per hour and the predictor variables were the % of the hour the focal subject spent feeding or spent traveling.  This does not measure calling rates during feeding or traveling; calculating those measure would require counting the number of calls the individual gave while he or she was actually feeding and dividing by the time they were feeding, and doing the same for calls they gave while they were actually traveling.  It is feasible, for example – if not terribly likely – that a focal individual calls 10 times in an hour during which they feed for 30 minutes, but none of the calls occur while they are actually feeding.  If you re-did the analysis to examine call rates per hour spent feeding or traveling (instead of instead of rates per hour in relation to the 5 of the hour they were feeding or traveling), the results might change little, but this seems to me to be the proper way to address the question.
Validity of the findings: I don’t have any concerns regarding the validity of the findings per se other than my question above about rates of calling while individuals were feeding or traveling.

A couple of comments about the Discussion, though:

238: Delgado did not actually show that male orangutan long calls “advertise fitness”.  Fitness is a measure of relative reproductive success (plus any indirect benefits derived via kin selection).  Data on long calling are consistent with the hypothesis that the calls provide information about male quality, and the very limited paternity data show that flanged males, who give the calls, generally have higher reproductive success than unflanged males, but this does not mean that calls advertise fitness.

245: Why call pant hoots “agonistic calls”?

Also (see below), I think the authors need to more to show that their findings are novel – or perhaps better, to show why their results are important for conservation purposes.
Additional comments: My main general concern is that in the Introduction, the authors set up their manuscript by invoking the importance of having behavioral data to validate the use of PAM as a conservation tool (specifically to validate its use as a way to estimate population densities of species that give loud calls).  They then provide results of analyses of variation in pant hooting rates in relation to differences in activity state and to variation in vegetation types, etc., and they examine the question of sex differences.  They don’t tie this in with the over-arching question regarding the value of PAM.  Most of the results are not surprising.  The extensive literature on chimpanzee loud calls (pant hoots, specifically) is replete with statements that chimpanzees call most often when they are traveling, when they arrive at food sources, especially fruiting trees, and during feeding bouts.  Kallan might have been the first to publish comparative numerical data on male and female calling rates, but Clark (1996, Amer. J. Primatol. 39: 159; cf. Clark & Wrangham, 1994) stated that adult males called significantly more often than subadult males, and that females called at rates comparable to those of subadult males.  He further stated that “most adult females called to infrequently to be analyzed statistically” and, in Clark & Wrangham (1994), that “[All-] Female parties called rarely” and did not call on arrival at unoccupied feeding trees.  Goodall also referred to sex differences in pant hooting, as did Marler in his early work on vocalizations, albeit without providing the kind of quantitative data the authors have provided in Table 1.  For that matter, the data presented here do not confirm that male chimpanzees “vocalize” more than twice as often as females, contrary to the statement on 222-223.  That may be correct, but the authors have explicitly not included data on low volume, short-distance calls, so we don’t know whether a sex difference exists for those calls and, if so, what its direction is.  Also, while we could expect to find a sex difference in pant-hooting rates (as noted above), the authors have pooled data on these with data on screams and barks, so we don’t actually know the extent of sex difference in each of those three kinds of vocalizations.  On 243, the authors note that the bimodal temporal pattern of calling (pant-hooting?) was “similar to what has been reported previously at Issa”.  What is really new, then, besides the examination of calling rates in relation to vegetation type?  Even there, the authors can only speculate about why the differences might exist.

Back to my initial point: what are the lessons for those concerned with chimpanzee conservation (or conservation of other mammals) on a landscape/habitat scale, especially when the animals in question are not habituated and are difficult to find and follow?  What do we gain from the call rate data when it comes to assessing the status of chimpanzees in fragmented habitat outside of protected areas in Tanzania and planning conservation strategy, or for that matter, what lessons does this work have for those working on the conservation of “savanna” chimpanzees in West Africa (or chimpanzees in general)?


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: VOCAL COMMUNICATION IN WILD CHIMPANZEES: A CALL RATE STUDY
Review round: 1
Reviewer: 2

Output:
Basic reporting: Overall, the paper meets the reporting standards.

I found the paper easy to read and thought it was written well. I spotted a few minor typographical/grammar errors, which I note in the general comments to the authors.

As a statistician rather than an ecologist, I am not particularly qualified to comment on the completeness of the cited literature, but I found it interesting to read nonetheless.

The figures, and tables are well presented, and the raw data have been shared.

The paper sets out the aims of the paper nicely in Lines 102-118. This section does a great job of describing the specific hypotheses tested, along with the expected findings in light of the existing literature. The discussion and conclusion return to these hypotheses, providing answers based on statistical analysis of the data.
Experimental design: The description of the statistical analysis would benefit from more detail; I don't think I could replicate the analysis despite being very familiar with generalised linear mixed-effect models and the software they used. I thought the results could also involve a more rigorous interpretation of the parameter estimates. Please find more detail in the general comments to the authors.

Otherwise, the paper meets the experimental design standards. The research questions were well-described, and their surveys appear well-designed.
Validity of the findings: The raw data have been provided. I have a few suggestions regarding the statistical analysis; see my general comments below.

The conclusions appear to be well-stated and interesting to me, although as a statistician rather than an ecologist I am not really qualified to comment.

Speculation is clearly identified throughout the discussion and conclusions.
Additional comments: I thought this was an interesting, well-written, and well-motivated paper. Estimating animal density with acoustic spatial capture-recapture (SCR) methods becomes relatively straightforward once a call rate estimate has been established, which is a central objective of this work. The authors made convincing arguments about why understanding chimpanzee call rate is important in its own right.

I was glad to see the authors use a generalised linear mixed effects model to analyse the call-rate data, and I agree that this is probably the best approach to take. In particular, they have used an appropriate response distribution (negative binomial, to account for overdispersion relative to the Poisson distribution) and have done a good job of checking the model's assumptions.

