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: DEVELOPING A MACHINE LEARNING MODEL TO IDENTIFY PROTEIN–PROTEIN INTERACTION HOTSPOTS TO FACILITATE DRUG DISCOVERY
Review round: 1
Reviewer: 2

Output:
Basic reporting: See 'general comments'
Experimental design: See 'general comments'
Validity of the findings: See 'general comments'
Additional comments: The authors presented a method for predicting protein-protein interface hotspots using supervised machine learning algorithms trained using an extensive number of sequence and structure derived features. The viability of the developed method has been demonstrated using a virtual drug screening analysis of the predicted hotspots on the EphB2-ephrinB2 complex.

Dataset: The final dataset is slightly different from the original SpotOn dataset. Therefore, the authors need to report the number of hotspot and non-hotspot residues in their dataset.

Performance evaluation: Since the dataset is imbalanced and have less number of samples compared with the number of extracted features, the estimated performance metrics might be sensitive to the portioning of the data into train and test sets. To address this issue and have a fair comparison with SpotOn, the authors need to evaluate their models using 10 runs of 10-fold CV experiments.

Table 3: The authors claim that their model outperforms SpotOn (based on MCC, F1, and sensitivity) is not accurate. First, SpotOn has AUC score of 0.83 while the authors model has an AUC score on only 0.75. Second, while the authors model has a better sensitivity, its specificity is 0.08 lower than SpotOn. Third, MCC and F1 tend to be higher for the model with the highest specificity. Fourth, the authors model has been evaluated using a single run of 10-fold cv while SpotOn model has been evaluated using 10 runs. Ideally, the two models need to be compared by inspecting the ROC curves. Since it is challenging to get the ROC curve for SpotOn, the authors might modify the threshold of their model to achieve an 0.88 specificity and show that at this specificity their model has a better sensitivity and hence better F1 and MCC metrics.

Feature importance: Lines 248-251 showed that Relative Complex ASA and Complex ASA features are among the top 15 features. These two features will assign the same value to hotspot and non-hotspot residue in the complex. If this is the case, how can these features have any discriminative signal? Moreover, to demonstrate the improvement in the predictive performance by the newly added features, a sensitivity analysis to quantify the change in the model performance before and after adding these novel features is 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: DEVELOPING A MACHINE LEARNING MODEL TO IDENTIFY PROTEIN–PROTEIN INTERACTION HOTSPOTS TO FACILITATE DRUG DISCOVERY
Review round: 2
Reviewer: 1

Output:
Basic reporting: ok
Experimental design: ok
Validity of the findings: ok
Additional comments: No more comment. Thanks for revision


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: TRIALS IN DEVELOPING A NANOSCALE MATERIAL FOR EXTRAVASCULAR CONTRAST-ENHANCED ULTRASOUND TARGETING HEPATOCELLULAR CARCINOMA
Review round: 1
Reviewer: 1

Output:
Basic reporting: 1.	This article is to develop the image enhanced materials for the ultrasound examination especially specific tumor targeting such as hepatocellular carcinoma.
2.	The article is written in English using clear and unambiguous text and complies with the professional standards of courtesy and expression.
3.	This Article is reviewed at the standpoint of an oncology clinician and the experimental pathology of malignant tumors.
Experimental design: 1.	This experimental study was executed in the adequate animals evaluation, data processing including statistical analyses scientifically and ethically.
2.	Materials and Methods, Line 70. In this study, there is only one image intensifier material, and there is no description of the selection from multiple candidates during the development process.
3.	Materials and Methods, Line 71. There is no description about the reason for selecting the gas(SF6) to be enclosed in the microbubbles.
4.	Materials and Methods, Line 72. There is no detailed description of GPC3 antibody and peptides that bind to the microbubbles.
5.	Materials and Method,s Line 130. There is no description of the experiment on the particle size of liposome microbubbles filled with SF6, considering only the change in the solution concentration.
Validity of the findings: The following issues need to be fixed.
1.	Figure 4.  The concentration in the right column is unknown.  After 72 hours, there is a slight decrease in the Cell Index. In the safety evaluation, it is necessary to extend the observation period.
2.	Figure 5-8. Please insert a size bar on each image.
3.	Figure 6. The explanation of “The fluorescence on the cellular membrane appears stronger than that in cellular plasma. ”.  This findings cannot be recognized in the image.
4.	Figures 7.  Fluorescence images cannot be recognized in photographs. It is better to replace images with Supplement Figure images.
5.	Figure 8,  There is no explanation for 3rd row.  And the initial elapsed time is missing in this figure.
6.	Figure 10.  In the safety evaluation of systemic administration (intravenous administration), evaluations of the pulmonary embolus are necessary.  A histopathological image of the organs including the lungs should be presented.
7.	Supplement Figure.  Image of xenograft tumor of the mouse obtained by duplex ultrasound with a scanner of frequency of 12 MHz
8.	HCC tumors are rich in blood flow. However, the Doppler echo image of the xenograft tumor in the Supplement Figure does not show a blood flow signal. There are problems in selecting the xenograft tumor.
Additional comments: 1.	It has little advantage over existing intravenous image intensifier materials for ultrasonography.
2.	Since this is negative final results, it is necessary to describe details above-mentioned problems for the future developments.
3.	As a result of the peer review, a major revision is required.


