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: RELATIVE CONTRIBUTION OF ECOLOGICAL AND BIOLOGICAL ATTRIBUTES IN THE FINE-GRAIN STRUCTURE OF ANT-PLANT NETWORKS
Review round: 2
Reviewer: 1

Output:
Basic reporting: The quality of the writing is greatly improved relative to the previous version, but the article is still confusing in places. The authors still tend to use very complex sentences with nested clauses and many colons, semi-colons and commas; these complex structures mean that any small grammatical errors or unusual word choices take a great deal of reader energy to untangle. I suggest that the authors continue to simplify their writing, particularly in the discussion which is noticeably less polished than the rest of the manuscript. I have identified many small grammatical points (listed at the end of this review) that the authors can quickly address to improve the manuscript. In the same list I identify a few points in the discussion that are confusingly written and need revision.

Most of the authors claims are sufficiently referenced. Only line 601 seems to reference previous work that is not explicitly identified. The R statistical software and vegan package, however, should be cited each time they are referenced (the R base package was not cited at all). This discounts the vast amount of work that went into developing these publicly-available resources and is quite discourteous.

Raw data were shared, but do not include sufficient information to replicate the study. In particular, there is no key to species names or attributes (either the identity of the attribute or the factor levels). It is impossible for me to say which plants appear in which forest types or what types of nectaries they bear. A metadata file explaining the raw data should be provided. Ant head lengths, and abundances were not provided.

While not necessary to replicate the analysis in this paper, to provide the highest value raw data for future re-use it would also help if the raw data included the number of surveys on which an interaction was observed as well as the overall interaction frequency. Ideally, the network provided by each survey would be provided as a unique sample of the overall network. This would provide important information on the variance in interactions over time as well as the variance in frequencies the authors address here.

The manuscript is self-contained and the results reflect the hypotheses.

Detailed comments:
Abstract
-line 18: "showned" should be "shown", "species" should be "species'", and "counterpart" should be "counterparts"
-line 34: should be "highly-nested and non-modular"
-line 36: "higher dependences on ants" seems like it needs "than vice versa"
-line 39: "plants species" should be "plant species"
-lines 39-42: sentence is not grammatically correct. I suggest splitting it into several.
-lines 42-44: How does spatialtemporal heterogeneity depict the effect of abiotic factors? Heterogeneity in what? This sentence is very unclear.

Introduction
-line 50 and elsewhere : should be "in a community" not "at a community"
-line 60: should be "species' dependence on their counterparts"
-line 62: mismatched quotes around "nested"
-line 75: first word should be "Another"
-lines 79-81: This sentence is confusing. Something like "Like nestedness in mutualist networks, modularity is thought to increase the persistence of species in antagonistic networks." It also seems like the sentence starting in line 81 should begin a new paragraph.
-line 98: typo in reference
-line 104: suggest replacing "importantly" with "significantly". Odd word choice.
-line 106: start a new sentence after the reference. Missing a "to" in "refer to here".
-line 110: should be "structuring the fine-scale patterns"
-line 123: "its" should be "their" since networks is plural
-line 130: no hyphen in "network level"
-line 140: should read "include the social..." since you are about to give a partial list of attributes
-line 147: no comma after "behavior"
-line 148: "influence" should be "influences" and "given that" should be "since"
-line 156: remove "-either addressing a couple of biological or abiotic factors,". Not necessary since you state that abiotic and biotic factors were explored separately in line 155.
-line 164: "Which" should probably be "What"

Materials and Methods
-lines 197-199: this ordering is confusing. Maybe "between "open" and "shaded" habitats, with the first three habitats () being included in the former physiognomy and the other three habitats () included in the latter".
-lines 200-201: Define "floristic similarity". How is it calculated? What aspects of flowers are included?
-line 210: reagent stripes or reagent strips?
-lines 220-221: remove commas. "disperse" should be "dispersed"
-line 224: should be "a widely dispersed location and that this could result"
lines 231-242 and 244-256: These two sentences span over 10 lines each and are quite difficult to parse. It may be better to list the attribtes briefly first and then, in separate sentences, give the details about each attribute.
-line 294: "values up to 1" is confusing given that values >1 are reported in the results. Do you mean "values close to 1"?
-line 299: repeated ")."
-line 314: should be "Bray-Curtis dissimilarity"
-lines 320, 323, 324: missing citations for the R base package and a citation for vegan on the second reference
-line 331: the "2" should be a superscript
-lines 331-332: it isn't clear how floristic similarity relates to network structure, especially since the raw data includes only a single network.

Results
-line 339 and elsewhere: should be "consisted of"
-line 340: It isn't clear what the difference is between "quantitative interactions" and "species associations". I infer that quantitative interactions are single observations and species associations are plant-ant pairs, but I'm not sure.
-line 342: should be "detected in"
-line 352: should be "Regarding 'species strength'"
-line 353 and elsewhere: "standout" should be "stand out". "standout" is an adjective and "stand out" is the verb.
- lines 353-355 would be clearer as 2 sentences.
- line 355 and elsewhere: it looks like "EFnectar" should be two words
- line 358: "peculiar" is an unusual word choice. Peculiar in what way? Are these ants morphologically or behaviorally distinct?
-line 366 and elsewhere: "disperse nectaries" should probably be "dispersed nectaries" to match "circumscribed nectaries".
-line 367: "amount of" should probably be "number of"
-line 375: it seems like a word is missing here. Maybe "by attracting a distinct group of associated ant species"?
-line 377: "considerable" should be "considerably"
-lines 382-383: "being an acceptble value" doesn't make much sense. I suggest shortening this line to "... (0.17) and suggests that ..."
-line 390 and elsewhere: "cero" should be "zero"
-line 392: "superior part" should be "upper part"
-line 408: "were significant to explain" doesn't make sense. You could simply say "explained". "network fine-grained structure" should be "network's fine-grained structure"
-line 413: "differential associations form" should be "different associations from"

Discussion
-line 421: "occur" should be "occurs"
-line 422: "relative" should be "relatively"
-line 423: "exist" should be "exists"
-line 424: "components of" should be "components for"
-line 428: "to forage" should be "for foraging"
-line 442: remove "mere", "explain" should be "explained"
-line 444: "possible" should be "possibly"
-line 448: again, "peculiar" is an odd word and makes me think that there is something wrong with the ants the interact with highly-specialised plants.
-line 460: "of rapid up taking" should be "to rapidly take up"
-line 474: subheading is not grammatically corred. Maybe "Relative contributions of attributes to the assemblage of pairwise interactions"?
-line 475: remove comma. Seems to be missing a word - maybe "associations grouped according to habitat"?
-line 477: "seems" should be "seem"
-line 490: "carbohydrates" should be "carbohydrate"
-lines 491, 498, 505, 508, 510: remove commas
-line 508: should be "among-habitat" and "provided in"
-line 510: "display" should be "displays"
-lines 512-516: This sentence makes me miss a brief description of the network as well, just to tie it all together.
-line 519: should be "considered in our analysis but rendered"
-line 521: remove comma
-line 525: should be "more plant species with EFNs"
-line 527: "being" should be "was"
-lines 527-528: the clause after the comma doesn't clearly relate to the first clause.
- lines 528-529: This isn't clearly shown... did you also find this in your study? Wasn't clear in the results.
-line 530: "Other potentially..." seems like it could start a new paragraph.
-line 538-539: it's not clear whether you mean that the intensity and duration of your sampling could demonstrate the spatial and temporal variation, or that it was not sufficient to do so? As written, it could be either, and the raw data are a single web so give no information about any sort of variation.
-line 543: should be "more thoroughly explains"
-lines 546-549: I suppose - but since your study did not show any such effect this does not seem particularly interesting.
-lines 549-553: This is the first time plant defense platicity has been mentioned, and it seems to come from out of nowhere. The use of "plasticity" makes me think of developmental flexibility but seems to be used as a synonym for "variability" here. It is also not immediately clear how EFNs relate to plant defense. I assume the nectaries attract ants which do the actual defending, but it is probably worth briefly stating this so that your paper is clear to network ecologists who do not work with ants. There aren't enough details in the data and results to let me evaluate this claim for myself (I can't find which plants have EFNs or whether they have more diverse sets of ant partners).
-line 557: Order confusing. "attributes, such as species degree, within a mutualistic network" would make more sense.
-lines 555-563: This paragraph seems to be saying that bigger ants tend to lose competitions and therefore need to forage more widely. That is very counterintuitive.
-line 564: Add comma after "Overall"
-lines 566-567: remove commas.
-lines 564-568: Very long. Split into two sentences.
-line 572: "seems in accordance to" is a very odd phrase to have here. "makes sense in" or something similar would be better as it does not invoke any sort of laws/rules in human-altered systems (that have not been mentioned)
-line 574: "do" should be "does"

Conclusions
-line 581: "result" should be "was"
-line 582: "show" should be "showed"
-lines 583-586: not grammatically correct and confusing. Ending with plant defense and ant foraging adaptations pins this sentence on the weakest, least-supported part of the discussion and undermines your point.
-line 591: "in network topology" and "in the fine-grain network structure"
-line 592: "being thus further evidence" is strangely legalese phrasing and a bit confusing. I suggest ending the sentence at "interactions" and starting a new one with "This provides".
-lines 594-599: Are these things common knowledge? They were not clearly demonstrated in the present study.
-line 598: Add "and are" before "more effectively" to make this grammatically correct.
-line 601: Add a citation for previous work where you show that other factors could affect network structure. Only habitat type seems to have an effect in the present study.