My main source of confusion about the model is linked to Lines 173-174, regarding the random effect for each 'follow'. What exactly is a 'follow'? Are two observations part of the same 'follow' if they are associated with the same individual chimpanzee? Or only if they are observations associated with the same individual within the same day? Overall, I think random effects in a two-level hierarchy are required: you might expect two observations taken on the same individual on the same day to be more similar than observations taken on the same individual but on different days, because an individual might be more vocal on some days than others. At the same time, you might expect two observations taken on the same individual to be more similar than two observations taken on different individuals, because some individuals might generally be more vocal than others. I suspect having random effects for both days ('follows'?) and individuals will probably do a better job at modelling the dependencies between observations. Moreover, putting some sort of temporal structure on observations taken within the same day would probably be advisable, because two observations taken at similar times are more likely to be similar than observations taken at different ends of the day, over and above the fixed effects due to time of day. I believe adding both levels of random effects, along with the temporal covariance structure, can be achieved easily enough with R package the authors have used, so hopefully this doesn't require too much more work. I think this approach might provide some answers to questions posed in the "limitations" section (Lines 313-315), because it allows explicit estimation of both between-hour variance and between-individual variance.

Here are a few more minor specific comments:

Line 67: Because "calls" is a countable noun, I think "fewer" is better than "less" in this sentence.

Line 79: Although I don't think it was made clear in the Marques et al (2012) paper, they actually estimated call density (calls per unit time per unit space) rather than animal density (animals per unit space).

Lines 96-97: I think "... at different rates depending on sex" sounds a little cleaner than "in function of sex".

Lines 102-118: I thought this section did a great job of describing and summarising this study.

Lines 131-134, and elsewhere: I haven't grasped exactly what the plus/minus symbols mean throughout this paper. In some cases I get the feeling they are proving a total range of the data; for example here I think "mean monthly rainfall of 118.4 +/- 92mm" means that the driest months have 26.4 mm of rain, whereas the wettest have 210.4 of rain. In other cases I got the feeling they were indicating the margin of error of an estimate to provide a (presumably 95%) confidence interval (e.g., Lines 225-226). Providing a range to cover all the data is a very different thing to a range of plausible values for a parameter. I'd recommend avoiding using the plus/minus symbol, and describing in words with more clarity, for example by using sentences like "monthly rainfall in the wet season ranges from 26.4 to 210.4 mm of rain", and "a 95% confidence interval for the mean call rate of adult females is...". Also, the lower limit of the range given for rainfall in the dry season (Line 132; "0.6 +/- 0.9mm) goes negative, which may introduce confusion.

Lines 151-152: There's a missing right parenthesis somewhere in here.

Line 157: The term "scans" first appears here and it isn't ever defined. I'm sure what it means, but it seems to be important. What is a "scan"?

Line 180: Linear models assume the errors are normally distributed, not the residuals. Additionally, GLMs and GLMMs don't even have an assumption that the errors are normally distributed anyway, and so even when the model is correct the residuals may not appear to come from a normal distribution. Although I've never used the package, I think DHARMa is plotting randomised quantile residuals, which are a clever transformation of the residuals that should appear to have been generated by a normal distribution. I recommend rewording this sentence accordingly.

Line 190-191: The fact that call rate appears to vary between individuals verifies that a individual-level random effect should be incorporated in addition to the 'follow' random effect (assuming I am correctly interpreting what a 'follow' is).

Line 201-202: If I understand the model correctly, then the difference in vocalisation rates between males and females is a parameter explicitly estimated by the model, but this statement saying that males call about "twice as often" is quite vague. How about presenting the actual estimated relative difference, along with a corresponding confidence interval? I think this can be achieved as follows: Table 3 suggests that females call exp(-0.817) = 0.44 times as often as males (which indeed roughly matches the "males call twice as often" wording above). A 95% CI for this would be exp(-0.817 +/- 1.96*0.216), or (0.29, 0.67). In other words, with 95% confidence, males call between 1.5 and 3.5 times as frequently as females. Communicating uncertainty around estimates like this in a statistically rigorous way would really strengthen this section.

I also felt that the other parameters estimated by the model could have been interpreted with similar rigour; this section states which variables were significantly related to  call rate, but it avoids reporting the size of the effects along with corresponding confidence intervals, which I think would greatly strengthen the results. Fitting a nice model but failing to interpret the estimated effects feels like the story is being left a little unfinished.

Lines 209-213: I wasn't sure why a Kruskall-Wallis test was being used to determine the significance of time-of-day. Couldn't the GLMM model be used to achieve this? Something like likelihood-ratio tests could be used to compare the three time levels to one another, with a manual correction used to account for multiple comparisons. A possible weakness of the Kruskall-Wallis approach is that it ignores dependence between observations made in the same follow, which may invalidate its assumptions. The GLMM deals with this dependence, and therefore seems like a more robust model from which conclusions should be drawn. Perhaps I'm misunderstanding things, in which case I think more detail is required to justify the use of a Kruskall-Wallis test around Lines 184-186.

Line 276-277: I don't think "call rate did not change with feeding behaviour" is quite the correct interpretation; a large p-value fails to reject the null hypothesis, but it does not imply the null hypothesis is true.

Line 297-289: Same as above; a non-significant effect of a swollen female in the party does not imply that there is no difference, only that there isn't evidence a difference exist.

Line 317: I think saying that the data might have "been biased" is a bit negative, and may lead to readers unfairly questioning the validity of this study. As far as I can tell, the data set is great! I think the point being made is that bias can be introduced by a researcher overgeneralising the results of this study to other points in time; this isn't the fault of the data at all, but rather inappropriate conclusions being drawn based on perfectly valid data. I'd recommend rephrasing this to say that conclusions shouldn't be generalised to other scenarios other than that which the data were collected.

Lines 360-361: The common terms are either "spatial capture-recapture (SCR)" or "spatially explicit capture-recapture (SECR)", rather than "spatially capture recapture".

Line 373: Typo; should be "... for their help in the field" rather than "... for their help a in the field".


