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: DETECTION OF BACTERIAL CONTAMINANTS AND HYBRID SEQUENCES IN THE GENOME OF THE KELP SACCHARINA JAPONICA USING TAXOBLAST
Review round: 1
Reviewer: 3

Output:
Basic reporting: No comment (see below).
Experimental design: No comment (see below).
Validity of the findings: No comment (see below)
Additional comments: The authors describe in the manuscript a pipeline named “Taxoblast”, which uses a BLAST-based approach and predefined taxa to identify contaminants, as well as hybrid sequences, in assemblies of genomes. The pipeline is publically available. As a case study, the authors examined the genome assembly of the kelp Saccharina japonica and identified some bacterial contaminants as well as bacteria–kelp hybrid contigs.

The manuscript is well-written and easy to follow. The described approach is simple, and it is laudable that the pipeline is freely available.

However, the manuscript reads rather like a case study. It remains uncertain whether the kelp genome harbours particular problems, or whether similar problems occur in other datasets. For example, the authors state that internal tests found very little differences between blastn and blastx searches. However, this may not apply for other genomes, in which the possible contaminants are less well-known, or less diverged from the host (for example eukaryote parasites). I expect that in these cases, a blastx-based approach has clear advantages.

It would also be useful to compare the present approach with some other programs or platforms, e.g. KRAKEN, which are commonly used for identification of contaminants.

Other issues:

l. 40: I would not call a paper published in 2011 as “recent”. Moreover, it would be useful if the authors cite a study showing that contaminants are an even more eminent issue in NGS-derived genomes.
l. 50 (and other places throughout the manuscript): Something went wrong with the in-text citations, for example here: “(e.g. [3,4])” .
l. 98-99: I think the authors mean: "blastx searches against the National Center for Biotechnology Information (NCBI) _protein_ nr database and blastn searches against the NCBI _nucleotide_ nr database". nr = non-redundant.


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: DETECTION OF BACTERIAL CONTAMINANTS AND HYBRID SEQUENCES IN THE GENOME OF THE KELP SACCHARINA JAPONICA USING TAXOBLAST
Review round: 2
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: See below.
Validity of the findings: no comment
Additional comments: The authors have submitted a revised version of their manuscript that describes the pipeline named “Taxoblast”, a BLAST-based approach to identify contaminants and hybrid sequences in contigs.

The authors have added a brief benchmark estimate and have corrected minor errors. The manuscript has significantly improved, and I acknowledge the rationale behind the study. I would have preferred a broader approach that compares, e.g., the results obtained with different methods and with different data. However, the paper is sound as it is.


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

Input:
Title: GINSENOSIDE RK1 BIOACTIVITY: A SYSTEMATIC REVIEW
Review round: 1
Reviewer: 1

Output:
Basic reporting: .
Experimental design: .
Validity of the findings: .
Additional comments: This manuscript handles Ginsenoside Rk1 bioactivity: A systematic review. This is an interesting and well-written manuscript. However, the manuscript contains some weakness that should be revised before it is considered for the journal publication:

Line 66: Genus Panax can be classified by origins. Chinese ginseng is Panax notoginseng and Korean ginseng is Panax ginseng “traditional Chinese medicines” should be changed or rephrased to “traditional medicines”.

Comparison with other ginsenosides will be needed.


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: GINSENOSIDE RK1 BIOACTIVITY: A SYSTEMATIC REVIEW
Review round: 1
Reviewer: 2

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: Authors curated 21 original research papers showing biological and pharmacological effects of ginsenoside Rk1 and summarized findings of each paper in the manuscript. Due to the style that seems to be common in medical fields, authors carefully enlist endless effects of Rk1 on endless lists of disease without providing any conceptual framework on how Rk1 works. This could be due to original papers that hardly provide any mechanism. Ideally, authors should have tried to make a sense out of all those different effects by extrapolating signaling mechanisms behind all those diseases. Nevertheless, the manuscript is resourceful and  should help readers who want to know reported effects of Rk1 on various disease.


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: GINSENOSIDE RK1 BIOACTIVITY: A SYSTEMATIC REVIEW
Review round: 1
Reviewer: 3

Output:
Basic reporting: 1)	The manuscript needs to be checked and re-edited for grammatical and punctuation errors. I would suggest that the authors revise the abstract, as lines 51-58 seem repetitive in nature.
2)	The manuscript summarizes all relevant papers and provides a good overview of the literature but this study has only been performed till Aug 2015 and not till the present day (in this case May/June 2017). Thus, it is recommended to revise this manuscript to include all the literature that has been reported till present date.
3)	How many different types of ginsenosides are there in total? It would be better to include a brief description along with a figure in the introduction, which talks about all the different ginsenosides, and how they are isolated. This will help the readers understand better, how Ginsenoside Rk1 is an entirely different class of compound and exhibits unique bioactivity when compared with its sister analogues.
4)	The results section on the anti-cancer activity including sub-titles liver cancer, melanoma and gastric cancer is very descriptive, repetitive and needs to be re-edited.  The authors have already provided similar information in table 2. I recommend thoroughly revising this particular part of the manuscript to reflect critical findings. Avoid using the word “they” excessively within the anti-cancer section. The other half of the bioactivity section (anti-platelet, anti-inflammatory and so on) is well-written.
5)	The references are adequately cited within the manuscript
6)	Figures and table provide enough relevant information and conforms to the publisher guidelines.
Experimental design: 1)	The research presented within the manuscript is original and within the scope and aims of the journal.
2)	The systemic review or meta-analysis is carried out in a detailed and systematic manner.
3)	The GRADE method followed by the authors is a good example of methods that need to be employed to avoid bias in reporting findings. The grading system has also been adequately described by the authors in Supplemental Table 2.
Validity of the findings: The authors tend to be quite positive about the findings published in other articles, without critically assessing related risks and disadvantages. This may in some cases sketch an unrealistic view of Ginsenoside Rk1 bioactivity. For example, the authors describe Rk1 as anti-cancer drug, however, not a whole lot of work has been conducted with this particular compound and majority of the work published has been primarily carried out in vitro. How would the drug behave in vivo when it is administered to tumor bearing mice or cancer patients ? Is the compound stable ? What are the solubility and bioavailability parameters that need to be taken into account to develop them into anti-cancer therapeutics? Such statements would greatly contribute to the novelty and originality of the manuscript, and I strongly recommend revising this in the conclusions/discussions section.
Additional comments: Line 46: Introduce space before we
Line 55: Shouldn’t the word be “on” liver cancer, melanoma and gastric cancer?
Line 71: Re-edit the sentence
Line 128: Change to includes or included rather than including
Line 268: “in the” is repeated twice
Line 272: Remove space between I and t
Line 301: Use “by” instead of “through”
Line 391: “Confirmed the anti-cancer effects” is a strong statement to use considerably due to all the in-vitro studied conducted. Please revise this statement.


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 AND GENOME-CENTRIC ANALYSES OF KOALA AND WOMBAT FECAL MICROBIOMES POINT TO METABOLIC SPECIALIZATION FOR EUCALYPTUS DIGESTION
Review round: 1
Reviewer: 1

Output:
Basic reporting: No comment
Experimental design: The majority of this project is excellent. This is the first in-depth culture-independent metagenome-based analysis of koala and wombat. A weakness relates to the use of fecal pellets as the sole source of microbiome analysis and then relating the microbiota to koala and wombat digestion. Eventhough there was an early publication in the koala, this only had a very low number of animals. The digestive microbiota communities in the gut and caecum and likely to be different to those in the fecal pellet. Can the authors perhaps include the comparisons of the metagenomes of a single animal.
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 AND GENOME-CENTRIC ANALYSES OF KOALA AND WOMBAT FECAL MICROBIOMES POINT TO METABOLIC SPECIALIZATION FOR EUCALYPTUS DIGESTION
Review round: 1
Reviewer: 2

Output:
Basic reporting: The basic reporting in this paper is fine.  Overall it is clearly written.  My most substantial comment is that sometimes information appears in the wrong section and is sometimes redundant with what has already been stated.  For example: Lines 80-90 present a summary of the results.  This does not seem appropriate for the introduction.  I have flagged several of these instances but the authors are encouraged to conduct a thorough review.

The figures for the most part are highly useful. Some required additional explanation.  E.g.,
Fig 2: indicate the significance of the different number of circles in panel A.
Experimental design: The design and analysis of the results are well defined.  The knowledge gap being addressed is articulated. The only criticism of the methods is that it would have been better if there were more control over the origin of the samples (ie more samples from the same zoo), but given the difficulty in keeping these species, the design is reasonable, and did not appear to impact the outcome.
Validity of the findings: Overall the findings seem valid, robust and statistically sound.  The notion that there are members of the koala microbiome that function to degrade PSMs is likely true, and the authors for the most part acknowledge that this is a hypothesis yet to be experimentally tested.
Additional comments: In this manuscript, the authors present a comparative study on the gut metagenomes of two iconic mammals from Australia, the koala and the wombat. This is the first investigation into these metagenomes.  They found that while there are many similarities in these metagenomes, the koalas is larger with greater representation of metabolism of plant secondary metabolites.  The authors propose some key players in the microbiomes of these animals related to PSM metabolism, lignocellulose degradation and urea recycling.