Figure legends
- Figure 1: It's a bit confusing to have core ants and plants both be blue. Maybe dark/light red and green to separate core/peripheral species would work.
- Figure 2: lines 870-871 seem copied from the results and have the same English errors. This is not a complete sentence.
Line 873: What does "Distant ellipses" mean? They seem to be in the middle of the plot and not particularly distant from anything. How was it determined which attributes explained the pattern? No attributes are presented on the plot, so it is impossible to check this. It would also help to include your interpretations of the axes in the figure legend.
Experimental design: This original research fits the journal aims and scope.

The research question is now well-defined and meaningful, and the introduction now clearly establishes the knowledge gap the authors intend to fill.

The experimental design is generally well described and rigorous, with the exception of analyses relating to abundance. It is not clear how plant and ant abundances were measured (were they counted using the honey-bait traps that were briefly mentioned in line 202? If so, it is not clear that this information was used in the analyses.) and whether they were explicitly controlled for in the analyses. No supporting data are provided, for example, that would let me see whether abundance relates to the positions of species on the NMDS plot. If ant and plant abundances were not measured independently of interaction frequencies, then it is not possible to make strong claims about abundance as abundance and interaction frequency may or may not be correlated (as the authors point out). If abundances were sampled independently, this information should be provided with the raw data. It is also not clear how floristic similarity was calculated.

The methods lack key details (abundance, floristic similarity) and the raw data are missing some elements that make it impossible to repeat the analyses used in this study. The study would be difficult to properly replicate without more information on how abundances were determined and what, exactly, floristic similarity means.
Validity of the findings: The association between different plants and habitat groups/structures is not clear enough to properly assess the conclusions. A table summarizing ant and plant attributes would be extremely helpful in interpreting Fig. 2 and would substantially strengthen the manuscript. It is currently impossible for a reader without expert knowledge of the system to judge the authors' claims about which traits structure the community. None of these claims are particularly unbelievable, so I am confident that the authors can support them with a more detailed presentation of their evidence.

Some of the conclusions are well-supported by the rest of the manuscript but the authors' claims about plasticity of defensive strategies are not. This may be speculation, but it is not clearly identified as such. Further revisions to the discussion may help to solve this problem.
Additional comments: I respect the work that went into assembling this dataset, but the analyses remain opaque enough that I am not confident in the conclusions presented. In particular, the theme of plasticity in plant defense appears out of nowhere in the discussion and does not clearly connect to the results. This is an interesting idea, so I suggest that the authors add plant defense to the introduction and make sure it is a clear thread through the entire manuscript.


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

Input:
Title: RELATIVE CONTRIBUTION OF ECOLOGICAL AND BIOLOGICAL ATTRIBUTES IN THE FINE-GRAIN STRUCTURE OF ANT-PLANT NETWORKS
Review round: 4
Reviewer: 1

Output:
Basic reporting: The English is still not perfect (see list of corrections in general comments) but is generally clear. There are only a few ambiguous sections (see list of corrections). The article is professionally structured, adequately referenced, and self-contained.
Experimental design: I have no further comments on the experimental design.
Validity of the findings: The findings seem to be valid and well-supported.
Additional comments: The authors have further improved the writing of the manuscript and it is now mostly clear, although a large number of small language errors and some run-on sentences remain, as well as a few places where a little more information would be helpful. These should be quite easy for the authors to fix, leaving quite a nice manuscript relating plant-ant network structure to habitat structure. However, I am still concerned that the data provided by the authors does not include all of the information that would be needed to re-create the study. There is still no key to plant and ant names, only abbreviated trait names are given in the "Attributes" tab, and I do not see ant head lengths, etc. Please expand the descriptions in the data to meet PeerJ's standard for raw data publication

Minor comments:

line 224: what was the minimum time before you determined that feeding had occurred?
line 252: it would help readers who are not ant specialists to briefly explain the difference between invasive and tramp species, if there is space.
line 288: NODF is less sensitive than what? I assume some other measurement of nestedness, but it would be good to be specific.
line 363: what was the value for Barber's Qn?
line 386: why don't you give d' for ants?
lines 530-539: these results only mention species strength. Did you examine the relationship between species strength and habitat?
lines 533-535: In the results it seemed like this was only the upper part of axis 2, not an overall trend. You may want to adjust the results or discussion so that they are consistent.
lines 571-572: by "simultaneous contribution", do you mean that each variable was significant on its own? This is somewhat confusing.
Table 2: Are these dominance categories hierarchical (i.e., A beats B, B beats C)?

Language comments:

Abstract

line 41: should read "species which, together with higher dependence of plants on ants, suggests potential...
line 43: "so prevailing that" should read "so prevalent that it"

Introduction

line 108: "evidence" should be "demonstrate". Evidence is only a noun in English, not a verb.
line 128: "render" should be "result in" or maybe "yield"
line 129: should read "plenty of variation in EFN..."
lines 205-209: This sentence is long and hard to parse. Can you break it up into a few shorter sentences?

Methods

lines 261-270: This sentence is extremely long and hard to read. I strongly suggest that you break it up into shorter sentences. Lines 267-270 are especially hard to follow.
lines 290-291: It is very helpful of you to include the names of the null models in two papers! That should make things much easier for your readers.

Results

line 364: "exist at" should read "exist in"
lines 367-368: "that is network still" should read "that the network is still"
line 373: "Regarding species strength" is a somewhat strange beginning to a sentence, and should probably be followed with a :
line 394: "within" should probably be "to have"
line 401: You give F=15.80 and F=15.79. This is a very small difference, but one of these values is probably wrong.
line 407: "having as well" should be "also had"
line 412? remove comma
line 433: "at our study" should be "in our study"

Discussion

line 442: should be "structure and is thus..."
lines 441-444: run-on sentence. Break into two or more. It is also not clear whether you mean that modules are a common feature of simulated networks or that the lack of modules is a common feature.
line 448: "community-level" should be "community level"
line 462: "at a community level" should be "at the community level"
line 473: "accordance to" should be "accordance with"
line 478: "species that constituting" should be "species that constitute"
line 486: "in nectar" should be "on nectar"
lines 486-492: run-on sentence. Break up.
line 492: "At our studied" should be "In our studied"
lines 492-495: I'm not sure what these lines are supposed to be saying. Do only the core ants provide biotic defence? If not, why are they specifically pointed out?
line 530: should be "outstanding as"
line 531: "transcending" should be "translating"
line 546: "At our study" should be "In our study"
lines 546-547: "in order to explore its effect in network structure" makes it sound like you excluded abundance in order to test its effect, which does not make sense. You could just remove this text.
line 561: "to account for interactions" might be more accurate as "to account for variation in interactions"
line 563: "capture" should be "captures" since you are talking about one set of censuses
line 583: "at plant species" should be "of plant species"
line 590: remove comma. It is also not clear whether you mean these plant-ant mutualistic networks, or some different ones.
lines 593-597: run-on sentence. Break up.
line 594: "resource in" should be "resource is"
line 599: "spectra" should be "spectrum"
line 603: "seems" should be "seem"
lines 607-610: I don't think this needs to be a new paragraph.

Conclusion

lines 627-628: Why did you change from EFN to EFnectaries here?
lines 946-947: "having as well" should be "also has". This text is also a direct copy of that in the main text.


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

Input:
Title: CORRECTING NUISANCE VARIATION USING WASSERSTEIN DISTANCE
Review round: 1
Reviewer: 1

Output:
Basic reporting: Well written paper, with clear explanations and easy to follow flow. Minor typos need to be fixed.
Experimental design: * I found somewhat confusing the procedure used to identify the early stopping parameter of the model. In section 2.4, the paper says "The MOA information is used for early stopping in model training", and in section 2.6.1 it reads "the hyperparameters are optimized over the compounds with known MOAs". Since the leave-one-compound-out validation is used for evaluation, how exactly is this evaluation separated from hyperparameter tuning?
* Boosting is employed to estimate variance of the results by sampling with replacement the embeddings in each well. Can you clarify if you mean single-cell embeddings, image-based embeddings or full well embeddings? This is necessary to understand the extent of the variation of the bootstrapping results.
* In this dataset, only DMSO wells appear in multiple batches, but the method can incorporate any treatments that have replicates across batches. Can you confirm that you trained the model on DMSO only for the experiments reported in the paper?
* Can you clarify whether the embedding transformation trained for the experiments in this paper is a neural network or an afine (linear) transformation?
* What is the difference between TVN and CORAL in the context of this paper? According to Ando et al. 2017, the TVN method is based on CORAL corrections as well.
* The WDN method is applied to embeddings after TVN. Why is TVN necessary? Ando et al. 2017 showed that TVN can effectively remove the unwanted variation from embeddings. The WDN method should be also evaluated on embeddings without TVN to assess the applicability as an independent strategy.
Validity of the findings: * It is surprising that CORAL performs better than TVN in the results presented in this paper. Can you comment on this? (also, see comment about difference between TVN and CORAL above).
* The method is trained leaving one compound out, and selecting the early stopping parameter for each compound. I find it confusing to train and evaluate the model at the same time, specially when one critical hyperparameter needs to be selected at each iteration of the evaluation. Can you comment on that?
* It would be nice to present a table with results reported by others to help the reader compare differences with other approaches that use the same dataset.
* Is the optimal stopping step the same for all metrics evaluated? Is it the same for all treatments? Can you give some more information and analysis about this? This is specially important given that the parameter appears to be a critical choice in the method.
* The batch classification plot (Fig 7) seems to indicate that the method keeps removing nuisance variation after the "optimal early stopping" point. Why is it that this improves but MOA assignment decreases?
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: CORRECTING NUISANCE VARIATION USING WASSERSTEIN DISTANCE
Review round: 1
Reviewer: 2