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: TRIALS IN DEVELOPING A NANOSCALE MATERIAL FOR EXTRAVASCULAR CONTRAST-ENHANCED ULTRASOUND TARGETING HEPATOCELLULAR CARCINOMA
Review round: 1
Reviewer: 2

Output:
Basic reporting: N/A
Experimental design: N/A
Validity of the findings: N/A
Additional comments: Medical imaging is an important approach for the diagnosis of HCC, and there is still scope for improvement to achieve accurate diagnosis. Therefore, what we need are novel and effective diagnostic tools with "better" abilities.
In this manuscript, Wu and Lin aimed to formulate a material that can combine with GPC3 of HCC for targeted enhanced ultrasound. A material of SF6 filled liposome microbubbles and conjugated with LSPMbs was prepared and assessed. Liposome microbubbles were made of DPPC, DPPG, DSPE-PEG2000, and SF6, followed filling SF6, and conjugating peptide. LSPMbs showed high affinity to Huh-7 cells in vitro, presented good enhanced ultrasound effects, did not show cytotoxicity. However, unfortunately they did not exhibit targeted fluorescence and enhanced ultrasound effects in xenograft tumors.
As the authors noted, the material described in this manuscript appears not to be utilized in clinical setting. Furthermore, the authors did not clearly show the reason of an unsuccessful attempt. In this point, the authors discussed it based on previous literature that suggest small amount of LSPMBs of 380 +/- 176.5 nm cannnot access to HCC cells.
This reviewer fully respects author’s efforts and the results of this experiment, though still wonders the fundamental meaning of this paper.


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: TRIALS IN DEVELOPING A NANOSCALE MATERIAL FOR EXTRAVASCULAR CONTRAST-ENHANCED ULTRASOUND TARGETING HEPATOCELLULAR CARCINOMA
Review round: 2
Reviewer: 1

Output:
Basic reporting: 1.	This article is to develop an image enhanced material for the ultrasound examination targeting specific tumor such as HCC.
2.	This Article is re-reviewed at the standpoint of an oncology clinician and the experimental pathology of malignant tumors.
Experimental design: 1.	This experimental study was executed in an adequate animal evaluation, data processing including statistical analyses scientifically and ethically.
2.	The response for each reviewers’ comments seems to be rewritten adequately and precisely.
Validity of the findings: 1.	The author carefully revised Figures according to reviewer’s comments.
Additional comments: 1.	Reviewers have already pointed out the poor experimental results.  And article authors are also aware of this issue.
2.	Although final results is negative, this article details the process of development materials for extravascular ultrasound targeting.
3.	The title "negative outcome" might lose the value of the paper, so it is better to rewrite the title in a different expression.
4.	A result of the peer review, acceptable except the title expression.


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: CHARACTERIZATION OF A PROFILIN-LIKE PROTEIN FROM FASCIOLA HEPATICA
Review round: 1
Reviewer: 1

Output:
Basic reporting: No Comment
Experimental design: No Comment
Validity of the findings: No Comment
Additional comments: This is a sound and interesting study.
I have some suggestions for the authors
First, they should consider making molecular models of the protein.  This is relatively straight forward using online servers such as Phyre2. This may help explain some of the results and differences between the fluke protein and ones from other species.

Second, they should consider measuring ATPase activity and comparing this to other species.


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: CHARACTERIZATION OF A PROFILIN-LIKE PROTEIN FROM FASCIOLA HEPATICA
Review round: 1
Reviewer: 2