S24-7 is mentioned in the abstract as being one of the unique members of the koala microbial community.  The role of family is not further discussed in the discussion, yet it has been implicated in the degradation of PSMs, particularly oxalate, which likely occurs in Eucalyptus. Some references of potential interest: Ormerod et al 2016 4:36 Microbiome and Millar et al AEM 82:2669;

Along these lines, the putative function of the two taxa common in the wombat were not mentioned in the discussion.

The discussion could be improved through the inclusion of additional recent literature.  recent experimental  Such as Kohl et al Ecology Letters 2014; Welte, 2016 AEM 04054; Haiser et al Science 2013 341:295, Spanogianopoulos Nature Reviews Microbiology 2016 14:273

Figure 1 is beautiful.  Does it include all genera in the Diprotodonts or is it an abbreviated tree?

Suggest removing “toxic” from Eucalypt. The concept of high levels of plant secondary metabolites can be explained in the text.

“’putatively” detoxifying bacteria

Lines 80-90 present a summary of the results.  This does not seem appropriate for the introduction

Lines 275-283 mostly contain information that should be in the methods.

Fig 2: indicate the significance of the different number of circles in panel A.

The term CDS seems to be defined long after its first use

Fig 3: add an explanation of the use of colored dots. I figured it out eventually but it would be useful as an aid to the reader.

Lines 348-359: I could not identify the results in this paragraph.  Seems like it belongs in the methods.

Interpretation that may be better suited to the discussion appears in the results, check lines 375-400 for examples.

The paper would be significantly shorter if the methods were not reproduced in the results

425: I think it would be more accurate to say microbe populations from koalas instead of koala populations; same comment line 471

443-455: most of this is methods

461-463: discussion

504-507 methods
524-527: discussion

619: restate the phylum’s name of Synergistaceae here

694-5: citation for phylosymbiosis?  See Bordenstein’s 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: ANTIMICROBIAL CONSUMPTION ON AUSTRIAN DAIRY FARMS: AN OBSERVATIONAL STUDY OF UDDER DISEASE TREATMENTS BASED ON VETERINARY MEDICATION RECORDS
Review round: 1
Reviewer: 1

Output:
Basic reporting: In general my opinion is that the manuscript is written in an extensive way. There is a lot information that is actually not really needed for the reader. I believe that the more interesting information that is presented in this study would/could be more convincing when it is presented as short communication. In its current version it contains too much side information that distracts the reader from the essential and good/nice results of the study. For instance, the introduction and discussion are rather long. The results focus quite extensively on the number of treating farms, while I believe the essential information is included in the nDDDvet results. Furthermore, the statistical comparison between acute and chronic treatments is nice, but in my opinion not necessary information.

L12-13: 89.4% and 10.8% does not sum up to 100%
Some sentences are formulated in a strange/difficult way, e.g. L27 “the highest mean number”. I suggest that the manuscript is checked by a native speaker. E.g. L262-263. I believe you mean “6334 single antimicrobial treatments of dairy cows were observed…”
L43 these products are actually “antibiotic-free”, since there is a withdrawal period. Consider rephrasing
L93 “population correction unit” instead of “corrected”
L183 per prescribing “veterinarian” or “practice”? The results are presented per veterinary practice.
L194 the multiplication sign “x” should not be in italic
L230 drop “the calculation of”
L268 drop “As would be”
L285-286 this information is not necessary in the text, below the table is sufficient.
L305-309 please rephrase, the test and its properties were already mentioned in the M&M section, so no need to repeat them here.
L310-314 Please rephrase, this is very technical and not clear what you mean. The sentence is 5 lines long and the message is unclear to me.
L327 there were no 6334 datasets. There were 6634 observations (= single antimicrobial treatments) in the dataset…
L350 “other” do you have an example of this?
L376 drop “that”
Please check the references. E.g. L523 Preventive Veterinary Medicine with capitals.
Experimental design: The study design is well described. However the authors should discuss the disadvantages of their design more thoroughly (see validity of the findings).
Validity of the findings: The discussion should stress more the external validity of these results. Although it is already present in the discussion, I would start the discussion with the novelty of the results (as actually present) and the drawbacks of the study design (currently mentioned at the end of the discussion):
-	Convenience sampling
-	No dry cow therapy
-	Only 30.6% of AM treatments in dairy cattle used
-	L216-217. Although this will probably only be a minority of the treatments, instead of not including these treatments, you could consider to estimate the AMC of oxytetracycline sprays, in analogy with Postma et al., 2015: “…1 mL will be sprayed per second and the fact that 3 s of spraying is the average duration mentioned in the SPCs for topical spray product…”
How do all these drawbacks influence the comparison with other studies?
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 CONSUMPTION ON AUSTRIAN DAIRY FARMS: AN OBSERVATIONAL STUDY OF UDDER DISEASE TREATMENTS BASED ON VETERINARY MEDICATION RECORDS
Review round: 1
Reviewer: 2

Output:
Basic reporting: Basic reporting and the use of English are good.
The authors explained the context of antimicrobial consumption in Austria well, and used relevant literature.
There are a few things that have to be addressed.

The are many acronyms in the article that are not explained, for example TGD (line 62 - it is explained in line 131), OIE (line 79), LKV (line 132),RDV (line 152), ESVAC (line 338), EDC, EFSA (line 423).

Line 207; "...., rather than per kilogram." (I assume you are referring to live weight?)

Lines 340-342. Are you referring to Germany (Merle et al), Austria (Obritzhauser et al), the EU, Europe or globally? Please state.

Lines 346-348 that refer to the New Zealand study: it is not only the calculation methods that distort the comparison, but in Austria dairy and beef production systems have been combined, while the NZ findings refer to dairy farms only. This makes a big difference and should be stated in the paper, or the NZ comparison should be deleted.

There seems to be an issue with one reference. Line 548. As it is, the authors have a mix of first and second joint report, and I believe the reference should be changed.
In APA 6th format it is:
ECDC (European Centre for Disease Prevention Control), EFSA (European Food Safety Authority), & EMA (European Medicines Agency). (2015). ECDC/EFSA/EMA first joint report on the integrated analysis of the consumption of antimicrobial agents and occurrence of antimicrobial resistance in bacteria from humans and food-producing animals. EFSA Journal, 13(1), 4006-4120. doi: 10.2903/j.efsa.2015.4006
In HARVARD is should be:
ECDC (EUROPEAN CENTRE FOR DISEASE PREVENTION CONTROL), EFSA (EUROPEAN FOOD SAFETY AUTHORITY) & EMA (EUROPEAN MEDICINES AGENCY) 2015. ECDC/EFSA/EMA first joint report on the integrated analysis of the consumption of antimicrobial agents and occurrence of antimicrobial resistance in bacteria from humans and food-producing animals. EFSA Journal, 13, 4006-4120.
Experimental design: Experimental design is sound and the method is described well.
The limitations of the research have been well described.
Using the standardised "Defined Daily Doses" for veterinary medicine of the EMA and use of AMC of active substances is a useful approach.
I believe the paper is within the scope and aims of the journal.
Validity of the findings: The findings are a good benchmark for Austria and a good comparison and contrast with other EU states.
Additional comments: I have gone through what the previous referees have said and I believe you have responded well to their 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: HRV-DERIVED DATA SIMILARITY AND DISTRIBUTION INDEX BASED ON ENSEMBLE NEURAL NETWORK FOR MEASURING DEPTH OF ANAESTHESIA
Review round: 1
Reviewer: 1

Output:
Basic reporting: This article introduces a heart rate variability based method to measure anesthetic depth.

In the introduction section, authors stated that EEG based indices are not suitable for deph of anesthesia (DoA) evaluation. They also stated that HRV would be more suitable to monitor DoA.  To my own knowledge EEG based indices like BIS or GE entropy are considered as gold standards and are widelly used worldwide for DoA evaluation.  I think authors should provide a more exhaustive literature references list to affirm that EEG is not suitable for DoA monitoring.

In the discussion, authors cited Jeanne et al 2009. This paper is related to nociception ; not to DoA.
Experimental design: The research question is well defined, relevant and meaningful.

Though the research question is well defined, the material and method section need to be described with more details.

Regarding the anesthetic procedure ;

- How anesthesia was performed ? Did all the patients received gas (sevoflurane, desflurane,...) ? How hypnosis was maintained (propofol ? gas ?) ? How anesthetic agents were adapted ?

- Were EEG indices like BIS or entropy measured during anesthesia?

 - Can authors list all the physiological/anesthesia parameters monitored during the procedure ?
Validity of the findings: The statistical analysis is not clear and should be completly rewrite ;

- How do authors justify the sample size ?
- Statistical analysis used for validation (ROC curve) are not clearly explain.
Additional comments: The statistical analysis needs to be explain more clearly.
I do not understand why authors didn't compared their method to the commonly use EEG based indices.


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: HRV-DERIVED DATA SIMILARITY AND DISTRIBUTION INDEX BASED ON ENSEMBLE NEURAL NETWORK FOR MEASURING DEPTH OF ANAESTHESIA
Review round: 1
Reviewer: 2

Output:
Basic reporting: The English language needs to be substantially improved in order for the paper to be accessible to a broad, international audience.  This is perhaps the biggest issue with the paper.