Output:
Basic reporting: The authors presented a method of correcting unwanted variation of cellular imaging data while preserving relevant biological information. The Wasserstein distance is used and evaluated the performance compared to other competing methods. Although some results show the advantage of this proposal, I have some concerns about target audience for application, novelty of method (selling points of 1-Wasserstein distance) and supporting examples.
Experimental design: Target audience: I was wondering while reading this article about who will read this paper and further use the proposed method in what situations. The author mentioned it is a general framework but I see this paper is an application to introduce a 'relative new' mathematical idea to a certain situation like cell imaging data in drug development. If you want to sell this method to a drug industry, the motivating example has to be concrete but at the same time popular enough to attract the audience. I suggest the author arguing the example presented here (e.g., Figure 2, page 7) that are very common, more than a case study, and promising datasets in the future as well, so that the readers have a reason to use this method or similar others. Including a second example is a good idea, too. Otherwise, this work is less valuable from an application standpoint. So far, there is a doubt what target audiences this paper can reach.
Validity of the findings: Novelty/Benefit of the method: The authors have to convince the readers why it has to be a Wasserstein distance. Two advantages explained (lines 90 - 92 ) is not fully convincing 1) non-linear removal of batch effects (multi-modal or non-Gaussian data); This advantage should be explained in the text (not only just saying it), theoretically or empirically, through data simulation, toy example, or real data example, which are not sufficiently provided in this work; 2) Adjusting the entire distribution more than two batches - This reason is misconstructed.  Some paper like Benito et al (2004) used only two batch problem but its application is not limited to two-batch. Other batch adjustments methods, as referred in Johnson et al (2007), Leek and Storey (2007) and Lee at al (2014) have the approach of multi-batch adjustment, either sequential or simultaneous way. The reason why some methods mentioned `target' batch or `source' of variation (lines 111-112) is rather a biological reason, not a mathematical reason, so it is the strength of their approach when extra information is available from the complex experiments, and importantly these methods could be used in a way of ``equal footing," when there is no biological information available to consider. Reversely, we could question the author if the proposed Wasserstein method can correct the nuisance variation with different weights across different quality of batches if any. Then, it could be a totally different argument, spinning around a weakness of the proposed method. I wish the authors could provide more convincing reasons over other methods.

Distance measures: One way of selling the Wasserstein method is the distance measure itself. You may consider rephrase (lines 143 - 155) or develop stronger argument against other distance metrics,  such as 2- Wasserstein distance, Euclidean distance, Mahalanobis distance (or regularized Mahalanobis distance for high dimension), or Cramer distance (as mentioned p.116). Perhaps the cell image data is high-dimensional. Can 1-Wasserstein distance overcome a large dimension situation? Again, the readers need to be convinced of why it has to be a 1-Wasserstein distance.

Philosophy of batch bias removal: The authors suggested three ways of evaluation, two of which measure how much biological signal is preserved in the transformed embeddings, and the other is how much nuisance variation has been removed. There is a philosophical concern for the first two approaches. We could always look at how much the biological signal of interest turned out to be, as a result of background noise removal. However, we should not be using this as a definite standard for the choice of batch removal because this potentially contains a double-dipping problem. The method developed so that preserve the biological difference are meant to display the biological difference, because it was preserved! It is ideal to blindside the biological information and it might result in removing the biological signal as well (and it could be the right thing). For this reason, I would rather recommend the authors to focus on the similarity among batches to evaluate the effectiveness of batch bias removal. The presented evaluation in Figure 8, displaying first two PCs is not enough (How much variation by first two PC are explained?; PC scatter plot in two-dimensional space is better than histogram). Maybe you could consider some statistical testing for the equality of the means, covariance matrices or distributions.
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: CORRECTING NUISANCE VARIATION USING WASSERSTEIN DISTANCE
Review round: 1
Reviewer: 3

Output:
Basic reporting: No comment.
Experimental design: No comment.
Validity of the findings: No comment.
Additional comments: The authors propose an approach for normalizing vectors derived from
high-content images monitoring cells subjected to chemical treatments
seeded in plates prepared across different experimental batches. The
approach exploits the fact that vectors from the same treatment
ought to be similar across batches. The approach accordingly minimizes
pairwise distances among the high-dimensional distributions entailed
by the vectors of different batches of the same treatment. The
distance calculation is approximated by a neural network. The authors
test their method on vectors derived
through both conventional and deep learning feature extractors on the
well-known BBBC021 image dataset

The problem of registering vectors onto a common space is an important
one, esp. given the fact that deep learning feature extractors are
susceptible to batch effects.

* One main suggestion is to acknowledge the fact that the idea of
assuming that feature vectors from the same treatment look the same
has been used before, esp. in the context of computing features for
high content images through deep learning approaches, see, e.g.,:
- Caicedo et al. Weakly Supervised Learning of
Single-Cell Feature Embeddings, CVPR, 2018
- Godinez et al. Unsupervised phenotypic analysis of cellular images
with multi-scale convolutional neural networks, bioRXiv, 2018

* Prior to feature extraction with conventional methods, an
illumination step is typically carried out (see, e.g., Singh, S et al. “Pipeline
for illumination correction of images for high-throughput microscopy.”
Journal of microscopy vol. 256,3 (2014)). Since the proposed approach
does not bring much of an improvement for conventional feature
extraction schemes, one wonders whether the reason for this is that
conventional features are applied on illumination-corrected images.
It would be great if the authors could comment on this. One also
wonders whether a) such an illumination correction was carried out in
this study prior to the application of the deep learning feature
extractors, and b) whether, after such a correction, the proposed
approach would still bring an improvement w.r.t. the alignment of the
deep learning-based vectors. It would be great if the authors could
show an improvement even after carrying out an illumination correction
on the images.

* The approach is certainly sensitive to the training time, as the
longer one trains, the higher the likelihood that the learned
transformation would remove subtle but important phenotypic
variations. The early stopping criterion proposed in the paper
requires some type of prior phenotypic knowledge regarding the
dataset, which in a more realistic setting, is hard to come by (often,
the MOA of most compounds in an exploratory scenario is not
known). Are there other early stopping criteria requriring no prior
phenotypic knowledge that could be used for this method?


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: CORRECTING NUISANCE VARIATION USING WASSERSTEIN DISTANCE
Review round: 2
Reviewer: 1

Output:
Basic reporting: The paper presents a theoretical framework for batch correction with one specific instantiation using the Wasserstein distance as the metric to match distributions of data points. The paper reads well.
Experimental design: The experiment is limited to one dataset, but the metrics used for performance evaluation and the experimental protocols are well designed.
Validity of the findings: The results seem inline with previous reports on the same dataset. The authors provide source code for reproducing the results on publicly available features. The methods seems to be better suited for deep learning features computed with pretrained neural networks.
Additional comments: The link to the code could be included in the manuscript.


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

Input:
Title: CORRECTING NUISANCE VARIATION USING WASSERSTEIN DISTANCE
Review round: 2
Reviewer: 2

Output:
Basic reporting: The usage of the TVN term is still confusing, as it only represents one step of the original TVN method. I suggest using the term 'PCA whitening' instead of TVN.

Line 128: "Our method incorporates all replicates of a given treatment on an equal footing, which can be advantageous over using only the controls."  I am now finding this contradictory since only the DMSO controls are used to learn the domain transformations. I suggest removing this sentence.

Lines 247 to 250 are duplicate sentences.
Experimental design: No comment
Validity of the findings: The inclusion of the bootstrap results is a great addition to the paper. The term 'vanilla' should be explained in the main text, though. It's not clear, for example, whether the vanilla result is just another sample in the bootstrap run? Or is the vanilla result using all embeddings? Also, the vanilla and boostrap results are pretty similar. It would aid for legibility to select only one type of result for presentation and discussion, esp. if the conclusions remain the same. The bootstrap ought to be more statistically informative, so I'd lean towards those results.
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: TENSIOMYOGRAPHICAL RESPONSIVENESS TO PERIPHERAL FATIGUE IN QUADRICEPS FEMORIS
Review round: 1
Reviewer: 1

Output:
Basic reporting: Manuscript entitled as: Tensiomyographical responsiveness to local fatigue in quadriceps femoris, have clear scientific idea and well organized research/experimental protocol. Results of this study are actual and applicable in sports and rehabilitation practice. According to my opinion, the article reach journal standards for publishing, but after minor technical corrections (marked in PDF format of the manuscript).
Experimental design: Experimental design is organized and realized on proper way, with well and adequately verify by relevant references.
Validity of the findings: Results of the study are clear and confirmed by applied statistical analyses.
Additional comments: Manuscript is written acceptable well and clear. Measurement protocol and statistical analyses was done on proper way. After suggested minor technical text improvement, manuscript should be accepted 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: TENSIOMYOGRAPHICAL RESPONSIVENESS TO PERIPHERAL FATIGUE IN QUADRICEPS FEMORIS
Review round: 1
Reviewer: 2

Output:
Basic reporting: I congratulate authors for an important contribution in understanding relation between TMG and fatigue. Although, i have several issues that needs to be addressed:

1.	It would be better to operate with peripheral and central fatigue, not local and general;
2.	Fourth section in Introduction is less clear to a reader. Furthermore, the reference Husted et al. (2000) is not reflecting its purpose. Husted did not publish TMG article;
3.	Instead of submaximal involuntary contractions put maximal elicited contractions. It would be clearer.
4.	Use equation editor to insert equations.
5.	The fourth paragraph of Statistics should start as: We assumed... Furthermore, was the external standard the same as external criterion (MVIC) ?
6.	Use abbreviations consistently after they are defined and define abbreviations before their first use (e.g. Abstract Tensiomyography -> TMG)
7.	Commas are missing or they are some extra (e.g. before respectively; ,, in discussion)
8.	Figure and Table with capital letter.
9. Avoid unnecessery abbreviations in Tables (VL, VM, RF and TMG could be written down). If there is no significnace remove data about differences. use only p instead of p.value
10. Figure 2 (no colours, no horizontal lines, only one legend)
Experimental design: 1.	Why did you use multiple t-test instead of 2-way ANOVA (or GLM)? What was the rationale for this? Did you compensate P-value for multiple comparisons?
2.	Was placement of sensor in line with the most recent finding of Šimunič (2019) Journal of Biomechanics?
3.	Move in Methods->procedures statement from Methods-Statistics (It was considered that the fatigue was achieved when the reduction of the MVIC...)
Validity of the findings: 1.	Figure 2: Why did you report only those fatigued and not all of them? What was the rationale for such an approach?
2.	How could you interpret Tc? And how decrease of Tc after fatigue? You found the opposite as Hunter et al. (2012). Any comments? Was your fatiguing protocol sufficient to fatigue fast twitch MUs?
3.	Could you report (somewhere in text) what was the average time until fatigue. This would be additional information that would allow interpretation of outcomes changes.
4.	Last sentence in Results: why did you at this point expect that TMG parameters must be discrimination tool with 100% Se and 100% Sp? In Discussion and Conclusions you've stated that it could be.
5.	Last sentence in Discussion: This could be applied only to 70% isometric exercise. Soften it up.
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: TENSIOMYOGRAPHICAL RESPONSIVENESS TO PERIPHERAL FATIGUE IN QUADRICEPS FEMORIS
Review round: 1
Reviewer: 3

Output:
Basic reporting: see below
Experimental design: see below
Validity of the findings: see below
Additional comments: ARTICLE REVIEW

TITLE: Tensiomyographical responsiveness to local fatigue in quadriceps femoris

Human participant/human tissue checks

Have you checked the authors ethical approval statement?
Yes, I checked.

Does the study meet our article requirements?
Yes, it does.

Has identifiable info been removed from all files?
Yes, it has.

Were the experiments necessary and ethical?
Yes, they were.

STRUCTURE

1. Basic Reporting

Although this is a well-constructed study, the English language needs to be reviewed. The introduction and discussion sections are well structured and follow a logical sequence. The materials & methods section needs to be better structured in order to enable the reader to understand the sequence of the experimental procedures. The authors support the information with relevant literature in the field. The structure of the manuscript conforms to PeerJ standards. The figure presented by the authors is relevant, of high quality, and is properly labelled and described. However, as I detail in the general comments, more figures are needed for a better understanding of the experimental procedures. The authors supplied the raw data.

2. Experimental Design

The present study is an original primary research, which falls within the scope of the PeerJ journal. The authors define the research question, which seems to be relevant in the field of fatigue evaluation, and clearly state how their approach to the problem fills an identified knowledge gap. The experimental procedures were performed to a high technical and ethical standard. The methods section needs to be further developed in order to better elucidate the reader.

3. Validity of the findings

The rationale and benefit to the literature was clearly stated by the authors, presenting both the strengths and limitations of the study. The data collected was robust and the analyses performed were appropriate to the research question. The conclusions were well stated, linked to the original research question and limited to the obtained results.

4. General comments

The aims of the present study were to examine and compare the tensiomyographical (TMG) responsiveness to quadriceps femoris (QF) fatigue by multiple statistical methods and to analyze differences between sex in the variation produced by fatigue in TMG parameters. The main findings were that TMG parameters, such as maximum radial deformation (Dm) and contraction velocity of the first 10% of the Dm (V10) of the rectus femoris (RF), showed both internal and external responsiveness, and both sexes exhibited similar strength change scores. This is a well-constructed study (valid and reliable methodological design), with novel practical findings related to fatigue measurement. In this regard, the authors should be congratulated for this interesting manuscript. The results presented here would be of interest to the readers of this Journal. However, there are some major comments and concerns that I will detail below in order to improve the quality of the manuscript, including spelling and grammar.

Abstract

- Line 17: Please rewrite the sentence “Fatigue influences athletic performance or increased risk of injury in sports…” to “Fatigue influences athletic performance and can also increase the risk of injury in sports…”

- Line 20: Please, change “Even so” to “However”

- Line 21-23: Please rewrite “The aim of this study was to examine and compare the tensiomyographical responsiveness to quadriceps femoris (QF) fatigue by multiple statistical methods.” to “Thus, the aim of the present study was twofold: (i) to examine and compare the tensiomyographical responsiveness to quadriceps femoris (QF) fatigue by multiple statistical methods and (ii) to analyze sex differences in the variation produced by fatigue in TMG parameters.”

- Line 33: Please, change “R2” to “r2”

- Line 24: Please, specify how many males and females participated in the study and the average age ± SD

- In results, I recommend the authors to report the statistical significance (e.g., p < 0.05)

- I suggest the authors to add a conclusion section with the last sentence (“Since the QF is the main strength contributor during multiple physical activities, clinicians and trainers will be able to discriminate the presence of fatigue and the magnitude of changes in the QF strength by TMG evaluation.”)

Introduction

- Line 47-49: Please rewrite the sentence “Since fatigue influences athletic performance (Thorlund et al., 2008; Ditroilo et al., 2011) or increased risk of injury in sports (Zebis et al., 2011; Liederbach et al., 2014), its study has been of interest.” to “Since fatigue influences athletic performance (Thorlund et al., 2008; Ditroilo et al., 2011) and can also increase the risk of injury in sports (Zebis et al., 2011; Liederbach et al., 2014), its study has been of interest.”

- Line 56-62: The sentence is too long and confusing. I suggest the authors shortening the sentence and be more specific.

- Line 87: Please rewrite “However, the external responsiveness of TMG has not been yet assessed for local fatigue…” to “However, to the best of our knowledge, the external responsiveness of TMG has not been yet assessed for local fatigue…”

- Line 89-91: I suggest the authors to rewrite the sentence for clarity. For example: “Furthermore, to our knowledge, TMG responsiveness has not been simultaneously evaluated in multiple muscle bellies, neither analyzed by multiple statistical indicators of responsiveness. At the same time, understanding the mechanisms behind the changes in TMG parameters caused by fatigue in both sexes, is also an area of research that needs further development.”

Materials and Methods

- Before the participants subsection, please consider to add a study design subsection, referring the following: type of design; a brief description of the subjects; a brief description of the experimental procedures (i.e., the study time period, the place where the experimental procedures were performed, as well as their environmental conditions, like temperature and humidity); the role of the evaluators during the tests (their experience administering the tests); the number of sessions needed to conduct the evaluations; and finally the ethical approval.

- I suggest the authors to rewrite the participants subsection, specifying the relevant information (e.g., inclusion and exclusion criteria) and removing what was already been referred in the study design subsection.

- Line 103: What the authors mean with “recreational athletes”? Please, specify with some examples of sports.

- Line 103-104: “…that was conducted from April to July 2018”. Please, move this information to the study design subsection

- In the procedure’s subsection, sometimes is hard to understand the sequence of the experimental design. Thus, for a better understanding, I suggest the authors to add a schematic representation of the procedures (e.g., see https://doi.org/10.3389/fphys.2018.01503 or https://doi.org/10.1371/journal.pone.0091754), and if possible, to insert new subsections between the following lines: 114-121 (e.g., Procedures); 122-144 (e.g., Tensiomyography measurements); 145-156 (e.g., Maximal voluntary isometric contraction test); 157-161 (e.g., Fatigue protocol test).

- Line 115: Please, specify the instrument used to measure the subject’s height.

- Line 115: Please rewrite “height was measured using a tape measure and body mass and body mass index (BMI) were registered…” to “height was measured using a tape measure, while body mass and body mass index (BMI) were assessed…”.

- Line 117-121: Please, rewrite these lines for clarity. The reader should easily understand the sequence of the procedures. What was the first procedure? It was the warm-up? If so, describe it. What was the second procedure? For a clear understanding, I suggest the authors to describe the procedures with a logical sequence.

- Line 119: “…at comfortable speed with low resistance…”. I suggest the authors to detail both the speed and the resistance, for replicate reasons.

- Line 121: “…the order of the tests was reversed…”. I recommend the authors to specify which tests were reversed and why they did that. What was the purpose? This is not clear for the reader.

- Line 144: “10% of the Dm (V10).”. After this line, I suggest the authors to present the relative and absolute reliability of the TMG parameters, through the intraclass correlation coefficient and the coefficient of variation, respectively. It is important to inform the reader about the consistency of the measurements performed.

- Line 149-151: For a better understanding, I suggest the authors to insert a figure demonstrating the procedures described.

Line 154-155: “After the warm-up, participants completed three MVIC for 5s, with a 60-second rest after each repetition.”. As mentioned above for the TMG parameters, here I also recommend the authors to present the relative and absolute reliability of the MVIC measurements.

- Line 163: Please, remove the end point in “Statistical analysis.”

- Line 173-175: As mentioned above, please consider moving the definition of internal responsiveness to the introduction section (see https://doi.org/10.1111/jan.12898). Include it in line 82, after the sentence “…reference measure or regression models) (Husted et al., 2000).”