Output:
Basic reporting: Wilkie, Cameron and Beddoe present the successful production of F. hepatica profilin in bacteria, purified using an N-terminal His-tag followed by size-exclusion chromatography. This protein is present in the tegument and thus thought to be important for parasite-host interaction. An protease site was present between the His-tag and profilin sequence, but not utilised to remove the tag.
The authors go on to show that the produced profilin binds to actin and keeps actin monomers soluble, preventing them from polymerising. Other profilins also do this. They also show the profilin binds to polyproline resin, does not bind the PIP variants they tried and is not recognised by Fasciola hepatica susceptible sheep or cattle immune serum.
In general, the manuscript is well-written and intelligible. However, the sentence on lines 245-247 reads a bit clunky in my opinion.
The manuscript does give the impression that all experiments were only performed once and not optimised (a bit "quick-and-dirty", although this sounds a bit too negative).
- line 285. the parasite-specific surface motif consisting of an acidic loop followed by a long beta-hairpin insert could be indicated in Figure 1. In that respect, another useful addition to Figure one would be a structural model, with the polyproline and PIP binding residues hightlighted. It should be possible to generate a useful structural model by homology modelling, even using a webserver.
- please provide full gels and blots for all figures as supplementary information, i.e. raw data.
Experimental design: - sonicated at 25-30% amplitude doesn't say very much without detailing the exact equipment and probe used.
- the polyproline resin binding experiment would be more informative with a range of profilin / resin concentrations, including proportions where the resin is limiting and where the profilin is limiting. In that way, it could be distinguished if a fraction of the profilin sample is unable to bind resin or if there was just not enough resin to bind all the protein.
- how likely is it that in the PIP mini-array the PIP(s) that profilin might bind are just not there? In this respect a control with a profilin known to bind PIPs would have been welcome. Or at least a summary on which profilins bind exactly which PIPs (and if these PIPs are in the array). In any case, the molecular identities of the 15 PIPs used in the micro-array need to be stated.
- Figure 6 is rather "ugly"
Validity of the findings: - replication of some of the experiments would have been welcome; or a statement as to how many times experiments were performed with similar results.
- after size-exclusion chromatography, the gel appears to show a double band. Was this analysed by mass-spec of N-terminal sequence analysis? Perhaps this was the result of spontaneous cleavage of the tag?
- please provide full gels and blots for all figures as supplementary information, i.e. raw data.
- conclusions are reasonable and unwarranted speculation is absent.
Additional comments: I could not locate the database ID quoted (accession number GEVX010111281, which database?), but the protein sequence appears to be the same as that of GenBank entries THD21072.1 (hypothetical protein D915_008168 [Fasciola hepatica]) and TPP67514.1 (hypothetical protein FGIG_07142 [Fasciola gigantica]). I think it would be good to add this information and/or where the original database ID can be found.
The last three amino acids in the sequence shown in Fig. 1 are GYL, while the above-mentioned GenBank entries have GLY. Could this be a typo?
Figure 5 is discussed before figure 4, so they would be better swapped.


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: CHARACTERIZATION OF A PROFILIN-LIKE PROTEIN FROM FASCIOLA HEPATICA
Review round: 1
Reviewer: 3

Output:
Basic reporting: Please see the general comment section.
Experimental design: Please see the general comment section.
Validity of the findings: Please see the general comment section.
Additional comments: The submitted manuscript, Wilkie et al, is an interesting read in which the authors have expressed, purified and characterized a profilin like protein in Fasciola hepatica. The paper is well organized and describes the introduction, the observations, and the methodology in explicit details. The results obtained are also reasonable. This paper represents an important contribution to the infectious disease community. However, there are major flaws with result interpretations that need to be revised before the manuscript can be considered for resubmission. My comments are given below:
Major concerns:
•	Please summarize the key findings and associated methods in brief in the introduction.
•	The authors claim that the immunized sera showed no reactivity to FhProfilin. However, the western blot experiment (fig 6) lacks a positive control showing that the immunized sera can detect any protein (at all). Please comment on it.
•	Phospholipid affinity assay: The authors claim that unlike conventional profilins, FhProfilin does not bind to phospholipids. To test their hypothesis, the authors performed Phospholipid affinity assay. To further support their findings, they incorporate a positive control that ,unlike the FhProfilin, binds to phospholipids.
However, there is a major flaw with the experimental design. The buffers used for FhProfilin and control protein are not identical. The  buffer used for the control protein contains additional tween 20. I suspect it may affect the experiment.
•	Immunization of animals: The manuscript lacks proper details of immunization of animals. For example, age, sex, etc. How many times the animals were immunized, days interval, what dose? Etc.
•	I highly recommend that the authors should include multiple sequence alignment file in the supporting information to show the conserved lipid binding and polypeptide binding site residues in different profilins across organisms. Currently the authors claim that FhProfilin contains 2 out of 5 PIP2 binding residues. Please show the residues in multiple sequence alignment figure.

Other minor issues:

Introduction
•	Please define the term ‘tegument membrane’ at its first occurrence in the manuscript.
•	In ‘FhProfilin’ Fh represents an organism and thus should be italicized.
•	Line 77: ‘….potential vaccine/drug targets due to their essential function of the Fasciola tegument….’
Please incorporate reference to show that FhProfilin is essential for the pathogen survival. If not available, please consider rephrasing the sentence.

Materials and methods:
•	Line 103: ‘50 mg ml-1’
I believe it is a typo. If yes, please replace mg/ml with ug/ml
•	Line 107: Please elaborate the term OD600
•	Line 119: I assume the Ni-sepharose resin was pre-washed with the loading buffer before adding the  supernatant. If yes, please incorporate the details.
•	Line 138: ‘FhProfilin (actin/FhProfilin)’
o	What does actin/FhProfilin represent here.
o	Please include the details of final volume of each reaction.
•	Line 163: ‘..according to the manufacturer’s instructions.’
Please provide a brief detail of the method.
Results and Discussion:
•	Line 199: ‘(accession number GEVX010111281)’
Is it uniprot id? Gene id? Please provide details.
•	Line 231: Please include references about recent studies on Plasmodium profilin. (PMIDs: 32034083 and 28552953)
•	Line 296: Please consider replacing ultrastructural with structural.
Figures and table
•	Fig 1A: Please highlight Fh in the tree
•	Fig 2B: Please replace ‘Fractions’ with ‘Fractions (ml)’
•	Fig 3: All the lanes in the SDS-PAGE image are smeary. Please comment on it.
•	Fig 5: If possible, please replace the current image with a single gel image showing all the lanes and the marker lane.
•	Fig 6: The gel lacks a positive control showing the immunized sera can recognize anything at all.