The literature review and structure of the paper is appropriate.

There are several details regarding the methods that are, however, opaque.  This includes (i) the criteria by which the experts scored the level of consciousness (i.e., the EACL); (ii) the construction of the ensemble neural network; (iii) the criteria for artifact removal in the ECG time series.
Experimental design: The experiment design is sound, and it is clear that substantial effort was put into execution. In particular, the effort to deploy five different anesthesiologists to obtain expert labels is commendable.  Also, the cross validation for the machine learning steps looks to be well designed.  Overall, the study seems rigorous.

However, there are methodological details that should be provided in the text in order to make the paper results more reproducible.  This includes being more systematic about the criteria used by the anesthesiologists to score the data; as well as providing better clarity on the design of the ensemble neural network (especially for readers who do not have a background in neural networks for classification applications).
Validity of the findings: The major contributions of the study are in the use of artificial neural networks to enable classification of depth of anesthesia from the so-called SDI, a measure of non-stationarity in the ECG time-series.  Here, non-stationarity is assessed by obtaining an empirical distribution over sliding windows of the raw data, minus manually removed artifacts.  I would encourage the authors to concisely state these contributions in the introduction of the paper, so they are clear up front to the paper.

As mentioned, I believe the results are overall interesting and sound.  My major concern is whether the correlation coefficient (between the SDI/ANN-derived classifier and the EACL) is the best way to measure performance.  From an application standpoint, what seems most important is determining when a patient is or isn't conscious, and thus I wonder why the authors didn't also do a more direct analysis on predicting these states in a more binary fashion (i.e., rather than attempting to obtain correlate against a continuous index).  As well, in understanding the power of the method it would be very important to compare performance to other DoA measures, though I accept the authors' argument that this is best reserved for future work.

There are also key questions about how the performance varies with different anesthetic regimes.  The authors provide the different types of drugs used, however do not disassociate the classifier performance in these terms.  For example, to the poorly performing cases all correspond to one particular drug regime?
Additional comments: This is a well-designed study that highlights the potential utility of ANNs to classify ECG-derived biomarkers for DoA classification.  However, since the main contribution of the paper is the use of the ANN, I think a more complete study may use other covariates in order to highlight why the SDI is the best one for this application; as well as how performance varies with different drug regimes.

The paper must be carefully copyedited in order for it to be accessible to a broad, international scientific audience.


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: HRV-DERIVED DATA SIMILARITY AND DISTRIBUTION INDEX BASED ON ENSEMBLE NEURAL NETWORK FOR MEASURING DEPTH OF ANAESTHESIA
Review round: 2
Reviewer: 1

Output:
Basic reporting: English editing is OK.
Literature references is more exhaustive.
Experimental design: Method is more clear. Authors added details about the anesthesia procedure and the statistical analysis.
Validity of the findings: Authors added comparison with the EEG based indexes as requested.
Additional comments: The reference to Jeanne et al. has not been deleted. This reference is about HRV and analgesia ; not HRV and DoA.


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: HRV-DERIVED DATA SIMILARITY AND DISTRIBUTION INDEX BASED ON ENSEMBLE NEURAL NETWORK FOR MEASURING DEPTH OF ANAESTHESIA
Review round: 2
Reviewer: 2

Output:
Basic reporting: The writing has been improved since the original submission.

There are some minor typos in the Figure captions that should be addressed (e.g., Figure 13 caption is truncated).
Experimental design: The experiment design is rigorous.
Validity of the findings: The findings appear valid and well-constrained.  I appreciate the authors' contextualization of their results.  The new figure 13 is helpful in understanding the merits of the proposed approach in contrast to BIS.  While the gains are modest according to certain metrics, it is clear that the proposed approach is accomplishing the intended goal of predicting DoA.
Additional comments: I recommend revisiting the sentence on p4, line 82 motivating neural networks.     We do not fully understand the mechanisms of ANNs and it is an overtstatement to say they operate similarly to manner to biological networks.


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: ADULT ZEBRA FINCHES REHEARSE HIGHLY VARIABLE SONG PATTERNS DURING SLEEP
Review round: 1
Reviewer: 1

Output:
Basic reporting: excellent throughout
Experimental design: query point re method is elaborated in general comments

it is an original and innovative design and research question
Validity of the findings: see below-- relates to method
Additional comments: The paper ‘Silent songs: What birds rehearse during sleep’ is both innovative and remarkable (in technique) and results and opens a window to mechanisms of control of error-coding in song learning practice in zebra finches. The paper is also beautifully written, clear in outline and outcomes and belongs to one of the best papers I have read in a while.

There are two important queries that I have for this paper-or really one major point. It concerns the syringeal muscles, or rather a missing explanation/ description in the method detailing which were used and why, ultimately affecting the results and conclusion.

1. To make the point, one can perhaps begin with a supplementary figure (Fig.S1), in principle the same as in the Larsen/Goller paper of 2002, but not as well marked in the muscle groups.
In this paper under review the caption might confuse readers.
Songbirds/oscines have at least 4 pairs of syringeal muscles—so the description saying that there are ‘at least six’ does not work well here. Why not give the exact number of pairs of syringeal muscles in the zebra finch and present this as a definite statement? In the Figure, four muscles are labelled (the largest, it says) leaving the reader wondering where the other muscles might be.
Moreover, as only three of these of these 4 identified muscles per side have been used for recording, it is not clear which ones. This is confusing for the reader.  It would make it easier if a reader were not to be referred to a Table in order to figure out which of the four muscles shown in the figure were, in fact, used for recordings.

My suggestion
1)	Provide and identify either all syringeal and tracheobronchial muscles of the zebra finch in this image if there are indeed more (as the caption suggests)—or the caption explains that the image showing only the largest has in fact x number more..
2)	Drop the ‘at least’ in the caption and stay with the number of muscles in the finch so the reader gets to know how many muscles zebra finches actually have.  Since muscle numbers vary between song birds and between oscines and sub-oscines (sub-oscines have three pairs) it is important to be told exactly how many there are in the study species.
3)	It would be helpful if, in the figure itself, information was provided and identified of those muscles of which recordings were made (especially since the number in the figure is not the same as in the text)

Under methods, lines 95-97, it is stated in each bird, recordings were obtained from either 2 different muscles on the same side, or from left and right muscles (Fig. S1; Table S1). Again, this is vague- so did it vary between finches whether recordings were made using q, 2 or 3 muscles and when using both sides, were the same muscles used on either side used or different ones?

It is best to spell out these methodological details.

If I remember the important Larsen/Goller paper of 2002 correctly, the various syringeal muscles have different functions. According to an older paper by Goller and Suthers 1996, the largest syringeal muscle, m. syringealis ventralis has no gating function but in its EMG activity was positively correlated with frequency modulation and with fundamental frequency so it would make sense to me if recordings were taken from this muscle but what about the other muscles?
Can the authors please provide a rationale for the muscles chosen and what the authors hypothesised specific muscles would be able to show, (what can adductors or abductors show?).
It would also be very useful to know whether the recordings were taken equally from left and right side (laterality is a consideration). It has been shown in a number of songbirds that song is not just produced bilaterally but certain frequencies (often at the higher end) may be produced exclusively by the left side (several publications especially by Suthers, but also by Suthers,Wild and Kaplan)..

I am concerned that, if the hypothesis of why certain muscles were chosen and what they are assumed to show is not presented and discussed convincingly, readers may not believe the results. The errors may be a result of the manner in which the recordings were sampled. Equally the authors may convincingly show why any syringeal or even tracheobronchial muscle will do for recording but, either way, any such explanation and detail, in my view, would strengthen the paper considerably.  I have no doubt that this is easy to fix, at least for these authors!

2. a smaller point but important
Lines 63,64—not all songbirds produce song that is highly stereotyped—but this sentence reads as if stereotyped song is universally applicable to all songbirds and, of course, that would be incorrect. The sentence simply needs and identifyer e.g., such “in zebra finches” or “in many songbirds, including zebra finches” --it’s stated clearly in lines 158-159 where it needs no addition or change.

I have no further comments to make other than to express the hope that this paper can address the method query satisfactorily. In my view, it is overall a very strong and innovative paper that should be published as soon as possible. It will be of wide interest to those working in neuroethology, psychology and in avian cognition.


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: ADULT ZEBRA FINCHES REHEARSE HIGHLY VARIABLE SONG PATTERNS DURING SLEEP
Review round: 1
Reviewer: 2

Output:
Basic reporting: In this paper Goller and colleagues exploit the muscle activity (EMGs) that coincides with neuronal activity of song control regions of songbirds during sleep in order to evaluate the potential meaning of this neuronal activity. The latter is thought being important for memory consolidation, and the activity of song control areas is one of the prime examples of rehearsals during sleep.
The authors computed EMGs that represent the various motor units (so called syllables) in the song of adult zebra finches. Then, they compared the envelop of the EMGs with the activities that they recorded during the night in the same muscles and animals. With this procedure they showed that most muscle activity during sleep does not correlate with the pattern found in singing birds. In particular, rarely the animals produced EMGs during the night that would represent the entire sequence of motor units (called the motif). From this, the authors conclude that it is unlikely that the birds replay the song during the sleep and conclude that the nightly neuronal activity of song control regions is not involved in song memory consolidation.
Experimental design: The methods are sound.
Validity of the findings: The methods are sound and the results are convincing. However, the discussion is somewhat disappointing and the data representation (figures and legends are somewhat careless) needs more work.
Additional comments: In general the methods are sound and the results are ok. However, the discussion is somewhat disappointing and the data representation (figures and legends are somewhat careless) needs more work. Further, the authors should replace/delete “silent songs” in/from the title. This wording is misleading since certain birds indeed produce silent songs.