- Line 175: “Of the current effect size statistics…”. Since the most appropriate effect size for calculating responsiveness remains controversial, why did you use the standardized response mean (SRM), instead of the Cohen’s d effect size (ES), or the standardized effect size (SES)? None of the formulas are influenced by the sample size. If the authors consider it appropriate, they can present the results of the three effect sizes and analyze possible differences between them (see https://doi.org/10.1111/jan.12898).

- Line 185-187: As mentioned above, please consider moving the definition of external responsiveness to the introduction section (see https://doi.org/10.1111/jan.12898). Include it in line 82, after the sentence “…reference measure or regression models) (Husted et al., 2000).”

- Line 198: Please, change “R2” to “r2”.

- Line 207: Before the sentence “All analyses were performed…”, please consider including the following sentence: “Statistical significance was set at p < 0.05.”

Results

- Please, put in italic the p-value

- Line 240: Please, change “97.1” to “97.1%”

- Line 242: Please, change “R2” to “r2”.

- In the tables, please also put in italic the p-value and change “R2” to “r2”.

Discussion

- Line 260-262: Please, rewrite “We used multiple statistical methods to evaluate the internal responsiveness (paired t-test and SRM) and external responsiveness (correlations, regression models and ROC) of the TMG, as recommended by Husted et al. (2000)” to “In our study, multiple statistical methods to evaluate the internal responsiveness (paired t-test and SRM) and external responsiveness (correlations, regression models and ROC) of the TMG were used, which is line with the recommendations of Husted et al. (2000).”

- Line 264-266: Please, rewrite “Thus, one strength of our study is that, as far as we know,, this is the first study evaluating various statistics from internal and external responsiveness.” to “Thus, to the best of our knowledge, this is the first study to use several statistical methods to assess internal and external responsiveness.”

- Line 265-269: Please, rewrite “An additional strength is that we evaluated TMG across multiple muscle bellies within the same study. Most of previous studies assessing fatigue by TMG have only evaluated isolated muscle bellies (García-Manso et al., 2011; Hunter et al., 2012; de Paula Simola et al., 2015, 2016; Giovanelli et al., 2016; Macgregor et al., 2016; Raeder et al., 2016; Wiewelhove et al., 2017)” to “Furthermore, since most of the previous studies assessing fatigue by TMG have only evaluated isolated muscle bellies (García-Manso et al., 2011; Hunter et al., 2012; de Paula Simola et al., 2015, 2016; Giovanelli et al., 2016; Macgregor et al., 2016; Raeder et al., 2016; Wiewelhove et al., 2017), our study presents novel findings in the evaluation of TMG across multiple muscle bellies.”

- Line 270-271: “Regarding the internal responsiveness, large and negative SRM of the TMG parameters were found in most of the muscle bellies.” Could the authors present a possible explanation for this occurrence?

- Line 275: Please, change “Changes” to “changes”

- Line 277: Please, rewrite “…other studies that induced local fatigue in other muscles…” to “…other studies that induced local fatigue in muscles…”

- When the authors state that their results are consistent with previous findings, I suggest them to provide additional explanations. The reader must be informed about these consistencies.

- Line 279: Please, change “finding” to “findings”

- Line 287: Please, change “according sex” to “according to sex”

- Line 290-294: Please rewrite: “According to previous studies (Clark et al., 2005; Lee et al., 2017; Ansdell et al., 2017), this was an unexpected finding, which was probably due to the use of higher intensities in our study (70% MVIC) compared to the strengths used in previous studies  (Clark et al., 2005; Ansdell et al., 2017), since sex differences in muscle fatigue decrease as the contraction intensity increases (Hunter, 2014).” to “Previous studies reported different strength change scores between sexes when intensities between 25-50% of MVIC were used (Clark et al., 2005; Ansdell et al., 2017). In our study, an intensity of 70% of MVIC was used, suggesting that as the contraction intensity increase, the sex differences in muscle fatigue decrease (Hunter, 2014).”

- Line 299: Please, change “authors” to “studies”

- Line 300-301: “Nevertheless, while AUC of V10 shown in our study was similar to their results, AUC of Dm was higher than theirs (Wiewelhove et al., 2017).”. Please, rewrite this sentence for clarity.

- Line 310: Please, change “had” to “has”

- Line 310-311: Why the authors consider a limitation using an effective protocol to induce local fatigue? This could be a future study suggestion, but not a limitation. Please explain for clarity

- Line 319: Please, change “evinced” to “evidenced”

- Line 319: Please, specify "them". Which variables showed external responsiveness?

- Line 328-329: Please, change “taking in consideration” to “taking into consideration”

Conclusions

- Please, rewrite this section. I recommend the authors starting with the main results of the study. Next, I recommend mentioning the practical applications of the findings, and finally, I suggest presenting some future research directions (see lines 327-331).


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: TENSIOMYOGRAPHICAL RESPONSIVENESS TO PERIPHERAL FATIGUE IN QUADRICEPS FEMORIS
Review round: 2
Reviewer: 1

Output:
Basic reporting: Authors do change all what was asked in the first review. In line 328 there is one extra comma as a simply typing error, which should be deleted.

According to my opinion manuscript is in full ready to be accepted for publication. Good work.
Experimental design: Design of the study is according with positive scientific practice.
Validity of the findings: Findings are at high level of applicability in sports and rehabilitation practice.
Additional comments: Excellent work.


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

Input:
Title: TENSIOMYOGRAPHICAL RESPONSIVENESS TO PERIPHERAL FATIGUE IN QUADRICEPS FEMORIS
Review round: 2
Reviewer: 2

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: Authors successfully corrected the manuscript to meet all reviewers comments raised.


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: TENSIOMYOGRAPHICAL RESPONSIVENESS TO PERIPHERAL FATIGUE IN QUADRICEPS FEMORIS
Review round: 2
Reviewer: 3

Output:
Basic reporting: The authors made a good job per instructions.
Experimental design: The authors made a good job per instructions.
Validity of the findings: The authors made a good job per instructions.
Additional comments: The authors made a good job per instructions.


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: GENE EXPRESSION PATTERNS OF NOVEL VISUAL AND NON-VISUAL OPSIN FAMILIES IN IMMATURE AND MATURE JAPANESE EEL MALES
Review round: 1
Reviewer: 1

Output:
Basic reporting: The authors reported the identification of opsin subfamilies in the retina and the whole brain tissue using RNA-seq. In addition, they examined the expression of these genes in sexually mature and immature male eels by qPCR. This article is very confusing both in how the experiments were conducted and how the data was presented and analyzed. They solely presented all their analyses without any emphasis or deep analysis. Their RNA-seq data did not provide much useful information and is lack of confirmation by qPCR. Therefore, this article is not ready to be published with my concerns listed below.
Experimental design: First, it is vague about how many samples/bio reps were sequenced and how did they pool the samples. The authors mentioned that they had 500m reads from RNA-seq. Was this a combination of samples from both brain and retinal tissues? I also did not find the raw sequencing data for this experiment. Therefore it is hard to evaluate this work statistically.

Second, how they identified and categorized the 18 subfamilies is a mystery. They mentioned that they constructed a phylogenetic tree and performed some kind of blast. However, they did not explain in detail how they recognize and categorize each subfamily.

Third, in the methods, the authors mentioned that they downloaded reference transcripts as the reference for mapping (L178). However, in the results section, they said they de novo assembled contigs. Therefore I was confused about what was used as the reference genome in mapping.

Fourth, for DEG analysis, it is unclear how the statistical test was performed. The authors mentioned using unpaired t-test (L230). However, it is more appropriate to use the adjusted p-value in DESeq.

Fifth, it is odd that the authors used a pie chart to illustrate gene expression levels. It is unclear how the percentage is generated using different bio reps. In addition, they only selected a proportion of genes and they calculated the percentage, it would cause bias in examining gene expression level and makes it hard for comparison. I would recommend the authors to use the normalized counts and generate a heatmap.
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: GENE EXPRESSION PATTERNS OF NOVEL VISUAL AND NON-VISUAL OPSIN FAMILIES IN IMMATURE AND MATURE JAPANESE EEL MALES
Review round: 1
Reviewer: 2

Output:
Basic reporting: This article is clear and unambigous. Only few details need to be improved:
L131: Maybe it is not necessary to repeat
L360: Please, correct immunohistochemical
Paragraph L312: Too much therefore, however, additionaly, ... This paragraph is unclear.
Experimental design: Please add more details in the methods described:
L137: How long did you replace freshwater by see water?
L211: What was the cDNA dilution use?
Validity of the findings: No comment
Additional comments: I consider this paper of high scientific quality. Results could contribute to broaden the knowledge about eel reproductive process and could help in developing strategies to improve their culture. Researchers proposed adequate techniques and research methods to reach their objectives and appropriate means for analyzing the results.


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: GENE EXPRESSION PATTERNS OF NOVEL VISUAL AND NON-VISUAL OPSIN FAMILIES IN IMMATURE AND MATURE JAPANESE EEL MALES
Review round: 1
Reviewer: 3

Output:
Basic reporting: This article is good to meet the topic.

The NGS rawdata and statistic value should be put in the supplements and/or online database before it can be published.