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

Input:
Title: VOCAL COMMUNICATION IN WILD CHIMPANZEES: A CALL RATE STUDY
Review round: 2
Reviewer: 1

Output:
Basic reporting: As per my original review, the paper meets the basic reporting standards.
Experimental design: The authors have greatly improved the description of their statistical model. I still have some questions/suggestions about their method, and I think an improvement could be made to strengthen their analysis. I provide further detail in the general comments below.
Validity of the findings: The paper largely meets the validity of findings criteria. I have a minor concern with the conclusion based on the Kruskall-Wallis test, which I outline in the general comments below.
Additional comments: The authors have done a great job with this revision, and the manuscripts has greatly improved. I have a few minor suggestions, listed at the end of this section.

My only substantial comment is about the use of the Kruskall-Wallis test. Thanks to the clear and concise description provided by the authors in their responses to my comments, I now have a much better understanding, which I'll summarise here to ensure I'm on the right page. Their initial attempt to analyse the data involved creating a categorical variable, with a level for every hour of the day, so for example if follows were conducted between 6am-6pm then there would be twelve categories (6-7am, ..., 5-6pm). Unsurprisingly, the large number of parameters required for this approach led to model identifiability issues and convergence failures during model fitting. For their GLMM, they therefore reduced the variable to only three categories. However, they still wished to compare data hour-by-hour, so they used the Kruskall-Wallis test separately.

My concern with this approach is that a Kruskall-Wallis test still assumes statistical independence, despite the authors acknowledging that data from two different follows are not statistically independent; their GLMM was constructed specifically to account for this dependence. If the independence assumption is violated, then it's possible for p-values to detect spurious effects at a rate higher than the selected significance level (i.e., a true null hypothesis will be rejected greater than 5% of the time), resulting in misleading conclusions.

Additionally, splitting a continuous explanatory variable (such as time) into a number of categories, be it three or twelve, is not generally consiered good practice; see textbooks such as Generalized Additive Models by Simon Wood and Vector Generalized Linear and Additive Models by Thomas Yee. A better approach in this situation is to fit the effect of time as a smooth function, for example using a spline or a generalised additive model. I think it's easy enough for the authors to do exactly this with their data set via the mgcv package using code something like the following:

fit.gam <- gam(y ~ s(individual, bs = "re") +
                   s(date, bs = "re") +
                   s(hour, bs = "cc") +
		   [any other fixed effects, like group size],
               knots = list(hour = c(0, 24)),
               family = "nb", data = df)

Then using plot(fit.gam) you can visualise the effect of hour on the underlying call rate as a smooth function, allowing you to see the spikes at dawn and dusk (for example). This model will also return a p-value for the significance of the time-of-day variable, just like the Kruskall-Wallis test, but is more appropriate because it also acknowledges dependencies among the observations. Note that using s(x, bs = "re") in mgcv is equivalent to using (1 | x) for random intercepts in lme4. I think this would be both more appropriate and more insightful than (1) the current GLMM with the three-level categorial variable for time and (2) the Kruskall-Wallis test. Generating conclusions from a single model is also conceptually neater than using two separate models.

To demonstrate this idea a little more clearly, I simulated data and fitted the model under a slightly simpler scenario (only individual random effects and no other explanatory variables other than hour of day), and the script is available here: http://bcstevenson.nfshost.com/chimpanzee-gams.r.

Minor comments follow.

L53: It might be best to define call density as the number of calls emitted per unit time *per individual* (just to avoid misinterpretation of number of animals emitted per unit time across the whole population).

L86: I think it's important to be clear about what you mean by a "loud call". How do you know if a call is "loud" or not? It seems problematic to define whether or not a call is "loud" based on the loudness of the call at its source (e.g., measured in dB), because you don't directly observe this variable. After reading on, I think the definition appears later on L160-162: a "loud" call is one that involved at least a scream, a pant-hoot, or a bark. Is that right? If so, I think the sentence here on L86 feels a little out of place, because using only "loud calls" and defining them in this way is particular to this very study, whereas this paragraph here is about PAM in general without focus on any particular species.

L106: Marques et al (2013) might be a better reference here because their study was about passive acoustic monitoring in general, whereas Stevenson et al (2015) is focused only on SCR.

L153-154: This is a nice definition and clarifies my confusion in my last review.

L180-182: I suggest "dependency among follows on the same day", because it's possible to misinterpret "at the same time" to mean the very same instant in time, rather than simply two follows in different hours of the same day.

L298, 305: In these lines the authors assert that they found that some explanatory variables "do not impact call rate", but, similarly to my previous review, these statements still sound like misinterpretations to me. I think they are being made due to large p-values, or possibly because explanatory variables were not included in the most parsimonious model. However, neither of these findings imply that the variable has no effect (or impact), only that there is no evidence that it does have impact. If the effect of a variable is very small it still has an "impact", but without sufficient data a model will not be able to detect its effect. Absence of evidence of an effect is not evidence that the effect is absent.


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: GENOME-WIDE ASSOCIATION STUDY AND POPULATION STRUCTURE ANALYSIS OF SEED-BOUND AMINO ACIDS AND TOTAL PROTEIN IN WATERMELON
Review round: 1
Reviewer: 1

Output:
Basic reporting: In this manuscript, the authors used a nature watermelon panel for GWAS analysis to detected the loci with seed-bound amino acids and total protein. The content was clear with professional English. References related with the content were also provided. The structure of manuscript was good and clear. The figures in the review version seemed in low-resolution.
Experimental design: The experiment design was good. I suggest the author make more analysis about the SNPs which you have got in the GWAS results for a further verification.
Validity of the findings: Some SNPs associated with the amino acids and total protein content in watermelon seeds were detected. Genes located in the mapping region were also analyzed in the discussion part. The speculation in the conclusions should be verified more.
Additional comments: 1. The authors used PCA results instead of population structure. For the GWAS panel, the population structure was not only PCA analysis. STRUCTURE and Admixture software would be used for population structure analysis.
2. The content from Line 255 to Line 314,was repetition of similar structures. As a suggestion, a table would be a better choice.
3. For the GWAS analysis, the Q-Q plot should be added.
4. According to the Manhattan plots, most traits exhibited some associated SNPs up the threshold line, this may due to the low re-sequencing depth. The seed amino acids and total protein seemed regulated by multiple genes as the quantitative trait . Due to the huge work for measurement these traits in multiple environments or years. I think the author should compare the SNP genotype (or the haplotype) with the phenotype to verify the accuracy of GWAS results. Maybe we can found some new information based on the above analysis. These SNP may only existed in some subspecies or variety.
5. For all the SNPs detected in the GWAS results? Is there some locus can used as the MAS tool ?
6. In the Results and Discussion part, the author  listed some candidate genes in the associated region. Is there some sequences variations in the candidate genes between different accessions ?