Abstract:
I suggest to delete the last sentence. Further, the data are not supporting the introductory sentence of the abstract. Thus, the authors might want to adjust the abstract.

Discussion:
Lines 281-296: This entire paragraph should be re-written. The authors make first the statement that the nightly activity does not resemble the song motif EMG and as such does not help to consolidate the song motor memory. But then they state that the nightly neuronal activity might produce noise that helps to avoid the decay of the motor memory. What is the difference between consolidation and avoiding decay? Actually, this reads as if the authors have no clue about the meaning of the nightly activity. So, either they should really explain what it might mean (not just using unspecified wording) or make the statement that the function of the nightly activity is unclear. Here, the authors should critically refer to previous evidence that nightly replays are indeed replays and important for consolidation.

Figure legends:
Line 475:  Include SLA.
Line 485: The authors should state that this example is a rare observation.
Line 489: explain what are the counts based on, time? Replays?
Line 503: What do you mean with timing of syllable like patterns during SLA (red)? I guess inter-syllable intervals. Please rephrase the sentence.
Lines 510-514: Numbering A-D seems wrong.  Please use the same number of motifs/SLA in both cases and state that the same colors were used so that the overall color difference would be meaningful. Further, state how many replayed “syllables” were indeed significantly similar to the song syllable of these 5 animals.

Figures:
Fig. 1B: Include correlation values of the various syllables
Fig. 3C: Include vSL, vSR.
Fig. 3D: Break down “2 or more syllables” and “2 or more syllables +unknown” in order to support your point that entire motifs were rarely produced.
Fig. 3E: Occurrences: Counts, percentage? Include 80, 40 in the upper panel.
Fig.4B-C: Please adjust the number of included data sets (See above).
Fig. 4C: include vSL, VSR.
Fig. 4D: Include syllables 1-5 and animals 1-5 into the figure.

Minor points:
Line 164: Move explanation of SLA to line 148.
Lines 174/175: Include statistic for this statement.
Line 183: Move this sentence to the discussion or delete.
Line 191: What do you mean with “visually categorized 457 SLA events into one to seven categories of activity?” Aren’t you using some statistical method for this?
Line 204: Is “3 ms longer” a statistically relevant difference. If so, include STAT or delete.
Lines 252-256: This statement seems out of place at this position?
Line 258: do you mean: during singing?, please specify.
Line 266-268: Please include the evidence for night-time replay in other systems?


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: 5-FLUOROURACIL AND IRINOTECAN (SN-38) HAVE LIMITED IMPACT ON COLON MICROBIAL FUNCTIONALITY AND COMPOSITION  IN VITRO
Review round: 1
Reviewer: 1

Output:
Basic reporting: Although the use of language is generally quite good, I would recommend asking a native English speaker to review the manuscript as some sentences are a little difficult to fully comprehend. Examples of this can be found on lines 114, and 150-152. Also, if the donors described on line 155 were the same used for the 5-FU experiment, it would be clearer if the text read ‘five of the six healthy human donors.’ and delete the remainder of the sentence.

Care should be taken with the use of abbreviations: abbreviated names are shown on lines 28-29 without previously being written, the abbreviation for irinotecan is written on line 35 but not always used later (e.g. lines 39, 41, 42), on line 58 it should read 'nuclear factor-kappa B (NF-κB)'.

Lastly, could the authors clarify whether changes described on lines 73-74 are proportional or actual numbers, consider changing line 363 to ‘after the simulated chemotherapy treatment’, and I couldn't find any reference to Figure S7 in the text.

I would like to commend the authors on Figure 1, which gives a very clear view on the experimental set-up.
Experimental design: In order to aid experimental replication, could the authors define 'mucus solution' (line 92), add suppliers/catalogue numbers to media components (lines 121-122), detail how the SCFAs were validated e.g. use of standards, or reference library (line 161), quantify the 'maximum speed' (lines 176, 180), and briefly describe or reference the phenol-chloroform method (line 177).

The research presented here is the logical next step to build on the previous research in the literature, and I strongly agree with the authors potential follow-up work described on lines 380-386.
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: 5-FLUOROURACIL AND IRINOTECAN (SN-38) HAVE LIMITED IMPACT ON COLON MICROBIAL FUNCTIONALITY AND COMPOSITION  IN VITRO
Review round: 1
Reviewer: 2

Output:
Basic reporting: The manuscript is written in an easy-to-follow English, with only a few typos and unclear statements (see section 4, general comments for specific things to change/consider). Relevant literature cited. Tables and figures generally of acceptable quality, though some improvement is needed (see section 3 for specific comments).
Experimental design: The manuscript reports primary research within the scope of the journal. Materials and methods are generally sufficiently described, though with some shortcomings (see below).

Specific comments to Materials and Methods section:
Line 92. The mucus solution is not defined (composition?)
Line 118 and elsewhere: Samples seem to be stored at -20C before further analysis. Storage beyond a few days at -20C is known to influence GM profiles as determined by high throughput amplicon sequencing. How long was the samples stored at -20C? Were some samples stored longer than others at -20C and do the authors have any data to support, that storage at this temperature did not influence the results?
Line 125-128: This information does not belong in Materials and Methods section (but rather in the results section)
Line 164-165: Lactic acid not detected? Sometimes present in rather high amounts. Please comment.
Line 176: FastPrep running at 1600 rpm? Does not make sense. Perhaps "oscilations pr. minute" is what is meant?
Line 176 and elsewhere: What is maximum speed?
Line 177: Provide more details or reference for phenol-chloroform extraction.
Line 189-206: Generally adequate details are given, but it is not clear how e.g. how the amount of 20 ng "amplicon" was determined (line 197).
Line 257-258 and 300-304 not described in Math&Meth. Leave information out?
Validity of the findings: Data generally robust, though there is some room for improvement (see below). One big concern is that it does not seem to be investigated how stable 5-FU and SN-38 is in the fermentors. One could speculate, that the main finding of the study (no direct effect of 5-FU and SN-38 on GM) could simply be a consequence of 5-FU and SN-38  being rapidly broken down. Please provide evidence (data or references) that this is not the case.

Other, smaller comments:
In the text examples of specific taxa differing significantly with treatment within the same donor are stated, but p-values are not stated in e.g. fig. 3 and 6. Further, was FDR or Bonferroni-correction carried out to avoid false positives?
Figure legends are not always as informative as they could be. P-values (and where relevant R-values) are rarely stated. It is not stated how GM-characterisation was carried out in the relevant figure legends - include information such as "as determined by 16S rRNA gene amplicon sequencing" etc.
Figure 4 is very messy and difficult to follow. Try to make it easier to understand. Perhaps by further dividing the figures into separate figures for mucus and lumen? Also state p and R-values.
Table 1: How was the confounding factors determined? By Permanova? Consider doing e.g. a canonical correspondance analysis (rCCA) also/instead.
Suppl. Fig. 4 indicate a strong run-effect. Was this taken into account (e..g by randomising samples to different runs) when doing the analysis (DGGE)? Was PCR1 for amplicon-sequencing done in one run or is there also a potential run-effect/systematic effect influencing data interpretation here?
Additional comments: Interesting study addressing a very relevant research question.

Specific comments, not already covered above:
Line 19: Add "dysbiosis" after "gut microbiota"
Line 31: Consider "softening" the conclusion to "our findings strongly indicate that..."
Line 72: What is meant by lower abundance? Absolute abundance? Relative abundance of certain taxa?
Line 251-255: Are these findings significant? Could there be an run-effect as discussed above?
All over the manuscript: "Illumina sequencing" is a very vague and unspecific term. Be more specific and call it e.g. "high-throughput 16S rRNA gene amplicon sequencing"
Several places: Phyla-names are not in italics


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: 5-FLUOROURACIL AND IRINOTECAN (SN-38) HAVE LIMITED IMPACT ON COLON MICROBIAL FUNCTIONALITY AND COMPOSITION  IN VITRO
Review round: 2
Reviewer: 1

Output:
Basic reporting: I am happy with the changes made to the manuscript, particularly regarding the improvement of the language, and enhanced clarity regarding methods.
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: 5-FLUOROURACIL AND IRINOTECAN (SN-38) HAVE LIMITED IMPACT ON COLON MICROBIAL FUNCTIONALITY AND COMPOSITION  IN VITRO
Review round: 2
Reviewer: 2

Output:
Basic reporting: All OK in the revised version of the manuscript.