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: CHARACTERIZATION OF A PROFILIN-LIKE PROTEIN FROM FASCIOLA HEPATICA
Review round: 2
Reviewer: 1

Output:
Basic reporting: Figures 6A and 6B are still unconvincing, low-res and they look like the contrast has been manipulated too much. I would recommend that somebody with more knowledge of Western blots looks at this, and at the raw data the authors may provide, so that the reliability of this information can be judged.
Experimental design: No further comment.
Validity of the findings: No comment.
Additional comments: The authors made a consistent effort to address all the reviewer concerns, although an important part of the additional experiments/controls were not performed.


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: CHARACTERIZATION OF A PROFILIN-LIKE PROTEIN FROM FASCIOLA HEPATICA
Review round: 2
Reviewer: 2

Output:
Basic reporting: The submitted manuscript, Wilkie et al, is an interesting read in which the authors have expressed, purified and characterized a profilin like protein in Fasciola hepatica. The paper is well organized and describes the introduction, the observations, and the methodology in explicit details. The results obtained are also reasonable. This paper represents an important contribution to the infectious disease community. However, there is major flaws with result interpretations that needs to be revise before the manuscript can be considered for resubmission. My comments are given below:
Experimental design: The paper is well organized and describes the introduction, the observations, and the methodology in explicit details. The results obtained are also reasonable.
Validity of the findings: The paper is well organized and describes the introduction, the observations, and the methodology in explicit details. The results obtained are also reasonable.
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: COMPARING MULTIPLE COMPARISONS: PRACTICAL GUIDANCE FOR CHOOSING THE BEST MULTIPLE COMPARISONS TEST
Review round: 1
Reviewer: 1

Output:
Basic reporting: The authors have reviewed the literature to show which types of multiple comparisons tests (MCTs) are being used by authors of papers in ecological journals (and just as important, which are not being used), and then present results of a simulation study to compare the performance of different MCTs.  The literature review provides nice context and seems to be done in a reasonable way, but by itself would not provide sufficient content for an interesting publication.  The simulation results are the more substantial part of the manuscript.  I have looked at the authors’ code and results, and see no flaws.  Their main results repeat the findings from some earlier papers; a few methods for MCTs are fundamentally flawed and should not be used, whereas a few others perform well.  Nonetheless, I am not aware of any previous papers that are as thorough or clear.  Thus, I think this work would provide a useful reference for many researchers, including those beyond the ecology researchers that are the focus of the literature review.

The manuscript is well written.  I found the text clear and free of grammatical errors.  The authors did a good job proof-reading and preparing the manuscript, which made the task of reviewing simpler. Thanks.

My main recommendation for improving the manuscript would be to expand the discussion of methods to at least touch upon the methods for false discovery rate (FDR) adjustments.  The Benjamini-Hochberg method for family wise error rate adjustments (FWER, line 116) has become extremely common in other sub-disciplines of biology over the last couple decades, and is used at least as commonly as MCTs.  I teach graduate biostatistics and draw students from a range of fields.  Because of its importance in “-omics” analyses, I have had to add discussion of FDR approaches alongside older MCTs such as Bonferroni, Tukey HSD, Scheffé, and Dunn-Šidák (the only MCTs I covered the last time I taught biostatistics).  Despite being dominant in a slightly different field, I think that most biology researchers and students are likely to be familiar with (or at least have seen reference to) the Benjamini-Hochberg FDR approach.  Thus it would greatly improve the relevance and utility of the present manuscript to include some discussion of the relationship between FDR and MCT methods.
Experimental design: The simulations are well documented as I saw no errors in the code.
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: COMPARING MULTIPLE COMPARISONS: PRACTICAL GUIDANCE FOR CHOOSING THE BEST MULTIPLE COMPARISONS TEST
Review round: 1
Reviewer: 2