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: GENOME-WIDE ASSOCIATION STUDY AND POPULATION STRUCTURE ANALYSIS OF SEED-BOUND AMINO ACIDS AND TOTAL PROTEIN IN WATERMELON
Review round: 1
Reviewer: 2

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: This manuscript by Joshi et al. reports on the GWAS and SNPs for the genes associated with the variation in amino acids and total protein contents in the watermelon seeds. It also provides information on genetic resources that would be useful for breeding targeting the improvement of watermelon seeds for dietary purposes. The significant SNPs and candidate gene information explored in this study would be fundamental for understanding molecular networks controlling variations in the accumulation of specific amino acids and proteins.

Although the manuscript was written excellently and appropriate experiment procedures and analysis were demonstrated, I would like the authors to check the following recommendation to improve their paper.

The introduction is breaken into so many paragraphs and they need to be merged into maximum of 4 -5 paragraphs. The sentence from L71 to L74 may be moved to the next to L62 . The sentence in L78-79 seems not complete.

Please, start L122 as the second paragraph in M&M

There many errors in writing with uniformity for IS unit, Table and Figure in the text, such as L133, L134, L185, L195L237 (figure, Fig, Figure?). L253, Table S3 must be corrected to Supplemental Table 3.

The paragraph extending from L252 to L314 ('Genes under association' section) needs to be broken into several paragraphs.

In Discussion, sentences need to be rearranged accordingly to their contexts. For examples, L323-330 needs to be moved to the next to the sentence on L342

Finally, some information for other approaches and their results for improving protein contents in other crops need to be provided in the 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: GENOME-WIDE ASSOCIATION STUDY AND POPULATION STRUCTURE ANALYSIS OF SEED-BOUND AMINO ACIDS AND TOTAL PROTEIN IN WATERMELON
Review round: 2
Reviewer: 1

Output:
Basic reporting: The author have revised the manuscript seriously. Thank you for the author efforts.
Experimental design: No comment
Validity of the findings: No comment
Additional comments: No comment


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

Input:
Title: IDENTIFICATION AND VALIDATION OF A NOVEL ZINC FINGER PROTEIN-RELATED GENE-BASED PROGNOSTIC MODEL FOR BREAST CANCER
Review round: 1
Reviewer: 1

Output:
Basic reporting: Clearly written
Experimental design: Reasonable
Validity of the findings: Mostly fine
Additional comments: This study explores the clinical significance of the expression profiles of Zinc finger proteins in breast cancer. The authors identified 14 Zinc finger proteins that are differentially expressed in breast tumors as compared to normal cells and associated with patient survival. The authors focused on these 14 genes and generated a gene signature. This newly built signature is associated with patient prognosis, immune score, and drug sensitivity. Overall, the manuscript is carefully written, and the detailed discussion of the identified 14 genes is very valuable for future studies. Particularly, the identification of 8 zinc finger proteins (ZBED2, ZBED3, ZNF219, TANK, IKZF3, PARP12, ZNF239, and MECOM) that are not appreciated before is an important finding in this paper.

However, the authors heavily focused on the 14-gene signature and most of the findings are descriptive. Breast cancer subtypes are well established, and it is not clear how the 14-gene signature is unique and what aspects are better. Although the authors confirmed the prognostic value with another data cohort, it would be important to confirm such a prognostic value with other large data cohort such as METABRIC.

The expression profile of the 14 ZNFs-related gene prognostic signature (Figure 3C) is not clearly showing the differential expression profiles among these 14 zinc finger proteins. This also suggests that 14 genes are not necessary to build such a gene signature. Since these 14 genes are independently associated with prognosis, it would be more interesting to see common and distinct clinical features among these 14 genes based on the expression or mutations. For instance, how frequently are they co-expressed? What kind of breast tumor subtypes are associated with these genes?

It is also unclear why one of the well-known zinc finger proteins in breast cancer, GATA3, is excluded from the analysis. GATA3 expression is known to be correlated with patient survival.

Minor comments

Line 168 “Have an early than low-risk patients”
Please clarify this statement.

Figure 1 B: Since many plots are hidden, it would look better if the scale of X-axis is adjusted.


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: IDENTIFICATION AND VALIDATION OF A NOVEL ZINC FINGER PROTEIN-RELATED GENE-BASED PROGNOSTIC MODEL FOR BREAST CANCER
Review round: 1
Reviewer: 2

Output:
Basic reporting: The article is well written and the methodology is well presented. I would suggest a schematic representation of the methodology since the mere description is difficult to grasp. I got a single sentence that is not clear: line 168, apparently, a word is missing.

The literature references are comprehensive and adequate.

The article structure, figures, and tables are clear and relevant. The article used publicly available raw data. But the generated data are well presented and completed. I would suggest the authors to share the R scripts used in the article.
Experimental design: The article is within the journal's scope.

The question is well defined, is relevant, and meaningful. The question is interesting and has clinical applications and also is relevant from a basic research perspective.

The investigation used a well-supported methodology and follow ethical standards.

The methods are well described with reasonable details. I am suggesting the inclusion of the R scripts to guarantee replication.
Validity of the findings: The article proposes a score based on zinc fingers proteins as a predictive tool to use on clinical set to detect patient OS and drug response. The justification of the proposed score is not justified, but it seems to work quite well on this problem. The methodology is adequate and the discussion is well presented.

The basic data is public and the results are reasonably robust, although some AUC values are below 0.7.