One small remark: In line 76 "microbiota" should be exchanged with e.g. "taxa".
Experimental design: All OK in the revised version of the manuscript.
Validity of the findings: All OK in the revised version of the manuscript.
Additional comments: The revised version is much improved and acceptable for publication 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: LOWLAND EXTIRPATION OF ANURAN POPULATIONS ON A TROPICAL MOUNTAIN
Review round: 1
Reviewer: 1

Output:
Basic reporting: I found this manuscript to be well-written, with the discussion flowing particularly well. The introduction, however, is a bit too brief and needs another paragraph that focuses on anurans in the tropics and what we know about their spatial distributions and how those have changed from historic records (and highlight potential reasons why). As is currently written, the transition from second to third paragraph is too abrupt.
Experimental design: The methods could use a bit more detail, as noted in my line-by-line comments. In regards to the analysis, it is unclear why the authors modeled detection probability as a function of the linear and quadratic effects of elevation. Unless sufficient justification can be provided, I think this needs to be eliminated and the analysis re-ran for each species. Displaying AIC tables with AIC values, model weights, and log likelihoods would also be helpful to the reader.
Validity of the findings: The scope of the authors current sampling is far from all encompassing. Making specific conclusions on species extirpation and range shifts from spatially and temporally limited data is slightly concerning, and I would suggest that the authors identify this issue.
Additional comments: Introduction:
The shift between the second and third paragraphs is very abrupt. There is no lead-in about anuran issues in the tropics. Another paragraph should be added before the last paragraph that focuses on what we know about how anuran distributions in the tropics and how they have been changing and, briefly, some hypothesized reasons for this change.
Methods:
L189-192: How much of an attempt was there to match up current sites with historic sites? How were current sites selected? Looking at Figure 1, there are 4 or so northern historical sites that don’t have current counterparts, which is slightly concerning. In addition, why were these months selected to do surveys?
L246: Did you look at model fit and examine assumptions for these models? This needs to be addressed in the methods and evidence of model fit should be provided.
L256: Should be “closed” rather than “close”.
L258: What is this average and SD referring to? Please make clearer.
L264: There needs to be some reasoning as to why the authors thought detection probability would vary with elevation. Is there any reason why other factors on detection probability were not considered (e.g. date, precipitation, temperature, etc.)?
L267-268: Why would there be a quadratic effect of elevation on detection probability? I cannot think of a reasonable explanation for this and the authors have given no justification.
L278-279: Same comment as above regarding model fit and assumptions.
Results:
L312: Table 1 does not show any AIC values. It is unusual not to include this somewhere in the manuscript if you are using model selection. Including AIC values and model weights would allow the reader to see what the top models were and how much better they fit than the null model. Also allows the reader to see if there are any issues with pretending variables.
L325: What about E. coqui occupancy probability? Did it increase from historic levels?
Discussion:
L343-344: I do not feel entirely confident that the range of sampling was thorough enough to state that certain species suffered extirpation or experienced range declines. At lower elevations, only about 6 sites were surveyed, and the range of the acoustic recorders was only 50 m. Have you really covered enough ground to make these definitive conclusions?
L373-383: This is good supporting information and does make me feel more confident in your results.
Figure 1: I would suggest changing the colors on this figure. I do not see the dark gray and am having trouble distinguishing between the two orange colors on the legend.
Figure 4: Based on how you did your sampling, I am not fully convinced that species are extirpated from certain elevations. I also think this figure is somewhat redundant given what you’ve already presented.
Table 1: The cumulative weights for many of these species are not very high. I would be curious to see model sets for each species with AIC values, weights and log likelihoods included. This could be included as a supplementary table but I do think it is important for the reader to get a better picture of what is going on. Typically if you are using AIC, you do include an AIC table with the above-mentioned items (and it doesn’t necessarily have to be a full model set, perhaps only the top models).


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: LOWLAND EXTIRPATION OF ANURAN POPULATIONS ON A TROPICAL MOUNTAIN
Review round: 1
Reviewer: 2

Output:
Basic reporting: ok
Experimental design: Needs more detail about the comparison between historical and current data to determine range shifts.
Validity of the findings: Historical data should encompass the same timeframe as the current data.
Additional comments: This manuscript examines changes in anuran distributions in the mountains of Puerto Rico by comparing historical data from museum specimens and previous studies with current data gathered with audio recorders.

I have a few concerns about the paper:
1)	Current data is temporally biased to the months of March-May, while historical data includes the full year. The authors could resolve this problem by limiting their historical dataset to include the same months as the current data.
2)	The method to estimate range shifts seems novel, how does it differ from other published methods?

Minor edits
Line 41: Remove comma after ‘diseases’.  Add comma after ‘species’.
Line 46: Replace ‘contrasting’ with ‘comparing’.
Line 49: Please provide more info about historical data, what are the data? Occurrence points? Transect counts?
Line 57: Instead of saying three lines of evidence, authors can say that Land use change is not responsible for these changes because LM has been a protected reserve for the past 80 yrs. However, previous studies indicate that 1) climate change has increased temperature/dry periods, and 2) Bd is present in X # of species.
Line 98: fix typo ‘chytridiomycosis’
Line 99: fix typo ‘characteristic’
Line 105: remove comma after ‘regions’
Line 110: start new sentence with “The flora”.
Before going into the goals of the paper, I feel like the authors should add a paragraph about the general patterns of amphibian declines in the Caribbean and Puerto Rico.
Line 153: Please provide list of species.
Line 157: replace ‘vocal’ with ‘active’
Line 159: add ‘of’ after ‘one’. Should read ‘one of the most studied…’
Line 161: all are endemic to Puerto Rico/ Puerto Rican bank.
Line 166: I would suggest to limit the historical data to those records collected during the same time as the current data (March-May).
Line 256: add ‘d’ to word ‘close’
Line 278-285: This seems like a novel way to estimate range shifts. How does it compare to other methods presented in literature?
Line 324: What do you mean with ‘the level’? please clarify.
Line 342-353: I think this summary paragraph is missing previous studies documenting the decline of some of these species (e.g. Burrowes et al. 2004). In addition, it seems that the authors are solely attributing range shifts to climate change-temperature, but then as the discussion progresses Bd and dry periods are also invoked as potential causes.
Line 355: Historical data is also problematic because includes all records throughout the year, vs current data only focuses March-May.


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: LOWLAND EXTIRPATION OF ANURAN POPULATIONS ON A TROPICAL MOUNTAIN
Review round: 2
Reviewer: 1

Output:
Basic reporting: No comment
Experimental design: No comment
Validity of the findings: No comment
Additional comments: L70: Should anura be a key word as well?
L124: Should be an “and” between Central Africa and Australia.
L184: “Comprehensive” rather than “comprehensible”
L 191: remove  “ in addition we”
L192: What does GBIF stand for?
L202: “transect” rather than “transects”
L316-219: Is site the same thing as a unique sampling locality? So E. coqui was found in 27/51 sites? It would be easier for the reader if you put the percentage of sites rather than raw number (i.e. 53% of sites).
L340: Should be “an increase in elevation” rather than “the increase of elevation”
L343: I would recommend consistently using either occurrence or occupancy rather than both terms.
L360-363: There could be a little more discussion on detection probability given the emphasis on modelling detection as a function of elevation, quad effect of elevation. I wonder—did elevation indeed influence detection probability for most species?
L459-460: I think you could expand slightly on how climate change acts directly on the pathogen to trigger outbreaks. What is it about climate change that causes chytrid to be more of a problem?
L445-463: You could combine these two paragraphs by moving the sentence of “While Bd may pose a threat..” to the last sentence of the entire paragraph.
L464-475: This paragraph can be eliminated. It doesn’t add much to the discussion and it seems tangential to the main issues at hand. You also never brought up the hurricane threat in the introduction, so there doesn’t seem to be much stock in this as an actual contributory threat. The two papers cited here by the same author stating that amphibians “may have been extirpated” by hurricange hugo is not very convincing.

Table 1: The cumulative weights for the current models do not seem to always match what is reported in Table S7. Particularly for E. brittoni and L. albilabris. This is concerning and all should be checked.

Table S6: It should be noted in caption that this is for historic dataset. Also, the formatting of this table should be consistent with S7 (i.e. this table doesn’t denote occupancy, it has superscript, it includes loglikelihood whereas Table 7 does the opposite.)

Table S7: Include in the caption what Psi and P stand for. The authors should include deviance values in AIC table too, as they help determine how much your model is explaining. #Par is usually referred to as K. It should also be noted in the cpation that this is for the current dataset.


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: VIRUS LIKE PARTICLES AS A PLATFORM FOR CANCER VACCINE DEVELOPMENT
Review round: 1
Reviewer: 1

Output:
Basic reporting: The summary highlights the importance and qualities of VLP's, however, I suggest improving the wording on lines 23-28 and creating a single paragraph, to avoid repetition of "VLP's" in each sentence.

In line 88, I suggest that more information about the VLPs should be included in the text, for example: classification, synthesis method, architecture (wrapped or not wrapped) or chimeras. This will give a greater impact to the review.