Output:
Basic reporting: This is a useful study, but is narrowly focused, so that results are limited in scope. The English is mostly very good, but there are a few poorly phrased sentences where the meaning would not be clear to the intended reader, and there are also some erroneous statements that need to be corrected. A wider range of relevant literature could have been used.
Experimental design: The design of the simulations is adequate, but limited in scope, and as a result the results are not as general as they could be.
Validity of the findings: The results are sometimes not interpreted well, with some minor errors. Much of the paper is advice drawn from previous literature, rather than based on results presented here. I consider that the advice should be more balanced, covering the various interpretations of best practice that are present in the literature.
Additional comments: The paper first shows that despite good advice in published papers and data analysis texts written for ecologists, there are still many ecological papers published in which the multiple comparison tests used are poorly chosen. It then provides advice to ecologists as to which tests should be used. This is actually the most difficult part. Perhaps the visual presentation in the paper of the way these tests behave will grab attention to the issue and further research by future authors – I hope so. But to quote from Oaten (1995) Ecology 76: 2001-9: “The difficulties arise from the need to present the methods concisely and, as far as possible, painlessly. …. assertions tend to rely on proof by authority and to take the form of exhortations and instructions rather than statements of results…In time, some judgments become accepted as laws. These can be hard to challenge”. Further, simulations can be misleading, by showing what happens only under particular conditions and with particular types of data.
The best starting point is probably to note that most statisticians would encourage estimating confidence limits for the size of effects (e.g. differences between means), rather than use significance tests, because using tests encourages readers to mistakenly believe that effects are real if (and only if) the test is significant. MCTs can be seen as an attempt to maintain this belief even when researchers are actually data-snooping rather than testing pre-set hypotheses. Thus, I would have preferred to see this paper start with a brief summary to point out that ideally ecologists should be setting out to estimate the size of hypothesised effects, rather than testing whether their data provides a significant result for an effect. They should be especially cautious if the study design involves questions about which of several groups may differ, and try if possible to ask pre-set questions about particular differences, on the basis of the focus of the study or the theory that drives the study. Often these planned questions will not relate to differences between pairs of means, but more complex differences. The best approach is to estimate the size of these differences (with confidence limits). MCTs appear to be appropriate when either the investigator has little idea of which groups may differ when setting up the study, or the results of a study are surprising, so that new, unplanned questions arise after examining the data. At this point, one could enumerate the comparisons that have reasonable biological interpretations and set up MCTs based on this set of comparisons. However, an alternative view is that the PCER could be used for all comparisons, with a warning to the reader that the overall error rate would be >0.05 as each question is considered in isolation from the others.
My other general concerns with this paper are that:
1. The data used for simulations is all drawn from the normal distribution, with equal variances. This is a missed opportunity, as it is very unlikely that most data from ecological experiments (or in many other types of experiments or surveys) would be normally distributed (in many cases, not even very well behaved), or have equal variances in each treatment, as authors often do not consider the likely distribution of their data. A study that included the robustness of the MCTs to modest differences between variances and modest variations in data distribution (modest meaning those which would not be obvious in small samples) would have been more useful, as it would show the behaviour of tests in the context of the kind of data that they are used on in practice, and as previous studies have examined the error rates of most of these tests on normally distributed data with equal variances. Further, the issue of unequal variances is not covered in the recommendations, especially in regard to the use of non-parametric tests (see my specific comment below).
2. The authors essentially dodge the issue of what the criteria are for using non-parametric tests. Many researchers feel that if they are not sure if the data are normally distributed, they should use a non-parametric test, and this is not good practice, partly because the issue of unequal variances requires a different analysis process, and partly because parametric tests (which actually rely on the distribution of the sample mean being approximately normal) are valid for moderately non-normal samples even for small sample sizes. Tests on samples that have different skewed distributions are a case where rank-based tests such as the MWW-U test are useful because the use of ranks reduces the effects of extreme observations, but there are also other ways to reduce the effect of extreme observations when using parametric tests. The advantage of this is that estimates of differences are possible.
3. I initially found it hard to understand why the simulated type 1 error rate of the parametric tests in Figure 3 is far below the 5% level in all cases. Then I realized that the error rates shown are per comparison rates, although this is not specified in the text or in the figure captions. If you want to know how well a test procedure works, it is more useful to show how well it does what it is designed to do, which is to ensure the EER rate does not exceed the 5% rate. So I would want to see the Type 1 EER for each test. I suggest this ought to be shown, and in any case the caption should make clear which error rates are shown. Obviously, the tests control EER by reducing the PCER rate below 5%. The results report that the study designs with many groups had the lowest per comparison error rates, with no explanation. This is what the MCTs must do, to control the EER.
4. The investigation of type 1 error only when a group of means are all equal misses the problem that occurs with the SNK test, where the EER becomes greater than the chosen significance probability when there are two groups of means, such that the means within each group are equal, but the groups differ (see Quinn, G.P. and Keough, M.J. (2002) Experimental design and data analysis for biologists. Cambridge University Press, Cambridge, p200). This leads to a false view of the performance of this test in Figures 3 and 4. As this was a known problem with this test, I suggest the authors should have demonstrated this problem.
5. It seems strange that the paper records the type 2 error rates of the various parametric MCTs (when one group mean differs, using normal distributions, for 4  study designs), but these results are not used when discussing which tests are recommended for various situations. The results (in Figure 5) show for example, that there is consistently less Type 2 error for Tukey’s HSD test than for the Bonferroni and Dunn-Sidak tests that are used for equivalent situations. While the difference is small, this suggests that the HSD tests will often be more powerful and thus a better choice. Exceptions might occur if the data are not of the type simulated, but this result conforms to that shown in Day and Quinn 1989, where the pattern of differences between groups was different, which suggests this is likely to be a consistent advantage of the HSD. On the other hand, the HSD can only be used with pairwise comparisons, while the Bonferroni and Dunn-Sidak methods were designed for any (potential) number of comparisons, whether pairwise or not.
6. The penultimate part of the discussion discusses using MCTs to interpret interactions in complex ANOVA designs. “MCTs can be very useful in disentangling statistical significance and differences among parameter estimates” (Lines 354-5). Many statistical texts would disagree (for example, Quinn and Keough 2002, p252), and I think the alternate approaches should be mentioned, rather than imply this is always the best or only way to untangle interaction effects.  The problem here is that there will often be a large number of means (so that the MCTs are not powerful), and the results of the many MCTs can be ambiguous. A simple example is where there is a significant interaction shown by the ANOVA, yet no pairwise comparison is significant. This manuscript itself points out that the MCTs may not be consistent with the ANOVA (lines 311-3). I suggest it makes more sense to try to understand why the interactions occur using interaction plots of means (a line joining the means of level 1 of factor A at each level of factor B, another line joining the means of level 2 of A at each level of B, etc). Three way or more complex designs can be broken down - for example plots of each two-way interaction at each level of a third factor. Another method is to construct sums of squares to test the effect of factor A separately at each level of Factor B (or the effects of factors A and B at each level of C where there is a three-way interaction, etc). These tests would use the mean-square error of the original ANOVA, which is the best estimate of the error variance, with more degrees of freedom (and thus more power) than if the data were separated to run these analyses. Tests of fixed factors would normally be run at each level of a random factor (for example ‘river’ in the example in lines 403-7), but with two fixed factors one might test the effect of A at each level of B, and the effect of B at each level of A. Plots of the means for fixed factors at each level of the other factors would aid in interpretation. As these are exploratory analyses looking for significant effects in a series of tests (especially if there are many levels of a factor), a Bonferroni or Dunn-Sidak adjustment of the significance levels may be appropriate, depending on how the results are reported to the reader. But the number of tests involved would almost certainly be less than MCTs on all the means involved in an interaction.
7.  The Abstract does not include some of the most useful conclusions presented in the discussion. I suggest these may include that Planned comparisons are overwhelmingly recommended (lines 262-3) and an unadjusted significance level can be used, that for planned non-parametric comparisons the Mann-Whitney -Wilcoxon U test is recommended, and that if planned comparisons are not used, Scheffe’s test may be used for any linear combination of the means, and Tukey’s HSD, the Bonferroni or the Dunn-Sidak tests are most commonly used for pairwise comparisons of groups. Other tests are recommended for particular types of data.