The conclusions are well supported by the results and answer the stated problem.
Additional comments: In my opinion, the article presents interesting results that could be used in other clinical contexts. If the authors could furnish the scripts this could evolve to a powerful workflow to be applied to other cancer types.


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: IDENTIFICATION AND VALIDATION OF A NOVEL ZINC FINGER PROTEIN-RELATED GENE-BASED PROGNOSTIC MODEL FOR BREAST CANCER
Review round: 2
Reviewer: 1

Output:
Basic reporting: Clearly written
Experimental design: Well-considered
Validity of the findings: Sufficient validation was performed
Additional comments: The authors addressed all concerns.


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: DIFFERENTIAL TRANSCRIPTOME ANALYSIS AND IDENTIFICATION OF GENES RELATED TO RESISTANCE TO BLIGHT IN THREE VARIETIES OF BAMBUSA PERVARIABILIS × DENDROCALAMOPSIS GRANDIS
Review round: 1
Reviewer: 1

Output:
Basic reporting: The Ms was well writeen in English, technically correct text, the situation of bamboo and the research progress were described in detail.
Experimental design: By sequencing the transcriptome of three hybrid offspring before and after infection, the experimental design is basically reasonable. And I think that the phenotypic changes before and after infection must be added, especially the differences in disease resistance, so as to lay a foundation for transcriptome sequencing and differential gene screening.
Validity of the findings: The sequencing results was analyzed and differentially expressed genes were screened, but which of these DEGs are related to disease resistance? In addition, how can the authors exclude the gene expression differences caused by the genetic background differences of the three hybrid offspring 3,6,8#? Therefore, gene expression differences caused by genetic background differences should be excluded in differential gene analysis.
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: DIFFERENTIAL TRANSCRIPTOME ANALYSIS AND IDENTIFICATION OF GENES RELATED TO RESISTANCE TO BLIGHT IN THREE VARIETIES OF BAMBUSA PERVARIABILIS × DENDROCALAMOPSIS GRANDIS
Review round: 1
Reviewer: 2

Output:
Basic reporting: In this manuscript, comparative transcriptomics using RNA-seq data were applied to analyze different expressed genes (DEGs) in three different species of B. pervariabilis × D. grandis with variable resistance and in the same specie under different treatments.  The functional genes identified here are important genetic resources to improve the resistance of B. pervariabilis × D. grandis to shoot blight.
Experimental design: Generally, the A. phaeospermum inoculated materials were compared with their respective water controls, and then compared with each other to identify DEGs. Why did you perform two kinds of transcriptome comparison to identify DEGs? One is between A. phaeospermum infection and sterile water inoculation in the same species. The other is in different A. phaeospermum inoculated varieties. You can discuss this.
Validity of the findings: No comment
Additional comments: For introduction
Too many descriptions about the infection symptoms of A. phaeospermum in bamboos. Author cited many research on host-pathogen interaction and application of transcriptomics which were performed on other crops. No related studies on hybrid bamboos?

For Materials and methods
1.	Line113, how were spores cultured?
2.	Please provide the link or accession number for your transcriptome raw data in the manuscript.
3.	Differentially expressed gene analysis section. I think it is better to describe in detail how are compared among various materials or various treatments, and write clearly what J3, J6, J8, S3, S6, and S8 stand for. This would make reader to understand the results easier.
4.	Line 154, The authors do not adequately state their procedures for qRT-PCR. Please add the appropriate details or add the literature.

For results
1.	Line 167. “Comparative analysis of transcriptomic expression data showed that 4387, 6316 and 4159 DEGs were obtained from the #3, #6 and #8 B. pervariabilis × D. grandis varieties inoculated with A. phaeospermum or sterile water, respectively”. It's not easy to understand what you mean without reference to Table 2. I suggest the following changes: “Comparative analysis of transcriptomic expression data was determined between A. phaeospermum infection and sterile water inoculation in the same species. The results showed that 4387, 6316 and 4159 DEGs were obtained in the #3, #6 and #8 varieties, respectively.”
2.	Line 175-178. “In the #3, #6 and #8 B. pervariabilis × D. grandis varieties after inoculation, a comparative analysis of differences in transcriptomic expression between varieties #6 and #3, #6 and #8, and #8 and #3 found 22053, 22464, and 4638 DEGs, respectively, including 7295, 7936, and 1778 upregulated genes and 14758, 14528, and 2860 downregulated genes.” I suggested reword this long sentence to “When comparing DEGs from different A. phaeospermum inoculated varieties, 22053, 22464, and 4638 DEGs were identified between varieties #6 and #3, #6 and #8, and #8 and #3, respectively, including 7295, 7936, and 1778 upregulated genes and 14758, 14528, and 2860 downregulated genes.”
3.	Line 179. “In total, 2154 DEGs were shared among the three varieties (Fig. 2); of them, 23 DEGs were upregulated in the three species, and 143 DEGs were downregulated (Table S2)”. Were all 23 genes up-regulated and 143 genes down-regulated in all three varieties? Please provide the expression of each gene in each treatment in Table S2.
4.	Line180, ‘By comparing these DEGs, ------’, these DEGs referred what?
5.	Line 186-188. ‘In the COG database, 1513, 2776, and 1421 and 1467, 7517, and 7417 DEGs were found for #3, #6 and #8 B. pervariabilis × D. grandis after inoculation with A. phaeospermum or sterile water, respectively.’ this sentence was more confused. Which genes were used for COG annotation, and how many genes were annotated in COG database?
6.	Line 189, I couldn't understand what was said in Fig. S1 due to its notes vague in the figure. What does a, b, c, d, e and f stand for?
7.	Line 238-241. “Candidate genes were screened from the group of common differentially expressed genes from multiple comparison groups. Based on the enrichment of upregulated DEGs in the GO and KEGG databases and combined with functional annotation information, 21 candidate genes for disease resistance were selected”. Why screening the candidate genes from the common DEGs? How selected 21 candidate genes based on the enrichment of upregulated DEGs in the GO and KEGG databases? Please discussion this.
For discussion
1.	The discussion mainly discussed the genes listed in Table 3. You can add discuss about the materials and methods used in this research.
2.	Line 257, please give the full name of PEG.