The revision would benefit from the inclusion of a figure where the production of VLPs is schematized, for example as shown by Antonina Naskalaska et al. (Polish Journal of Microbiology 2015, Vol. 64, No 1, 3-13), Dan Yan et al. (Appl Microbiol Biotechnol (2015) 99: 10415-10432) by mention some authors.
Experimental design: Does not apply
Validity of the findings: Does not apply
Additional comments: - Has the field been reviewed recently? If so, is there a good reason for this review (different point of view, accessible to a different audience, etc.)?
I consider the review to be of great interest since it exposes the use of VLPs as a prophylactic platform for the prevention and treatment of various types of cancer of importance in the world. The last review that I could find about VLP's as a vaccination strategy is 2014, so I consider that this work brings an update in this field so nove.

- Does the Introduction adequately introduce the subject and make it clear who the audience is/what the motivation is?
The summary highlights the importance and qualities of VLP's, however, I suggest improving the wording on lines 23-28 and creating a single paragraph, to avoid repetition of "VLP's" in each sentence.
I made this recommendation because I consider that from the first moment that the VLPs are mentioned it is clear that the information that will be read in the text will be referring to those particles so it is not necessary to repeat VLP's at the beginning of the sentences in lines 23- 28.

In line 88, I suggest that more information about the VLPs should be included in the text, for example: classification, synthesis method, architecture (wrapped or not wrapped) or chimeras. This will give a greater impact to the review.
In line 88, where the author begins to describe the VLP's, I suggested that the author provide more information on the origin of the VLPs because in the introduction little is said about these particles and their classification, although the revision is not focused on obtaining VLPs, I consider that to provide general information about the various types of VLP's will provide more information to readers

The revision would benefit from the inclusion of a figure where the production of VLPs is schematized, for example as shown by Antonina Naskalaska et al. (Polish Journal of Microbiology 2015, Vol. 64, No 1, 3-13), Dan Yan et al. (Appl Microbiol Biotechnol (2015) 99: 10415-10432) by mention some authors.
I cited these authors as an example because in their publications they showed schemes that explain very clearly how the VLPs act. I believe that these works can provide the author with a different and clearer perspective on how to outline the role of VLPs as a prophylactic or vaccine platform against cancer. But if the editor thinks the scheme is appropriate, please ignore my recommendation.

- Is the Survey Methodology consistent with a comprehensive, unbiased coverage of the subject. If not, what is missing?
The research platforms used to obtain the information allow to review several areas of research carried out with these VLPs, which is why I consider it to be an adequate methodology for this manuscript.

- Are sources adequately cited? Quoted or paraphrased as appropriate.
All references are properly quoted.

- Is the review organized logically into coherent paragraphs/subsections?
Yes


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: VIRUS LIKE PARTICLES AS A PLATFORM FOR CANCER VACCINE DEVELOPMENT
Review round: 1
Reviewer: 2

Output:
Basic reporting: The authors performed an excelente review of several paper about the potential use of VLPs as cancer therapeutic molecules. The review is well redacted and profound.

Minor changes.
Authors mention that VLPs are promising molecules to fight cancer, however, they must include a paragraph with the potential drawback of this approach. For example, several reports have showed that preexisting human immunity against several VLPs reduced the potential use of this particles. Authors should declare why only few of them has been successfully enter in clinical trials and less are have been licensed.
Experimental design: no comments
Validity of the findings: no comments
Additional comments: no comments


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

Input:
Title: VIRUS LIKE PARTICLES AS A PLATFORM FOR CANCER VACCINE DEVELOPMENT
Review round: 1
Reviewer: 3

Output:
Basic reporting: The review is well written and follows the guidelines of the journal. However, the manuscript has some deficiencies since, according to its title “VLPs as platforms...”, it should address the issue from two points of view: VLPs against cancer-causing viral infections (preventive) and VLPs developed against cancer (therapeutics). Nevertheless, most of the content is only focused on VLPs as preventive vaccines against cancer-associated viral infections such as those related to HBV, HCV, HPV and HHV-4 (EBV). It is also important to consider that these oncogenic viruses are associated to malignant processes, thus VLPs vaccines are developed to prevent viral infection that in turn has an impact on the incidence of cancer.
VLPs as therapeutic vaccines are only addressed in the section 2.3, about pancreatic, prostate, breast, skin and lung cancer. In the case of HCC, the information in that section is mixed.
On the other hand, authors do not discuss all the strategies that are being used for development of VLPs as a carrier of antigens, but only show in Figure 1, one of two main strategies for generation of VLPs (genetic fusion). It would be important to contrast at least with a second strategy; authors might consider Eriksson et al., 2011 to extend information. Another important point that the authors do not address is the viral origin of the VLPs, since origin is important from the point of view of the immune response that could be presented
Therefore, authors should consider dividing the review into two topics: preventive VLPs and therapeutic VLPs or should focus the review only on therapeutic VLPs.
Experimental design: No comment
Validity of the findings: No comment
Additional comments: Minor revision
•	In line 119 the word ("Table 2") would be better if it said ("see Table 2")
•	In line 207, the drug is Gardasil not Gadrasil.
•	The paragraph on lines 431-433 is confusing or repeated
Major revision
•	In lines 105-107, compared to that, do VLPs represent “a much lower risk of autoimmunity”? Which vaccines have autoimmunity problems or do they exhibit auto-antigens?
•	 In line 150 quotes the work of Pleckaityte et al. 2015, and it is mentioned that the VLP developed by this group has therapeutic potential, but in the work show in vitro results of viral neutralization. Can the vaccine cure hepatocellular cancer or only prevent HBV infection? Or control and eliminate chronic HBV infection in people but without hepatocellular cancer?
•	The paragraph of line 197 to 200, is a conclusion of the work of Ding et al. 2009? Or it is a contribution of the work of Su et al. 1998, if it is the latter, then that work should be better described.
•	The paragraph of lines 250-251, refers to the VLPs developed by Martin Caballero et al. 2012 and Jemon et al. 2013, these proved to be therapeutic, then, according to the paragraph would be prophylactic or therapeutic?
•	All section 3.7.2 EBV should go after section 3.2 HPV.
•	The finding of mRNA in the EBV VLP in the work of Russ et al. 2011 could be more discussed (Line 436).
•	Section 4.0 (Summary), needs more discussion about the different ways to obtain preventive and therapeutic chimeric VLPs. The works of Martin Caballero et al. 2012 and Jemon et al. 2013, can be used to extend the discussion. On the other hand, the conclusion is very general, because some VLPs have been promising, but many others have not.
In the figure it is not clear if what follows after the mouse are more viruses or are dendritic cells?


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: VIRUS LIKE PARTICLES AS A PLATFORM FOR CANCER VACCINE DEVELOPMENT
Review round: 1
Reviewer: 4

Output:
Basic reporting: This is a very interesting, original, and well-written review by Ong et al., regarding virus like particles as platforms for cancer vaccine development. The authors presented almost all existing literature in an innovative and entertaining form. However, I would like to see some more recent literature which describes extracellular vesicles and their close resemblance with VLPs. I would like to see authors critically reflect on how VLPs are different than exosomes/microvesicles and that role can these extracellular vesicles play in vaccine development platform ?

Please see articles such as
1)	Extracellular vesicles and viruses: Are they close relatives? Nolte-'t Hoen E, Cremer T, Gallo RC, Margolis LB. Proc Natl Acad Sci U S A. 2016 Aug 16;113(33):9155-61. doi: 10.1073/pnas.1605146113. Epub 2016 Jul 18.
2)	Microvesicles and Vesicles of Multivesicular Bodies Versus “Virus-Like” Particles. Albert J. Dalton. JNCI: Journal of the National Cancer Institute, Volume 54, Issue 5, 1 May 1975, Pages 1137–1148, https://doi.org/10.1093/jnci/54.5.1137
3)	Extracellular Vesicles and Their Convergence with Viral Pathways. Thomas Wurdinger,1,2 NaTosha N. Gatson,3 Leonora Balaj,1 Balveen Kaur,3 Xandra O. Breakefield,1 and D. Michiel Pegtel4. Advances in Virology. Volume 2012 (2012), Article ID 767694, http://dx.doi.org/10.1155/2012/767694
Experimental design: Original content within the scope of the journal
Validity of the findings: Conclusions are well-stated, and data has been presented in a very entertaining manner,
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: VIRUS LIKE PARTICLES AS A PLATFORM FOR CANCER VACCINE DEVELOPMENT
Review round: 2
Reviewer: 1

Output:
Basic reporting: No comment
Experimental design: No comment
Validity of the findings: No comment
Additional comments: The modifications made along with the information that was added and the figures positively increases the academic contribution of this article


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: VIRUS LIKE PARTICLES AS A PLATFORM FOR CANCER VACCINE DEVELOPMENT
Review round: 2
Reviewer: 2

Output:
Basic reporting: Without comments
Experimental design: Without comments
Validity of the findings: Without comments
Additional comments: Without 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: VIRUS LIKE PARTICLES AS A PLATFORM FOR CANCER VACCINE DEVELOPMENT
Review round: 2
Reviewer: 3

Output:
Basic reporting: In Figure 2, I think it would be best if the self-assembly was first and then the release and budding to continue with the VLPs
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: GENOME-WIDE IN SILICO IDENTIFICATION OF MEMBRANE-BOUND TRANSCRIPTION FACTORS IN PLANT SPECIES
Review round: 1
Reviewer: 1

Output:
Basic reporting: 1. I suggest having an English language editing service or an English-speaking colleague proofread your paper to ensure proper language usage and to improve logical flow.