Figure 3. I can’t identify the OTUs and values. It needs to redraw with the large size before it can be published.
Experimental design: It will be better to have a flowchat demonstrated the experimental design and NGS analysis.
Validity of the findings: The NGS rawdata and related statistic value should be put in the supplements and/or online database before it can be published.
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: GENE EXPRESSION PATTERNS OF NOVEL VISUAL AND NON-VISUAL OPSIN FAMILIES IN IMMATURE AND MATURE JAPANESE EEL MALES
Review round: 2
Reviewer: 1

Output:
Basic reporting: The authors have addressed most of my concerns. However, their Figure 4 is the duplication of Figure 5 which should be corrected. Meanwhile, I would suggest Figure 1,2 and table 1 could be moved to supplemental.
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: GENE EXPRESSION PATTERNS OF NOVEL VISUAL AND NON-VISUAL OPSIN FAMILIES IN IMMATURE AND MATURE JAPANESE EEL MALES
Review round: 2
Reviewer: 2

Output:
Basic reporting: This manuscript is improved. However, there are still some typos and need to be checked carefully before it can be published.

1. There are typos in literature, for example:
Lin JY, Wang FY, Li WH, Wang TZ >> Lin JJ, Wang FY, Li WH, Wang TY
Check and correct all the literatures again before it is published.

2. Figure 4 is not correct and I cannot see it.
Figure 4 and Figure 5 are the same one?
Experimental design: The experimental design is fine in this version.
Validity of the findings: Figure 4 is not right.
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: EVALUATION OF A NEW ARGOVIT AS AN ANTIVIRAL AGENT INCLUDED IN FEED TO PROTECT THE SHRIMP LITOPENAEUS VANNAMEI AGAINST WHITE SPOT SYNDROME VIRUS INFECTION
Review round: 1
Reviewer: 1

Output:
Basic reporting: The manuscript by Quiñonez et al. evaluated a new antiviral agent against the White Spot Syndrome Virus (WSSV) infection in the shrimp Litopenaeus vannamei. The propose was to incorporate Argovit, a formulation containing silver nanoparticles, in the food of L. vannamei as a prophylactic treatment for this important virus disease. The authors evaluated the viability of this proposal by performing several tests.
The article explored the literature regarding the importance and the advantages of using silver nanoparticles (AgNP) as an antiviral agent in those shrimps. Nevertheless, not even one reference was made to the toxicity of silver (Ag) and AgNP to crustaceans or aquatic organisms, despite there is literature reported about it (e.g. Fabrega, J., et al. Environ. Int. v 37, p 517, 2011 and George, et al., ACS Nano v 6, p 3745, 2012). You must consider and describe the disadvantages of the use of this AgNP food as well, especially because it is linked with one of the questions you were trying to answer (whether Argovit-4 supplemented in feed exhibits toxicity towards shrimp).
In general, the work is well written, clear and logically presented. The Figures and Tables presented are relevant to the content of the article and properly described and labelled. Though, all the figures are not presented in a sufficient resolution, particularly Figure 4, in which the legends of the y-axis are almost impossible to read. All the appropriate raw data have been made available.
Experimental design: Methods were described with sufficient information to be reproducible by another investigator. Basically, the authors divided the experimental design in 4 bioassays aiming to achieve 4 different answers.
	An experimental concern regarding the “Preliminary Bioassay 1: Toxicity test of Argovit-4 supplemented in feed” need to be addressed. This bioassay was performed to answer whether Argovit-4 supplemented in feed exhibits toxicity towards shrimp. The experimental design for testing the toxicity of Ag as AgNP was conducted to answer only a small piece of this important and complex question. Why did the authors choose to perform the experiment for only 192 hours? It may be not a sufficient period to look at effects on crustaceans after exposure via food. A longer period of exposure would be required to ensure the non-toxic effect of the Argovit-4 supplemented in feed, otherwise, the findings of this work are answering only some acute effects questions. What about the chronic effects?
Additionally, the endpoints chosen (“signs of toxicity”, such as lethargic movement, empty gut or atypical colour change and mortality) are the tip of the iceberg. We do not know, from the results of this experiment, if there are occurring internal and sub-lethal effects (bioaccumulation, distribution of Ag to the tissues, histo and/or physiological alterations). Adding this to the short time of exposure, I am afraid you cannot state that there are no toxic effects of Argovit-4 supplemented in feed to the shrimp.
Validity of the findings: The toxic effects of Ag and its nanoparticles should not be taken as a simple and trivial question, despite the successful results obtained using them against the WSSV. The highest concentration tested, and the most effective against WSSV, was 1000 µg/g. This is an enormous amount of AgNP. Taking this into account, there are some thoughts to be considered in the discussion:
-	What would be the satisfactory frequency of feeding with Argovit-4 to achieve the expected results against WSSV infection? Would it be occasional or continuous?
-	The observation of AgNP chronic effects should be considered.
-	Is there Ag accumulation by the shrimp? If there is Ag accumulation, what would be the impact of this accumulation in shrimp consumption?
-	It would improve your findings regarding AgNP toxic effects if you also looked at sub-lethal effects. For example, you already collect the haemolymph to perform the assay with haemocytes. It would be very useful to use this haemolymph also to determine Ag concentration. By doing that, you could investigate internal doses of Ag after feeding and whether this Ag is being absorbed - and distributed throughout the body - or is being excreted (Vannuci-Silva, M. et al. Environ. Toxicol. Chem, vol 38, n. 4, 806. 2019).
-	How could we avoid the contamination of the marine environment (water and biota) that surrounds the shrimps’ farms?
Additional comments: The benefits of AgNP as broad-spectrum antimicrobial can be used to solve many issues, however, we must be aware of its possible risks. A deeper discussion about the toxic effects of AgNP on the crustaceans is necessary. The study suggests the use of a substantial amount of AgNP (up to 1000 µg/g) to combat WSSV infection. I strongly recommend other ecotoxicological assays using longer periods of exposure (chronic) and observing also sub-lethal and “non-visible” effects. The possibilities to avoid contamination of the aquatic environment by the waste of AgNP would be valuable information too.


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: EVALUATION OF A NEW ARGOVIT AS AN ANTIVIRAL AGENT INCLUDED IN FEED TO PROTECT THE SHRIMP LITOPENAEUS VANNAMEI AGAINST WHITE SPOT SYNDROME VIRUS INFECTION
Review round: 1
Reviewer: 2

Output:
Basic reporting: Reviewer comments:

Overall and interesting paper in merit of publication; however, the statistical assessment and description are greatly lacking. Please re-analyse your data using adequate statistical models for the mortality assays.
The introduction and methods are well written, but I think the results and discussion may change once the data are correctly analysed. The use of an ANOVA may be correct but the data types need to be explicitly mentioned. If parametric statistics are required, I suggest modelling the data in a glm or using a basic Kruskal test.

Major comments:
The statistical analysis is really the only part of the process that seems to be lacking. All the experiments seem to be well planned and rigorously executed. You should apply a form of mortality model, such as is available from the coxme package in R. The data type (normal?) is not reported, so it is difficult to know if the correct tests have been applied, but I doubt in this case that they are sufficient to analyse the data. In addition, the result of all statistical tests are not adequately represented in the text. You should be explicit in saying which test was used and the resulting output such as degree of freedom and supporting statistics like r2 or others depending on the test you use and the exact p-value.

Minor comments:
General English needs to be corrected.

There is no mention about whether this antiviral might have negative impacts on humans after consumption? Could this be elaborated upon in the discussion?

Fig 3 – this should be re-plotted using a better statistical model, possibly using the R package coxme and related packages.

Figures: Please keep your colour system the same throughout. You use blue triangles for different controls across graphs and it is confusing.
Experimental design: See report
Validity of the findings: See report
Additional comments: A great paper, but your analysis needs work. I can suggest it is accepted once the analysis is sound.


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: EVALUATION OF A NEW ARGOVIT AS AN ANTIVIRAL AGENT INCLUDED IN FEED TO PROTECT THE SHRIMP LITOPENAEUS VANNAMEI AGAINST WHITE SPOT SYNDROME VIRUS INFECTION
Review round: 3
Reviewer: 1

Output:
Basic reporting: A discussion regarding the toxicity of silver (Ag) and AgNP to crustaceans or aquatic organisms was made. Figure 4 was rebuilt and separated into 3 figures, which improved the visualization and comprehension of them.
Experimental design: The authors have addressed the answers regarding bioassay 1 satisfactorily.
Validity of the findings: The authors have addressed the answers regarding the environmental concerns of using AgNP fo shrimp treatment. The questions that could not be solved by this work pointed out for the next studies.
Additional comments: The authors considered the reviewers' comments and suggestions. The questions that could not be answered by this study were pointed out in the discussion and further studies were proposed aiming to solve them.


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: ANTIMICROBIAL RESISTANCE AND GENETIC RELATIONSHIPS OF ENTEROCOCCI FROM SIBLINGS AND NON-SIBLINGS HELICONIUS ERATO PHYLLIS CATERPILLARS
Review round: 1
Reviewer: 1

Output:
Basic reporting: see entire review below
Experimental design: see entire review below
Validity of the findings: Manuscript. No.: #41227

Title: Antimicrobial resistance and genetic relationship among enterococci from sibling and non-siblings Heliconius erato phyllis caterpillars

Authors: Rosana Huff, Rebeca Inhoque Pereira, Caroline Pissetti, Aldo Mellender de Araújo, Pedro Alves d’Azevedo, Jeverson Frazzon, Ana Paula Guedes Frazzon

Overall, the manuscript is well written- clear, professional and scientific English language is used throughout. The study’s experimental design needs more work. Specifically, the authors will need to provide more information on a) why only one antibiotic resistance gene was tested, and b)why their results don’t correlate between the susceptibility testing results (particularly erythromycin resistance) and the detection of the erythromycin resistant gene ermB (see detailed comment below under results section of this review).