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: DIFFERENTIAL TRANSCRIPTOME ANALYSIS AND IDENTIFICATION OF GENES RELATED TO RESISTANCE TO BLIGHT IN THREE VARIETIES OF BAMBUSA PERVARIABILIS × DENDROCALAMOPSIS GRANDIS
Review round: 1
Reviewer: 3

Output:
Basic reporting: The authors sequenced the transcriptomes of three varieties of hybrid bamboos with different degrees of pathogen resistance, and compared differentially expressed genes before and after inoculation, and between different varieties.
Experimental design: Generally, the experimental designs are feasible. But the processes should be clearer. For example, the tools used to identify DEGs.
Validity of the findings: The findings are reasonable.
Additional comments: Line 24 specie？
Lines 28-30 This sentence is unclear.
Line 31 another expression of the former sentence
Line 41 what’s the difference between species in title and varieties here？
Line 75 Liberibacter asiaticus, should be italic?
Line 109 use species or variety should be uniform across the paper
Line 134 HISAT2, version and parameters should be provided
Line 136-138 Is this the link to the reference genome? you can cite their reports or the database. Can hisat2 be used to align short reads to transcriptome assembly? Moreover, you should provide the Transcriptome Shotgun Assembly (TSA) accession.
Line 141 generally, FPKM is superior than RPKM and was widely used at present. What is the tool you used to identify differentially expressed genes?
Line 170 species? Or varieties?
Lines 173-174 you said 394 DEGs were shared, but why only 74 genes were un- and downregulated? How about the 320 shared genes?
Lines 175-178 you should think about the constitute differences between these varieties.
Line 179-180 similar to lines 173-174
Lines 185-235 should be separated and placed after the suitable results of differential gene analysis.
Table 2 what is J and S? should be included in the table legend.


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

Input:
Title: DIFFERENTIAL TRANSCRIPTOME ANALYSIS AND IDENTIFICATION OF GENES RELATED TO RESISTANCE TO BLIGHT IN THREE VARIETIES OF BAMBUSA PERVARIABILIS × DENDROCALAMOPSIS GRANDIS
Review round: 2
Reviewer: 1

Output:
Basic reporting: No other comments
Experimental design: No other comments
Validity of the findings: Through bacterial inoculation, transcriptome sequencing and differential gene analysis, the MS finally screened a series of key DEGs, and analyzed the expression of five DEGs genes. The MS has been modified according to advices of the reviewer, so the MS can be accepted.
Additional comments: No other 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: DIFFERENTIAL TRANSCRIPTOME ANALYSIS AND IDENTIFICATION OF GENES RELATED TO RESISTANCE TO BLIGHT IN THREE VARIETIES OF BAMBUSA PERVARIABILIS × DENDROCALAMOPSIS GRANDIS
Review round: 2
Reviewer: 2

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: All points have been well addressed.


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: VARIATION OF SOIL MICROBIAL CARBON USE EFFICIENCY (CUE) AND ITS INFLUENCE MECHANISM IN THE CONTEXT OF GLOBAL ENVIRONMENTAL CHANGE: A REVIEW
Review round: 1
Reviewer: 1

Output:
Basic reporting: The review is dealing with soil Microbial Carbon Use Efficiency (CUE) variation as a result of Global Environmental Change.
CUE is an important concept for understanding the future trajectory of soil-climate feedbacks which was not sufficiently reviewed.  CUE is thus increasingly recognized by microbiologists, ecologists, and modelers alike as essential for understanding the causes and consequences of microbial C cycling. Accordingly, the present review is interesting to understand the consequences of environmental change on CUE  and C cycling in general.
Despite the importance of the topic covered in this review, however, there are many drawbacks in this work.
First,  the review was not well organized and could be improved in term of structure and content. In fact, CUE reflects a collection of numerous processes (physiological, ecological and community dynamics) that influence C metabolism across varying scales of time and space. The responses of these  processes to environmental changes are very important to understand the relevance of CUE at different scales from a single microbial cell to entire ecosystems. However,  these processes were not well presented in this review. For example, the environmental sensitivity of microbial physiology and microbial community structure and activity are very important to determine the impact of environmental changes on CUE. However, authors have not well developed these aspects in their review and instead they focused on quantifying CUE methods which were previously reported in many reviews.
Figures and tables should be added to better illustrate the impact of environmental changes   on CUE and to give more information about the possible interaction between the most influencing factors and microbial soil communities.
Experimental design: CUE depends on a complex processes linked together at different scales which makes the study of the CUE  variation a challenging task. Thus, the impact of each environmental changes   on these processes should be investigated separately. Authors are invited to firstly define CUE and indicate the parameters influencing  microbial carbon metabolism in relation to 1) soil characteristics (pH, soil moisture, nutrients availability, organic matter composition, organo-mineral interaction, physical properties such as porosity ...) and 2) soil microbial community structure and dynamic to determine the interaction between different microbial groups involved in C cycling. Soil microbial activity change from one ecosystem to another according to carbon source, microbial community and ecological environmental factors. For instance, CUE is not the same in humid and semi arid zones. Thus, authors should highlighted the variation of CUE in different ecosystems and climatic zones as many researches were conducted on these aspects.  Investigating the impact of variation of global environmental change is more difficult as it is a dynamic process requiring mathematical models to analyze and to predict the evolution of CUE which depends on the interaction between different biological and physicochemical parameters at two different scales (time and space).
Validity of the findings: According to the purpose indicated in the introduction, Limits of ecological environmental factors  and Composition of microbial community are the most important sections of the review. Authors are invited to more develop these two sections and to analyze the possible correlation between the environmental factors  parameters on CUE and C cycling. Furthermore, a section on soil microbial adaptation and the impact of environmental factors on the dynamic and diversity of soil microbial communities should be added.
The conclusions were  not well linked to the purpose of this review.
Additional comments: No additional comments