2. There is sufficient background about the importance of MTFs in rapid response to stress. However, to aid the reader in interpreting the results and understanding their significance, more background is needed on the proteolytic processing of MTFs (and how this is related to the location and number of transmembrane domains), the alternative splicing of MTFs and the methods used to identify MTFs.

3. The Materials and Methods section is too brief. Most of the methods (including those presented in the Materials and Methods) are described in the Results and Discussion section, and these are not explained in sufficient detail.

4. The Titles/labels do not always accurately reflect what is shown in the figure or table, and in some cases more information is needed to interpret the figure.
-The x-axis in Fig 1d should be labeled “Arabidopsis gene families that contain MTFs”.
-In Fig 2b, I suggest providing the percentage of MTFs that have each topology.
-A more accurate title for Figure 3 would be “Alternative splicing of membrane-bound transcription factors”.
-In Fig. 3b, it is more accurate to say that alternatively spliced forms of AT5G10510 lack a TM domain. In addition, the colored lines/blocks should be explained.
-In Fig. 3c, The description  “ALS-dependent activation” is not appropriate because you have not shown that these TFs are activated by alternative splicing.
-Table 1: TOPCONS-single is not included.
-Table 2 title: It is not correct to refer to these transcription factors as the “MTF family” because they belong to several different TF families.
-Table 3. The title does not reflect what is in the table, which is the number of MTFs in each TF family.

5. The TM predictions from all seven programs are not provided, and it is not clear what predictions are shown in Supplemental Table S2. Are these the combined TM predictions?
Experimental design: 1. The need to identify MTFs in other species is stated (Lines 68-71), but I suggest more clearly articulating the gap in knowledge and your specific goals. For example, did you develop a new approach to identify MTFs rather than using an existing approach because there was a need for a more robust MTF identification pipeline?

2. There needs to be more discussion about the discrepancies between your results and those of Kim et al. (2010) (lines 133-134). What were the differences in your approaches that may have led to this discrepancy? You cite the fact that you identified all previously characterized MTFs as evidence that you have obtained high-confidence MTF predictions. Was this not the case for Kim et al. (2010)? If it is difficult to conclude which method is more reliable (line 134), would it be better to use both approaches when trying to identify all possible MTFs?

3. The methods need to be more fully explained.
-More detail about the TOPCONS-single algorithm (lines 117-118) and how it was implemented is needed (there is no mention of this program in the Materials and Methods). Based on Hennerdal and Elofsson (2011), only four prediction programs seem to be used by the best performing version of TOPCONS-single (http://single.topcons.net/). If this is the case, the workflow (Fig. 1) is incorrect.
-How the other prediction programs (lines 86-89) were implemented also needs to be described, including the criteria for calling a transmembrane domain.
-The reason for making TMHMM 2.0 predictions separately should be clearly stated (lines 119-120).
-Table 2 should be cited when mentioning the plant species included in the analysis (line 82).

4. The rationale for using the seven prediction programs (lines 97-101) is not fully explained. Why did you choose to use a different approach than Kim et al. (2010)?
Validity of the findings: 1. The conclusion that the location of transmembrane motifs in the N- or C- terminal regions of most MTFs supports the conservation of a proteolytic mechanism for MTF activation (lines 26-28, 74-76, 192) needs to be clarified. I interpret conserved in this context to indicate evolutionary conservation, but given that the MTFs belong to different gene families, this does not seem to be correct. It is more accurate to say that the location of TMs indicates that MTFs are processed by the same proteolytic mechanism. However, why the location of the TM domains indicates this needs to be explained.

2. The conclusion that half of the MTFs in Arabidopsis and cotton could be regulated by alternative splicing (Lines 29-31, 77-79, 207) also needs clarification. It is not clear whether your conclusion that MTFs are regulated by alternative splicing is based on the presence of an alternatively spliced transcript or on the fact that these transcripts have different protein domains. In Table S3 you list the alternatively spliced transcripts and note whether they contain a TM domain, but you don’t discuss how often alternative splicing has the potential to regulate the MTF. When you say that ALS was involved in the activation of MTF families (Lines 208-209), is this based on the fact that alternative splicing removes/adds a TM domain?

3. The conclusion that MTF activation involves at least two independent mechanisms (lines 218-221) was reached by other studies. The activation of MTFs was not directly assessed in this study, and this section should be revised to specifically summarize the conclusions of your study.
Additional comments: 1. In the abstract lines (31-33) and in the conclusion (lines 215-216) you say that you have done the first systematic analysis of MTFs in plant genomes. This wording is not correct because systematic analyses have been done in Arabidopsis and rice (Kim et  al 2010), which you appropriately cite in the Introduction (Lines 65-68).

2. The explanation for the discrepancies in TM prediction between algorithms is vague (lines 110-112). It would be helpful if the advantages and disadvantages of each method were explained.

3. I suggest not listing the plant names (Lines 146-150) because they are already listed in Table 2. Similarly, all of the gene families with MTFs (lines 162-169) don’t need to be listed in the text because they are already shown in Table 3.


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

Input:
Title: GENOME-WIDE IN SILICO IDENTIFICATION OF MEMBRANE-BOUND TRANSCRIPTION FACTORS IN PLANT SPECIES
Review round: 1
Reviewer: 2

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: This manuscript describes the identification of membrane-bound transcription factors (MTFs) using in silico computational analysis. 1089 MTFs belonging to 52 gene family were identified from 14 plant species, most of which contain one or two transmembrane region. The authors also found that approximately half of the MTFs can be regulated by alternative splicing. The manuscript provides an important resource for readers who might be interested in MTFs in plants. However, there are several major issues that need to be addressed.

1. A previous study (Kim et al 2010) has identified MTFs from Arabidopsis and rice, and the results were quite different from the current study. 50% of the previous identified MTFs were not recovered by the current study (Fig 1 e). What could be the reason for the difference, and which method is more reliable? How many of the MTFs with known functions were identified by the current study but not by the previous study? Further analysis and discussion on this topic should be provided.

2. It is recommended to include experimental verification for some identified MTFs, for example, the 21 MTFs identified only in the current study. Arabidopsis TF resources are widely available and the experiments should be easy to carry out.

3. Do the authors have an estimation of the false discovery rate among the 1089 identified MTFs?

4. A phylogenetic analysis should be conducted to see how many of the MTFs are conserved between different plant species? Do the NAC MTFs from different species belong to the same sub-group among all NACs? How about bZIP, SBP, bHLH, and other MTFs?

5. The analysis of MTFs regulated by alternative splicing  (Fig 3) depends on the accuracy of gene model annotation. While it could be assumed that the Arabidopsis gene models are quite reliable, the author should discuss how reliable are those for cotton and other plant species.

6. Table 2, Picea abies is not a Bryophyta.

7. The English language of the manuscript should be improved, such as those in lines 51 to 53.


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

Input:
Title: GENOME-WIDE IN SILICO IDENTIFICATION OF MEMBRANE-BOUND TRANSCRIPTION FACTORS IN PLANT SPECIES
Review round: 2
Reviewer: 1

Output:
Basic reporting: 1. The English has been improved, but there are still grammatical errors and awkward phrasing. For example:
Pg 2 Lines 49-51. By “attached” I think you mean “located adjacent to”
Pg 2 Line 59 “been” should be deleted.
Pg 3. Line 75 “that” should be changed to “those”
Pg. 3 Line 80 “database” should be deleted.
Pg 4 Line 90 “conserve” should be “conserved”
Pg. 6 Line 153 “researches” should be “studies”
Pg. 6 Line 160 should be “workflow for MTF identification established in this study can identify MTFs”
Pg. 7 Line 176. “families of MTFs” here should be “families containing MTFs” because not all members of these families are MTFs. Similarly, in lines 186-187, it is better to say “the six families with the highest number of MTFs”
On pg. 9 line 233, “nuclear transcription factors” is not correct. Do you mean “transcription factor domain” or “nuclear localization signal” or both?

2. In the Materials and Methods the in silico analysis of alternative splicing is still not included. On pg. 4 Line 93- the table containing the list of species should be cited.
On pg. 4 Lines 106-107- what is the “the strict standard”?

3. For Supplemental Table S2 a legend that describes the contents of this table is needed.
Experimental design: 1. The parameters/criteria used for membrane protein prediction are not described. If the default parameters were used, this should be stated.

2. My original confusion about how the analysis was done stems from the fact that 4 out of the 7 prediction programs in Table 1 are used by TOPCONS-single. From Hennerdal et al. 2011: “The best-performing version of TOPCONS-single, using four individual methods (Table 1), is available as an easy-to-use web-based prediction server at http://single.topcons.net/. It uses the globular protein filter of SCAMPI to weed out non-membrane proteins and then proceeds to run the rest of the predictors–HMMTOP, MEMSAT-1.0 and S-TMHMM—on the remaining set.” Thus the use of TOPCONS-single seems redundant. Why were the predictions from the 6 programs not simply combined or TOPCONS-single used directly? Note that there is a more recent reference for TOPCONS-single (Tsirigos et al. Nucleic Acids Research, 2015 6:21383).

3. On page 5 Lines 138-141, you claim that TMHMM 2.0 is the most accurate software available. However, the reference you cite (Flugge and Kunze, 2003) does not make this claim.  Is the wrong reference cited?