Please see below for my specific comments to improve the manuscript.

Abstract and Introduction:
The introduction needs more detail and background with relevant citations to establish a) why studying the antimicrobial resistance (AMR) and multidrug resistance (MDR) is overall so important considering the current global AMR crisis, and b) why studying AMR and MDR is particularly important in insects. Providing more background information on our current knowledge regarding the role of insects in the dissemination of AMR/MDR would also be very beneficial for the readers of the journal.

Line 26 – bacteria instead of bacterial.
Line 41 – instead of ‘submitted to’, use ‘analyzed for’
Line 79 – please elaborate on what ‘host’ you’re indicating here

Materials and methods:
Lines 128-129 – how are these eggs transported?
Line 130 – How many fecal samples were collected? I see you mention this in your abstract as n=12, you should also put this in the actual methods section

Lines 153 – 156 – the DNA extraction method needs more detail, only citing a paper isn’t enough. Was a kit used? What was the elution volume? Did you measure DNA concentrations? PCR methods also need to be far more detailed. What is the mastermix composition? What is the program used? Where were the mastermix components purchased from?

Line 154 – tuf gene – at first use, please explain what the function of this gene is. This helps readers with adequate information for easy understanding.
Lines 161 – 162 – PCR assay need more detail. I see you provide table 1, but this does not have the mastermix compositions, the thermal cycler programs. Please include this information either in the text or in the table as separate columns.

Lines 173 – 182 – Antimicrobial susceptibility testing – Please detail on the actual protocol used. Just a reference is not enough as this is one of the most important methods you have used for generating your dataset. What standard was used to assess whether the strains were susceptible, intermediate or resistant?

Lines 184 – 187 – Why was only one antibiotic resistance gene chosen? I see you tested for so many other antibiotics for your Kirby Bauer assay, but you have tested only one of the genes. There are hundreds of genes that could be tested not just for presence, as done in this study, but also for quantification of these genes in the starting sample collected. Therefore, selecting only one gene for detection does not make much impact as per a study like this is concerned.

Lines 184 – 191 – The PCR methods need more detail in this section as well as mentioned above.

RESULTS:
Antimicrobial Susceptibility – lines 225 – 233 – Instead of a table, I recommend the authors provide a figure of this for better understanding. You could divide your plot into the different strains of Enterococci tested, and make a stacked plot with the results from the different antibiotics tested.

Lines 235 – 236 –
•	Why only one antibiotic was tested for the gene presence need to be explained.
•	Authors also need to explain why the 55 strains tested negative for ermB, and all 55 strains tested positively resistant for erythromycin as per their antimicrobial susceptibility testing results.

Line 244 – I did not notice a table S1 in the submitted manuscript packet
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: ANTIMICROBIAL RESISTANCE AND GENETIC RELATIONSHIPS OF ENTEROCOCCI FROM SIBLINGS AND NON-SIBLINGS HELICONIUS ERATO PHYLLIS CATERPILLARS
Review round: 1
Reviewer: 2

Output:
Basic reporting: The article is generally well structured. Enough background information is provided to stress the importance of the topic. I have certain concerns considering English language that will be listed below in the field General comments for the author. Although I made specific comments and suggested corrections, a native speaker should check the manuscript. The figures and tables are appropriate, corresponding to the text well – I have made minor objections only.
Experimental design: The study matches aims and scopes of the Journal. The aims are clear and are logically related to the background information. The research question is meaningful and relevant in light of scarce data about the topic.
Validity of the findings: The results are valid. Their importance is unquestionable in light of serious problem of growing antibiotic resistance. Conclusions are clear, logic and based on the results.
Additional comments: Abstract

Line 26-27: In the sentence “Studies evaluating bacterial in insects could provide information about host-microorganism-environment interactions.” What do you mean by “bacterial”? Is it bacterial community, bacteria, microbiome or something similar? “Bacterial” is an adjective.
Lines 53-54: In the sentence “Resistant enterococci strains could be related to environmental pollution or linked to environmental resistome.” It would be better to erase “linked to”.
Lines 54-57: In the sentence “The PFGE analysis showed related genetic relationship among some strains, suggesting that the enterococci isolated from fecal samples of fifth instar sibling caterpillars might have come from common sources, by diet (herbivory) and/or via vertical transmission (through egg surface).…” I believe that the word “related” is redundant. If there is a relationship between the two, then it is obvious that those are related. In addition, I would write “…via diet (herbivory) or vertical transmission (through egg surface)”.
Lines 57-59: In the sentence “Further studies will be conducted to better understand the role of Enterococcus on the microbial gastrointestinal tract community of these insects…” I believe there should be “…in the microbial gastrointestinal tract community of these insects…”

Introduction

Line 62: please improve English. “Heliconius (Lepidoptera; Nymphalidae) comprises numerous widespread species of butterflies” or Heliconius is or represents a widespread genus of butterflies”. To comprise means to contain. So, genus can only contain species.
Line 80: please rewrite. I don’t understand “Gut microbial community provide an especially diverse range of benefits in insect nutrition, e.g. by providing xenobiotic metabolism? To provide means to give someone something that they need (Cambridge Dictionary). So, how is the gut microbial community related to xenobiotic metabolism”?
Line 83: to introduce an abbreviation „GI tract“, you should provide full term here when first mentioned, not in the line 85. In the line 85 the abbreviation is enough.
Lines 86-88: It is hard to understand this sentence. In addition, there should stand “one of the most frequent genera” instead of “the most frequently genus”. Anyway, the whole sentence should be rewritten. For example: Enterococcus is one of the most frequent bacterial genera present in the gut microbiota at different life stages of Lepidoptera (Brinkmann, Matens & Tebbe, 2008; Chen et al., 2016; Ruokolainen et al., 2016; Snyman et al., 2016; van Shooten et al., 2018; Allonsius et al., 2019).
Lines 92-94: I would write like this: Hammer et al. (2014) reported that Enterococcus was the most abundant genus found in immature stages and adults of H. erato from Panamá. First, if it is Enterococcus spp, as you wrote, then it is related to species of that genus. Second, Hammer et al. (2014) did not mention subspecies of H. erato, as I could see in the original article. If I am wrong, then please name that subspecies you referred to.
Line 105: Previusly was not mentioned that Enterococcus might play a fundamental role in the development of those insects. Perhaps you intended to state that implicitly earlier in the text, but here it sounds too explicitly without references. Please, rewrite or add references.

Results

In the Table 2 – I think it should be emphasized that it is about the strains. “Number (%) of enterococci strains isolated of caterpillars from”
In M&M (lines 118-122) you introduces three localities you collected butterflies from in this order: “The first female (HEAB2) was collected in a forest fragment located in Águas Belas Agronomical Station (30° 120 02' 18.1" S; 51° 01' 23.0" W), the second female (HES2) in a forest fragment located in São Francisco de Paula (29° 26' 34.1" S; 50° 36' 48.8" W) and the third female (HEV2) from a population in an intense urban area in Viamão (30° 09' 40.5" S; 50° 55' 01.5" W).” Then, in the lines 127-128 you change the order: “A total of 12 eggs were collected (five from HEAB2, five from HEV2 and two from HES2) with the assistance of a paintbrush. Also for example in the table 2 it is HEAB2, HEV2 and HES2. This is very confusing for a reader because the reader most probably is not familiar with the localities in your area; for example, I first remembered that the third locality was in urban area and later I noticed that I was interpreting results in a wrong way because you changed the order of localities with no reason. Please, try to be consistent throughout the manuscript as possible. And please check whole result section including tables and figures to fix this problem.

Discussion

Lines 296-297: “E. casseliflavus was more frequently identified in feces of H. erato phyllis caterpillars”. Compared to what?
Line 297: Perhaps it is better to write “The diet constitutes an additional source of microorganisms”
Line 299: with regard to; “since these bacteria are able to…”  instead of “since this genus are…”
Line 301-304: Unclear sentence; I would put it like this: “Since E. casseliflavus is frequently a part of microbial communities in plants (Byappanahalli et al., 2012; Micallef et al., 2013; Ong et al., 2014), the predominance of this species in fecal samples of H. erato phyllis caterpillars could be explained by the herbivory of juveniles.” I do not understand what it means “unleashing the predominance of these species”.
Lines 304-305: Please explain in more detail how enterococci may act beneficially for caterpillars.
Line 319: Delete “linked to”
Line 326: Delete coma after “Although” and before “may be associated”
Lines 341-342: “these bacteria improve quality of diets poor in nutrients and take part in development…”
Lines 344-345: …herbivory enables abundant supply…
Line 346-347: …those leaves could be the source…
Line 348: Besides the diet, the vertical transmission…
Lines 354-360: This is too complicated, it should be rewritten.
Lines 361-362: Teh et al. (2016) showed the route of transmission of E. mundtii in S.littoralis when administered in vivo; the authors reported the presence of E. mundtii at all life stages of this insect. In addition, they established the presence of this bacteria in oocytes and the muscle tissue in the first-instar larvae of the second-generation offspring, highlighting again the vertical transmission of enterococci in this butterfly species.
Line 365: despite our analysis does not include
Line 366: we do not discard the possibility that enterococci…

Conclusions

Line 371: E. casseliflavus was the dominate genus
Line 374: delete “also”
Line 376-377: via diet (herbivory) and/or vertical transmission (through egg surface).
Line 378: in the microbial GI tract


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: ANTIMICROBIAL RESISTANCE AND GENETIC RELATIONSHIPS OF ENTEROCOCCI FROM SIBLINGS AND NON-SIBLINGS HELICONIUS ERATO PHYLLIS CATERPILLARS
Review round: 1
Reviewer: 3

Output:
Basic reporting: On line 170-171, please elaborate on the quality of the sequences generated. Please mention: how many base pairs were the sequences? Did you use any filtering criteria? If so, how many sequences did you have initially and how many did you end up wit after filtering.