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

Input:
Title: VARIATION OF SOIL MICROBIAL CARBON USE EFFICIENCY (CUE) AND ITS INFLUENCE MECHANISM IN THE CONTEXT OF GLOBAL ENVIRONMENTAL CHANGE: A REVIEW
Review round: 1
Reviewer: 2

Output:
Basic reporting: Reviewers' comments:

This manuscript entitled “Variation of soil microbial carbon use efficiency (CUE) and its Influence mechanism in the context of global environmental change” has shown general description of CUE and its measurement methods, also reviewed and analyzed the research progress of soil microbial CUE variation and influencing factors. In addition, it provides a theoretical foundation for scientist, researchers and relevant stake- holders to predict future climate change. Therefore, it needs to be further modified to improve the quality of this article.
The main concerns about the paper are as follows:
Experimental design: 1. The sentence: “Water stress, for example, inhibits the …..”. Author should mention some detailed researchers on the spreading of wastewater on the soil.
2. In the other article paragraphs the author does not give details of the articles cited in effect of water in CUE. We would have liked to see some impacts of using wastewaters by providing a section that discussion the effect on microbial composition, CUE, substrate intake. Also, include in the review work; recently published work related to valorization of OMW
3. For the subject some references are old and not all them are very relevant, use newer and at least highly cited ones.
4. We would have liked to see representative table citing different carbon sources (acids, sugars, lignin, phenol, etc.) on the CUE.
Validity of the findings: None.
Additional comments: 1.	Line 70: interactions interactions (delete repeated word).
2.	Line 74: Full stop for the sentence “sequestration in soil ecosystems.”
3.	Line 141 “and methods based…” to Line 142: too long sentence.
4.	Author should add citation in the section of Substrate absorption rate method and Substrate concentration change method.
5.	Line 261 Delete repetitive references
6.Line 356: soil organic carbon (12.3). (18.2-75)….. More explanation??


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: VARIATION OF SOIL MICROBIAL CARBON USE EFFICIENCY (CUE) AND ITS INFLUENCE MECHANISM IN THE CONTEXT OF GLOBAL ENVIRONMENTAL CHANGE: A REVIEW
Review round: 2
Reviewer: 1

Output:
Basic reporting: 1. Authors should enhance the figure resolution.
2. Line 647:  surface surfaces (delete repeated word).
Experimental design: 1. Author should include in the review work; recently published work related to valorization of OMW: a. https://doi.org/10.1002/cbdv.201900608   ;  b. https://doi.org/10.1007/s13762-021-03145-0
2. Divide your article into clearly defined and numbered sections like numbered 1.1 (then 1.1.1, 1.1.2, ...), 1.2, etc so that the reader can follow and understand the tenor of the paper.
Validity of the findings: Good.
Additional comments: No additional comments


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

Input:
Title: EFFECT OF YEAST AND ESSENTIAL OIL-ENRICHED DIETS ON CRITICAL DETERMINANTS OF HEALTH AND IMMUNE FUNCTION IN AFRICANIZED APIS MELLIFERA
Review round: 1
Reviewer: 1

Output:
Basic reporting: Overall, the article was written in clear, unambiguous English.

1.	Minor grammar revisions are needed (e.g. lines 24, 47, 69-70, 71-73, 103, 110-111, 176-178, 206-209, 350).
2.	I suggest using ‘honey bees’ instead of ‘honeybees’ throughout the manuscript (Snodgrass, 1956).
3.	Line 60. Please consider including the reference for using yeast of S. pectoralis (Lizama, 2011), and perhaps use ‘bees’ instead of ‘honey bees’.
4.	In line 72, please list the four ‘informative variables’.
5.	Please consider the use of a specific stage of the bee development instead of ‘broods’, such as ‘worker pupae’ (line 154).
6.	I assume the authors refer to the role of glucose oxidase in social immunity, but the term ‘community defense function’ (line 197) is unclear, it would be helpful if the authors rephrase or define the concept.
7.	The authors use the term ‘house keeping primer’, I suggest following MIQUE guidelines (Bustin et al., 2009) and consider the term ‘reference gene’ (line 223, 230-231).
8.	Lines 287-289. Please check the use of brackets; the paragraph is unclear, please revise structure.
9.	In line 337, the authors used the term ‘nutritional status’, however, this variable was not previously defined in the manuscript, it would be helpful if the authors define the concept or use the term “food intake”.
10.	In line 413, it is unclear if by ‘markers’ you refer to gene expression.
11.	As a minor comment, it would be helpful if the authors present the scientific name the first time that the organism appears in the manuscript and use the abbreviation or common name after. For example, the scientific name of Apis mellifera is in line 370 but its firs mention in in line 45, ‘native bees’ are mentioned in line 87, but their scientific name was included until line 95, and the scientific name of the brewer’s yeast is specified in line 380 but previously mentioned in line 344.
12.	Also, I suggest revising the nomenclature of genes; italicize throughout the manuscript and be consistent to avoid confusion (e.g. line 497 had the name of the genes -or proteins- and not the italicized abbreviations.)

The authors provided information and references in the background section to show context. However, I would suggest revising the first paragraph of the manuscript and include additional information on why nutrition is important for immune responses and an appropriate reference (lines 46-48).