Specific comments.
Abstract:
Lines 19-20: The meaning of: “Due to the variable conditions of the data being analysed” is not clear, and this clause should be rephrased to make the meaning clear to readers.
Line 22: “including >40,000 reports of their use in ecological journals” does not convey any information – there is no time period specified. Perhaps a statement such as: “and we have documented extensive use of MCTs in ecological research” would be better.
Line 22: MCT should be MCTs.
Line 24: “We first reviewed the recommendations on their correct use.” There is nothing about this in the Results, Methods or the Background section of the Introduction. I assume that many of the recommendations in the discussion are taken from this review. I would have expected to see how this review was done – were a range of statistics texts consulted? Or were the initial descriptions of the tests checked to see what they were designed to do?  Or what? And some MCTs were designed or recommended for particular situations. The only reference to this is the statement “Principles behind matching a MCT to an experimental design are discussed below.” on line 174 in the Methods. ‘Below’ turns out to be in the Discussion. But this should be in the results before the results of simulations, to help the reader evaluate what the simulation results indicate in terms of what to use. One example is Scheffe’s test – see my comment below. Another is the Tukey-Kramer test, used to replace Tukey’s test when the sample sizes are unequal – see my comment below.
Introduction:
Line 34: The meaning of “Data analyses are crowded with factors of interest from experiments and observations” is not clear.
Line 51: “multiple comparisons evaluations” should be “the evaluation of multiple comparisons”.
Line 100: “creates more work for α” is poorly phrased. The non-statistician readers this paper is directed to will not understand what is meant.
Lines 112-113: “The EER reflects the adjustment in error rates to account for multiple comparisons” should be “The EER reflects the adjustment in PCER to account for multiple comparisons”  and “ ways to adjust for EER” should be “ways to adjust the PCER” (the EER is a chosen value, so “to adjust for EER” has no obvious meaning.)
Lines 114-5: The statement “EERs that reduce the power to detect differences are known as conservative, while those adjustments that are less strong are known as liberal” simply does not make sense. MCTs that control the EER to below 5% (by strongly reducing PCERs) are known as conservative, while those with less strong adjustments of the PCERs which do not control the EER to at or below 5% are known as liberal.
Methods:
Lines 121-132: The search method is useful, although it has the limitations described by the authors, and the Table of search results interesting. However, there are other issues here. The frequencies with which these methods are used does not tell us whether they were used for the correct types of data. For example, it would be useful to know how often the 3 common methods used to compare adjusted means after an analysis of covariance, as this is a likely error.
Lines 190-192: See my general comments on using normal distributions with equal variances, and on the misrepresentation of type 1 error rates for the SNK in practice, where means are not all equal.
Lines 192-193: Setting only 1 mean to be different from the rest to guage type 2 error may not provide a good indication of the differences in power between some tests, as in practice several means may differ in an experiment.
Line 207: “We excluded the Tukey-Kramer test and Dunnett’s test since they are only applicable for special cases”. But the Tukey-Kramer test is used in place of the Tukey test when sample sizes are unequal (‘unbalanced group study designs’). Does this mean that even for the unbalanced designs the authors evaluated Type 1 and 2 errors for the Tukey test?
Results:
Lines 225-6: Treatment with higher sample sizes did not always have lower per comparison error rates than equivalent designs with lower sample sizes. This is not true of the LSMG versus HSMG for balanced designs, nor of the HSFG versus LSFG in unbalanced designs (for most tests). Nor is it true that unbalanced designs reduced the proportion of per comparison type 1 error for all tests – the reverse is true for many tests in the HSFG design and in the LSMG design.
Lines 228-231: As I would expect, the SNK does not exceed an error rate of 0.05 in Figure 3. But Fig 3 shows it does not always have a higher error rate than the other tests as indicated here. Further, in the p-value density plots of Figure 4, it is Scheffe’s test that is relatively constant from zero to one (for some sample conditions), not the SNK test, which has a clear peak near 1. This error should be corrected.
Figure 4 and Figure 6: The four parts of each of these figures are labelled a, b, c, d and thus the caption statement in both figs that “Simulation group abbreviations can be found in the Figure 2 caption” is of no use to the reader. The simulation group abbreviations should be used in place of the a-d.