On line 204-206: What parameters were used to construct this dendogram? For example, did you construct a maximum likelihood tree? Describe again how many sequences you used. Also describe, what was the outgroup used for the tree. How should the branch lengths of the tree be interpreted. Right now the methods in this section seem very vague and they need more details.
Experimental design: No comments
Validity of the findings: No comments
Additional comments: I really enjoyed reading this paper. I think this paper adds an additional dimension to microbiome research in Lepidoptera. The missing details mentioned above should be added before Acceptance.


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: ANTIMICROBIAL RESISTANCE AND GENETIC RELATIONSHIPS OF ENTEROCOCCI FROM SIBLINGS AND NON-SIBLINGS HELICONIUS ERATO PHYLLIS CATERPILLARS
Review round: 2
Reviewer: 1

Output:
Basic reporting: The authors in their "Rebuttal letter" have answered the questions and comments I had previously made on the earlier version of the manuscript. The authors, however, do mention in the letter on few instances (some of the methods and results/figures comments) that they did not make some of the changes to the manuscript as they thought it was not necessary.

I still do think that making those changes/ adding those elements into the main manuscript will enhance the quality of the manuscript as a whole, and that based on PeerJ standards it is necessary to do so.
Experimental design: see comment above
Validity of the findings: see comment above
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: ANTIMICROBIAL RESISTANCE AND GENETIC RELATIONSHIPS OF ENTEROCOCCI FROM SIBLINGS AND NON-SIBLINGS HELICONIUS ERATO PHYLLIS CATERPILLARS
Review round: 2
Reviewer: 2

Output:
Basic reporting: The article is revised well and the english is clear now.
Experimental design: The authors have done a great job in revising the methods and they are more articulate now.
Validity of the findings: Overall the paper looks great now after all the revisions.
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: COMPREHENSIVE PHENOTYPING AND TRANSCRIPTOME PROFILING TO STUDY NANOTOXICITY IN C. ELEGANS
Review round: 1
Reviewer: 1

Output:
Basic reporting: The manuscript had many typos and instances where words were entirely missing. One highlight is "in contract" on line 258. I recommend the author go over the manuscript again with basic grammar and syntax in mind.

There was nice background and context provided.

One issue I had was with some acronyms such as NES. Which was mentioned multiple times in the results section without an explanation of the acronym. I might have missed the declaration of it before.
Experimental design: I thought the experimental design was well planned and seemingly well executed. The authors presented novel research that has relevance for basic biology as well as relevance in with regard to translational medicine in humans. I thought the methods were well explained and I was not at a loss when considering the motivation and execution of the experiments.

I think that some tools and annotations used by the author are out of date such as GSEA since they are relying so heavily on the gene set enrichment analysis of the project it would be nice to use an additional tool (Such as GOAtools https://www.nature.com/articles/s41598-018-28948-z) to see if the see the same results.

The wbcel235 annotation is also quite old and the authors would find potentially more genes of interest if they use the current wormbase version (WS271).
Validity of the findings: I do not doubt the validity of the findings.

I think that the amount of differential expression (DE) of genes found in the two RNA-seq experiments should be put in the paper and not put in a supplemental. The amount of DE genes found has a high impact on which biological pathways are reported.

In addition, it is easy to copy and past 20 enriched biological pathways but it is hard to make sense of the results. I commend the authors for doing their homework and interpreting their data but with so many pathways it is easy to be speculative about the validity of their findings. I think RT-PCR validation on the important genes mentioned by the authors would do well to boost the RNA-seq data.
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: COMPREHENSIVE PHENOTYPING AND TRANSCRIPTOME PROFILING TO STUDY NANOTOXICITY IN C. ELEGANS
Review round: 1
Reviewer: 2

Output:
Basic reporting: Basic reporting:
In general manuscript is well written with a few changes suggested.
Line 1: Remove “of”
Line 32: Consider rephrasing as not clear what “the nanotechnology” is defined to include.
Also imprecise language is often used such as around, or ~ and this weakens the impact of the manuscript. This will be explored further in the experimental design section also.
Line 165 & 166: Unnecessary repetition from methods.
Experimental design: This research matches the aims and scope of this journal.

Research question is not very well defined and this is reflected in the experimental design.
Whilst the methods are well described and could be replicated, the overall research question is very general and chosen methods do not follow a clear line of questioning and argument despite still providing some useful data.
Validity of the findings: The major issue is centred on questions of uptake of these nanoparticles. In table 1. Particle size is shown to vary between each treatment. Previous literature has demonstrated the importance of particle size and uptake of particles in C. elegans (eg. Fang-Yen, C., Avery, L., & Samuel, A. D. (2009). Two size-selective mechanisms specifically trap bacteria-sized food particles in Caenorhabditis elegans. PNAS, Flavel, M., et al. (2018) "Growth of Caenorhabditis elegans in defined media is dependent on presence of particulate matter." G3: Genes, Genomes, Genetics. The variation of size introduces a variable to the experiment that needs to be addressed by the authors. Furthermore, if there is a variation to the uptake the relative concentrations into the worm could vary significantly and concentration of these particles and compounds inside the worm should be measured.
Addressing the variation of size in these particles would also include at least some speculation or ideally experimental data on how these particles are being taken up. At 100 micron, these particles are not far outside the edible/ bacterial size range for C. elegans. Therefore, even though steps have been taken to prevent aggregation, aggregates of as little as 2-5 particles would be likely to be ingested at a similar size to food and therefore the mode of uptake is no longer analogous to toxicity in larger animals such as humans. If these particles are being ingested as food this study becomes less about the effect of nanoparticles and more about the toxicity of ingesting silver, titanium etc which reduces the novelty of this work significantly and also as already discussed the concentration of these compounds inside the worm has not been controlled for. If the uptake is likely to be across the cuticle some imaging would be helpful to see where these particles accumulate and if they are able to be excreted. Overall, it is also unclear from the evidence presented whether the relative size difference between a worm and these particles is different enough to justify the claim that  this is a useful tool for profiling nanotoxicity, if the ultimate application of this research is to compare the toxicity effect of these same sized particles with other much larger animals in the environment (fish, humans etc) .
The secondary issue is that whilst the RNAseq data is useful other assays both phenotypic and more general could have been conducted to align with this data. Other general phenotypic measures such as lifespan, response to stress and behavioural assays would strengthen this manuscript. A dose response to these particles would have also been useful to understand the effect or potential benefit of reducing these nanoparticles in the environment.
Line 87 & 91: Numbers of worms used for each assay varies and is not reported accurately. For experiments of this size it is not clear why a set number of worms was used and reported. Especially as assays are short and worms are unlikely to be censored. If they have been censored this does not appear in raw data or discussed in text.
Line 108, 119: Unclear why more precise worm numbers were not used
Additional comments: I commend the authors for preparing this manuscript. The data provided is a good indicator of some relevant toxicity parameters. The use of advanced techniques, particularly the RNAseq strengthens the manuscript and provides novel data that would be interesting to both the C. elegans and wider research community. However, there are currently some major issues with the manuscript that would need addressing, before it could be accepted for publishing in PeerJ.


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

Input:
Title: COMPREHENSIVE PHENOTYPING AND TRANSCRIPTOME PROFILING TO STUDY NANOTOXICITY IN C. ELEGANS
Review round: 2
Reviewer: 1

Output:
Basic reporting: The authors have made all necessary improvements to basic reporting required.
Experimental design: Experimental design has not changed since last revision.
Validity of the findings: I accept the arguments put forward by the authors and agree it is valid to infer that uptake of nanoparticles has occured. The lack of control or measurement of this process still causes me concerns.

The authors speculate in their rebuttle that there is unlikely to be uptake across the cuticle.This again raises the question of how reliable a model for nanoparticle toxicity this presents, if this critical uptake pathway for nanoparticles in higher animals is not present in this model. I accept that there is disease and cellular homology that makes C. elegans a useful model for general toxicology, but the authors should include more discussion of what makes C. elegans a useful model for nano-particle focused toxicology.

If uptake is entirely through the pharynx, then clearer comparison to other studies where these same elements and comparable concentrations have have been conducted on C. elegans, but not using nanoparticles is required. It is unclear currently whether the toxic effects are related in any way to the delivery method of the compounds being through nanoparticles or if it is only a consequence of an increased dietary intake of silver and silica. Because the article is framed around investigating nanoparticle toxicity, not a toxicity experiment using traditional methods and therefore this distinction needs to be fully explored to meet the aim of the study.

If the uptake is mediated by co-feeding with OP50 E. coli, is there any effect on the viability of E. coli?  Whilst uptake may have occurred it is also unclear still if there is evidence that this uptake is constant between treatment groups.
Additional comments: Most of the feedback from the first submission of this manuscript has been addressed. However, there are some remaining issues with the manuscripts fundamental aim that need to be addressed. These are described in detail in the validity of findings section. Overall, I accept the findings are of interest to the C. elegans field, due largely to the RNAseq data. However, I do not recommend publication unless the short comings of this experiment are overcome, or at least thoroughly justified in the manuscript text.