Also, it would be clearer for the reader if the authors use one definition for your variables. For example, you are using ‘critical markers’ and ‘critical regulators’ as synonyms, and ‘defensive traits’, ‘immune traits’, 'immune defensive traits’ and ‘social defensive characteristics’ interchangeably, which makes the reading hard to follow. ‘Defensive traits’ and ‘critical markers’ are in the title, the authors are not defining or listing the traits or markers in the manuscript clearly.
Experimental design: The authors described the methods in detail, there is enough information to replicate the study. The research question is well defined. However, I recommend revising the following:

1. The reference of the software used to design the primers mentioned in line 226.
2. Please include the primer concentration (line 237) and the reference of the supplementary excel file, which complements the information of the section ‘Gene expression: RNA Extraction, First-Strand cDNA Synthesis, and qPCR’.
3. Schmittgen & Livak (2008) reported a method to calculate relative gene expression by normalizing the target gene expression to a reference gene and a calibrator. It would be helpful if you report the relative fold change of the treatment groups compared to the control in the ‘Results’ section, and not only the relative expression normalized to the reference gene (lines 301-303).
4. Please consider removing the information in lines 198-210, it is the rationale for using those genes in the study but it’s not part of the ‘Materials and Methods’ section, the authors could incorporate the information in the ‘Introduction’.
5. In the supplementary excel file, please include the units for food intake, and define the treatment key (A-I).
Validity of the findings: The statistical analyses seem appropriate, but I suggest the following:

1.  To include if the data was tested for normality and homogeneity of variance before subjecting it to a mixed model (1merTest, which to the best of my knowledge is an ANOVA).
2. In the 'Results' section, please include degrees of freedom and the sample size (lines 277-281, 288-290, 296-301, 307-313, 322-327).
3. The authors report the variance withing each treatment (lines 281-285, 290-295, 303-306, 318-320, 329-33); if the authors want to sustain that there are is no significant variability between groups, I recommend considering an appropriate analysis, such as Levene’s test.
4. Honey bee immune responses are complex, and is difficult to make assumptions on individual and social immunity based on the expression of three immune related genes and two heath markers (survival and food intake). I suggest revising lines 88-90 to acknowledge the limitations of the study. The approach of the discussion is in line with the scope of the study.
Additional comments: The authors provide valuable information on the effect of diets enriched with bee associated yeasts (Starmerella bombicola, Starmerella etchellsii, Starmerella bombicola 2, Zygosaccharomyces mellis, and Sacaromyces cerevisiae) and essential oils of Lippia graveolens ( carvacrol, thymol, and sesquiterpenes) on health markers (survival, food intake, fat body percentage) and the expression of immune related genes (Vitellogenin, prophenoloxidase, and glucose oxidase) in honey bees. The article provides enough background and context to understand the research gap. Also, the methodology and statistical methods seem appropriate, and the discussion is in line with the scope of the study. However, I suggest including more information on the effect of nutrition on honey bee immunity in the ‘Introduction’ section; following the MIQUE guidelines to report the methods and results of the gene expression analysis; revising the gene nomenclature throughout the manuscript; using an appropriate statistical analysis to report differences in variability between groups; and to use the same definition for the measured variables throughout the manuscript.

I hope the authors find my suggestions 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: EFFECT OF YEAST AND ESSENTIAL OIL-ENRICHED DIETS ON CRITICAL DETERMINANTS OF HEALTH AND IMMUNE FUNCTION IN AFRICANIZED APIS MELLIFERA
Review round: 1
Reviewer: 2

Output:
Basic reporting: see below
Experimental design: see below
Validity of the findings: see below
Additional comments: The manuscript entitled “Effect of yeast and essential oils-enriched diets on critical markers in the immune system and defensive traits of Africanized Apis mellifera” aimed to analyze the effects of the dietary supplementation with native yeasts or EOs on workers bee in lab conditions. For that, worker bee survival, fatty body mass, and gene expression of vitellogenin (Vg), prophenoloxidase (proPO), and glucose oxidase (GOx) were measured to evaluate the effect of additive components.
Several native yeast strains obtained from Scaptotrigona pectoralis, not from Apis mellifera as is suggested by the authors, and three chemotypes of Mexican oregano, Lippia graveolens that differ in their main dominant component were tested.

In addition to the minor comments attached below, I have two main concerns that arise from the review of this manuscript:

First, I think the assays with yeast are independent of the EOs assays. The aims are completely different and the possible conclusions are unrelated to each other.
When yeasts are added to a normal bee diet as a supplement it is expected that the metabolism and immune system of bees remain optimal as with a normal diet. Maybe an immune challenge can be expected? In this case, the response variables chosen by the authors were correct to test that aim. However, when phytochemicals (as EOs) are added to a normal diet as an additive is expected that this compound acts as a xenobiotic, not as a supplement since EOs are not present in the diet naturally. In that case, to be metabolized after its ingestion, EOs possibly induce detoxification mechanisms. Thus, the variable response to be tested should be related to the search for transcripts associated with detoxification pathways, for example, not immune genes. (Berenbaum & Johnson. 2015. Xenobiotic detoxification pathways in honey bees. Current Opinion in Insect Science).
Moreover, for the determinations tested I think that the sampling size for fat body mass (three bees/replica every three days) and gene expression (three bees/replica pooled sampled every three days) are very low. Due to the high costs of molecular determinations, in a future experiment, authors could choose to include more individuals (without pooled) in fewer sampling times to increase the reliability of the results obtained.

Second, the results obtained on bee survival.  All treatments were made maintained worker bees in captivity for 12 days. This is a habitual procedure in honey bee research. It is normal and desirable mortality lower than 20% in the control group. In the present research, mortality obtained in all assays is abnormally high and I think that some of the measured variables are inconsistent (as Fat body scarcely developed in mature bees) due to this.
In control treatments, mortality rates should not be higher than 10-20 %. If this not happens, maybe some extra factor is influencing. Authors should check and rule out some factors before continuing or repeating these tests as the assays temperature range, humidity conditions, ventilation of the device, food availability, among others. For example, 36 C is an optimal temperature to achieve the emergence of bees from a capped comb but, for bioassays in devices with little ventilation, it is advisable not to exceed 32 degrees.

Specific comments:

 1. The section must be sorted in descending order:
-	Honeybee immune system (emphasizing the components that will be measured in this work)
-	Bee nutrition
-	Yeast background
-	EOs background
-	Aim/hypothesis/highlight

2. The aim is not well state

3. In M&M section minimize the background.

4. The groups were statistically compared with the control group, not with each other. Multiple comparisons were not made? Please, inform the N evaluated in each response variable.

5. The figures are many and individual (I suggest to group all yeast and all EOs). In addition, they do not show the statistical differences found nor the N. Authors should present figures with legend to be self-explanatory.