Line 231: Scheffe’s S test is designed to test any linear comparison of the means, not just pairwise comparisons, and this is why the test is conservative (reduces the EER below 5%) (and less powerful – see Fig 5) relative to tests designed for use when only pairwise comparisons are considered. It is also why it coherent with ANOVA results, as ANOVA F tests depend on whether any linear combination of the group means is larger than expected by chance. The reader should be made aware of this in the ‘Background’ section, or when the selection of tests was described (line 203), but also reminded of this when discussing its selection for unplanned comparisons (lines 307-315). It is not clear if the use of this test in the surveyed literature was for its designed purpose, or if it has been frequently used for pairwise comparisons. I assume the frequencies shown in Table 2 represent the sum of either use of the test.
Discussion:
Lines 254-259 (Parametric or non-parametric data). This section should also deal with the issue of unequal variances among the groups tested, This is a much more frequent problem in practice in terms of deciding whether a parametric general linear model analysis should be used, and non-parametric methods are also subject to an assumption that the distributions in the treatments compared are identical in shape and scale  (i.e. except for the median or mean)- see Johnson, D.H. 1995 Ecology 76: 1998-2000. Thus the variances must be similar (on some scale) for the test to function correctly. In other words, the use of non-parametric tests may be useful when the data is far from normally distributed, but they do not solve the problem of unequal variances – unless some transform of the data would equalise the variances but make the distributions very non-normal.  There are methods available that cope with unequal variances – there is mention of this later in the discussion, but no advice on when and why they should be used.
Lines 272-3: Note that orthogonal contrasts are not completely independent statistically (see the Oaten, 1995 reference above).
Lines 273-4: Orthogonal contrasts are not necessarily comparisons that do not include the same group in more than one comparison. In the example of A, B, C and D used, A to B, C to D and (A+B)/2 to (C+D)/2 are all orthogonal contrasts. I cannot see the point of such a “loose definition made operational for non-statisticians” (lines 276-7) nor do I see the value of a conflation of orthogonal comparisons and planned comparisons, even if ‘other literature’ has adopted this (I note that no reference is provided here).
Lines 282-3: “there is little practical difference between planned non-orthogonal comparisons and unplanned non-orthogonal comparisons”.  I do see a difference between these, and the key issue is whether the comparisons are planned or not (see Ruxton and Beauchamp 2008) . First, if the limited set of questions that are of interest to the ecologist in setting up the study define comparisons that are not all orthogonal, I see no reason why they should not all be tested using the PCER, provided the logical links between non-orthogonal questions are pointed out to the reader. I see the examination of whether comparisons are orthogonal mainly as a useful check for the researcher, to point out to her/him that the answer to one question may be linked to the answer to others, and as a brake on the number of comparisons made, as the number of potential orthogonal contrasts is the number of groups minus 1. Unplanned comparisons, on the other hand, by definition, may arise from any of the potential ways to compare a set of groups with each other. Only considering pairwise comparisons is one way to reduce the potential number of these unplanned comparisons, yet gain information about where any differences arise among the groups.
Lines 309-311: See my comments on Scheffe’s test above (Results, line 231). It would be useful here to explain when Scheffe’s test is designed to be useful – when a complex comparison is chosen for testing after the results have been examined.
Lines 318-319: Simultaneous testing using the Bonferroni adjustment of the PCER is known to be conservative – that is, it reduces the EER below 5%, and thus it often reduces the PCER below the level that other tests do (see Figure 3). The sequential Bonferroni should be less conservative than the simultaneous test (and the same is true for the sequential Dunn-Sidak test). As shown in Figure 3 the Dunn-Sidak adjustment of PCER is slightly less conservative in some designs, and Tukey’s HSD is less conservative in some cases than the Dunn-Sidak tests. The power of tests is reduced if the tests are more conservative than they need to be, but this is not well shown in Figure 5, because the pattern of differences between the means that was simulated does not show these differences well. One can see however, that the HSD often has slightly lower type 2 error rates than the Bonferroni and Dunn-Sidak tests.
Table 4: The difference in the 3rd row of the table should have j=2 for the first variable.
Table 5: The 9th row repeats the 8th row and should be deleted. There appears to be a 2-1-2 row missing below row 12.
Conclusions:
Surely Figure 7 should be mentioned in these conclusions?
 I disagree with the thrust of the recommendation regarding Scheffe’s test, which is designed for an unusual situation of a complex comparison, selected from all such potential comparisons after the results re available, and thus constrains the PCER to very low levels and has very low power compared to other MCTs. Several studies show Tukeys HSD is the pairwise test with the best power, so this is the best recommendation for all pairwise contrasts, unless sequential testing is to be done. While the authors may not agree with my view, I think they should explain the ways the MCTs were designed for different types of comparisons.