4. I still think the rationale for using these programs needs more explanation. The points you make in your response (that you wanted a method that uses resources that are freely available to the scientific community and are applicable to more plant species) are convincing and should be mentioned in the paper. Also, you don’t explain why you used multiple algorithms, which I think was to identify as many membrane proteins as possible. Finally, you argue that these prediction programs have been frequently used and have led to good results. But the references you cite (Che et al. 2010 and Li et al. 2011) do not mention these prediction programs. References directly supporting this statement should be cited.
Validity of the findings: 1. The discrepancies between your results and those of Kim et al. are still not discussed in detail. You don’t explain how the approach by Kim et al differed from yours and whether they also identified all previously known MTFs. You also don’t directly address why you found less than half of the MTFs found by Kim et al. in Arabidopsis. Do you think there are more false positive predictions in their dataset? That your datasets are complementary? What implications does this have for the discovery of MTFs in other species?
Your statement that the databases from other species are “highly reliable” (pg. 6 line 169) is not justified because you have not demonstrated that they are reliable. This does not mean that your analysis is not valid, but you should be clear about the limitations.

2. Pg 8 lines 210 and 212- The explanation for why the location of transmembrane domains indicates conservation of the proteolytic mechanism needs further clarification. For example, I think your point is that TM domains need to be located at the N- or C-terminal ends of the protein to allow a functional TF to be released by proteolytic cleavage, which occurs next to the TM domain. Are there references to back up this observation (eg. all characterized MTFs have this type of protein structure)? Does this mean that the putative MTFs with TMs located in other regions of the protein are not likely to be functional?
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: GENOME-WIDE IN SILICO IDENTIFICATION OF MEMBRANE-BOUND TRANSCRIPTION FACTORS IN PLANT SPECIES
Review round: 2
Reviewer: 2

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: The authors have addressed previous concerns to certain degree, though some of the points might not be resolved due to technical difficulty, such as the issue of FDR rate. As it currently stands, the manuscript is acceptable now 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: FITNESS IMPLICATIONS OF SEX-SPECIFIC CATCH-UP GROWTH IN NEPHILA SENEGALENSIS, A SPIDER WITH EXTREME REVERSED SSD
Review round: 1
Reviewer: 1

Output:
Basic reporting: This is a well-written paper and the results are interesting.
Experimental design: This is basically a well-designed laboratory experiment. Some methods need to be described with more detail (see comments for the author)..
Validity of the findings: I am concerned whether the results obtained under the artificial laboratory conditions can be used to infer selection of life-history traits in nature (see comments for the author).
Additional comments: This is a well-written paper on a basically well-designed laboratory experiment and the results are interesting. However, I am concerned whether the results obtained under the artificial laboratory conditions can be used to infer selection of life-history traits in nature.

The cost of delayed maturation on fitness, in which female spiders that mature slower in the field are more likely to be eaten by predators before they reproduce, cannot be assessed in a laboratory experiment. Although this cost is briefly mentioned in the Discussion, I am not convinced that it is unimportant in nature. Although large spiders may be too big to be eaten by invertebrate predators, they may be more attractive to larger vertebrate predators. Other factors like physical disturbance by storms may also add to the cost of delayed maturation. A more comprehensive view is that delayed reproduction at large size may be advantageous in a low-predation, low-disturbance environment but non-adaptive in a high-predation, high-disturbance environment.

The nutritional value of the flies fed to the spiders needs to be addressed. Drosophila may not contain enough nutrients for spiders to grow optimally and reach maturity. Although the methods are not clear, it seems that spiders in the low-food conditions were fed only or mostly Drosophila, whereas those in the high-food conditions may have received more of a mixture of Drosophila and Calliophora. Could the increased nutritional value of a mixed diet influenced the results? In particular, if males in the Low-High treatment were fed only or mostly Drosophila (because they were too small to capture Calliophora), this might have impeded their catch-up growth potential, whereas females in the Low-High treatment probably received a mixed diet during the second half of the experiment.

The first section of the methods states that the spiders were housed in small plastic cups. This may have been alright for small juveniles, but the cups would have been too small for larger individuals. Hopefully, at some point the larger spiders were transferred to larger containers, but this is not stated.


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

Input:
Title: FITNESS IMPLICATIONS OF SEX-SPECIFIC CATCH-UP GROWTH IN NEPHILA SENEGALENSIS, A SPIDER WITH EXTREME REVERSED SSD
Review round: 1
Reviewer: 2

Output:
Basic reporting: The ms is well written and all relevant raw data are available. The discussion is too spider-centered. There is a large body of literature on sex-specific growth in insects which is not sufficiently cited (e.g. papers by Stillwell & Davidowitz).
Detailed comments:
L246: These headings are awkward. With regards to the analysis, why did you not model the interactive effects of family and treatment here? Also, why is the sex effect not explicitly tested? See comment below.
L304-305: Again, why is not sex included as a predictor, especially in the interaction?
L313-314: But High-Low and Low-Low might still differ with regards to fecundity.
L317-318: Why are size or mass not included as covariates?
L377: Please inform us of the phenology of N. senegalensis.
L417: ...PROBABLY result from an…
L418: …is VERY LIKELY facilitated…
L458: Detecting variation in growth responses due to additive genetic variation or maternal effects in a population does not equate to a "range of developmental strategies".
L514: You do not formally test for sex-specific growth responses or for any interactive effects of sex.
Experimental design: The study can address the research question. The rearing effort is laudable. It is very painstaking work to rear that many orb-weavers.
There are design limitations; i.e. growth was not measured for each instar, which was probably prohibitive logistically. Hence our understanding of sex-specific growth remains pretty basic.
Validity of the findings: In this study, the sex-specific effect of food limitation on growth and size at maturity was measured. That is, the results provide information on sex-specific growth mechanisms, with implications for possible sex-specific selection processes. Hence, any discussion of patterns of natural and sexual selection must be speculative and should be treated as such. However, the discussion reads as if the study provides direct insight into selection processes, which it cannot. Thus, I recommend toning down the discussion throughout.
The statistical approach of multiple pairwise comparisons falls short of providing a comprehensive analysis. It is quite peculiar that the study aims to identify sex-specific growth responses to food limitation during early and late stages of development, but no model is built with sex as a predictor or part of an interaction with treatment. SSD is of course very pronounced in adults in this species and we don’t need stats to tell us that, but sex-specific growth responses should be tested explicitly.
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: FITNESS IMPLICATIONS OF SEX-SPECIFIC CATCH-UP GROWTH IN NEPHILA SENEGALENSIS, A SPIDER WITH EXTREME REVERSED SSD
Review round: 2
Reviewer: 1

Output:
Basic reporting: All my - and the other reviewer's - comments on the first version of the ms have been adequately and nicely addressed. I have have no further comments.
Experimental design: All my - and the other reviewer's - comments on the first version of the ms have been adequately and nicely addressed. I have have no further comments.
Validity of the findings: All my - and the other reviewer's - comments on the first version of the ms have been adequately and nicely addressed. I have have no further comments.
Additional comments: The paper is now in great shape and I recommend publication.

Best wishes,

Matthias Foellmer


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: MITOCHONDRIAL GENOMES OF THREE TETRIGOIDEA SPECIES AND PHYLOGENY OF TETRIGOIDEA
Review round: 1
Reviewer: 1

Output:
Basic reporting: Clear, unambiguous, professional English language used throughout but I noticed a couple of things worth revising:

Lines 128-129 “The positive relativity were found between A+T content and AT-skew, and also between G+C content and GC-skew.”

Replace “mt genomes” with mitogenomes (e.g. in line 132).

The graphic resolution of the main figures should be improved in the final submission for publication.
Experimental design: No comment.
Validity of the findings: No comment.
Additional comments: This is a well-structured and well written work reporting the near-complete mitogenomes of three Tetrigoidea species. The paper is adequate in its scope, and fits well in the standards of 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: MITOCHONDRIAL GENOMES OF THREE TETRIGOIDEA SPECIES AND PHYLOGENY OF TETRIGOIDEA
Review round: 1
Reviewer: 2

Output:
Basic reporting: Although the article is written in clear English, the authors should check again carefully for typos. Striking examples:
pdf line 37: "Batraehididae, Cladonltidae, Diseotettigidae, ..."
Fig. 3: "Tridactuloidea" instead of "Tridactyloidea"

The names of the species studied should be given in full at first mentioning with the genus not abbreviated.
Experimental design: pdf lines 30-38: The authors compare two systematic divisions of Tetrigidae (OSF versus Zheng) that - apart from the level subfamily versus family - is nearly identic except that two taxa are missing in the latter system. Is this due to different arrangement of species or only due to the two (sub-)families missing in China?

pdf lines 56-61: The exact purpose of the study, apart from adding new data, is not clear from the introduction. The authors state that they "further study of the phylogenetic relationships among Tetrigoidea". How will they reach this aim? Are the species selected representatives of different (sub-)families or do they represent (sub-)families, of which genome data are not yet available from genbank?

The aim of the study should given more precisely and in accordance with what is presented in results and discussion.
Validity of the findings: no comment
Additional comments: No additional comments