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

Input:
Title: IDENTIFICATION AND CHARACTERIZATION OF CRITICAL GENES ASSOCIATED WITH TAMOXIFEN RESISTANCE IN BREAST CANCER
Review round: 1
Reviewer: 1

Output:
Basic reporting: Zhang and coworkers investigated the potential mechanism of tamoxifen resistance in this manuscript entitled “Identification and characterization of crucial genes associated with tamoxifen resistance in breast cancer”. They utilized the already established bioinformatics approaches to re-analysis of microarray data from a public repository with accession no. GSE26459. The bioinformatics analysis demonstrated the candidate hub genes, gene ontologies, and pathways to discuss the tamoxifen resistance. The typical analysis leads to a set of prognostic hub genes. I commend the immunohistochemical assay showed 2 hub proteins as a significant drug target in tamoxifen resistance in breast cancer.
Experimental design: no comment
Validity of the findings: no comment
Additional comments: Some suggestions listed below for consideration by the authors to improve the manuscript:

1) Please clarify in the introduction how your work is different than previous works. I would suggest to include a few more sentences on line 97 to clarify the contributions of your work compared to previous work.
2) In the methods section, gene ontology and pathways analysis (line 138), could you please mention which statistical test was utilized to screen out the statistically significant terms?
3) In the method section, In PPI analysis (line 145), why you considered only the top 15 as hubs? What was the analysis methods of the PPI and parameters?
4) In Table 1, could it be possible to write the log FC in 2/3 decimal and scientific notation can be used for P-value instead of E notation?
5) I wonder why the authors only consider KEGG pathways, while there are other 6-7 data sources such as Reactome, NCI, etc. I would suggest, since the paper mostly discuss the pathway-based mechanism of tamoxifen resistance, to incorporate other pathways annotations in this manuscript.
5) I think Figure 1 is a very simplistic diagram with abbreviations, and adding much description in the legend would help the readers.
6) The resolution of fig 2 is not clear.
7) The survival analysis part lacks methodological descriptions.
8) The roles of hub genes can be explained in more detail in the discussion.


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

Input:
Title: IDENTIFICATION AND CHARACTERIZATION OF CRITICAL GENES ASSOCIATED WITH TAMOXIFEN RESISTANCE IN BREAST CANCER
Review round: 1
Reviewer: 2

Output:
Basic reporting: (1) Some descriptions of this paper are unambiguous. Please see Major Q1.
(2) Literature well referenced and relevant.
(3) Figures are low quality. Please see Minor Q1 to Q6.
(4) Raw data are supplied.
Experimental design: (1) The experimental method has some problems and needs to be redesigned. Please see Major Q4 and Q7.
Validity of the findings: (1) More research data are needed to support the conclusion of this study. Please see Major Q1 to Q7.
Additional comments: Breast cancer is one of the predominant types of tumors in women worldwide. Despite great progress in the early diagnosis and treatment recently, drug resistance and distal metastasis remain major causes of cancer-related mortality. In this manuscript, Kai Zhang and colleagues investigated the potential mechanisms of tamoxifen resistance through bioinformatics analysis. The study revealed a number of critical hub genes that might serve as therapeutic targets in breast cancer resistant to tamoxifen and provided potential directions for uncovering the mechanisms of tamoxifen resistance. Although, the current study is interesting but there are some major concerns that need to be addressed.

Major concerns:
1. “Title” “crucial genes” The present study only screened the differentially expressed genes associated with tamoxifen resistance and did not prove that the screened genes were involved in tamoxifen resistance. This manuscript contains a lot of loose language, which exaggerates the significance of the relevant results and must be carefully revised by the author.
2. “Cell culture” Line 125-130. Because the cells in this study were not purchased directly, the authors should provide STR identification results for the two cells.
3. “Immunohistochemistry (IHC)” Line 183-184. Please give a clear definition of endocrine therapy resistance and references.
4. “Statistical analysis” Line 194-196. The IHC clinical analysis lacked statistical methods.
5. “DEG Gene Ontology and pathway enrichment analysis” Line 228-231. In addition to expression verification, the authors should use siRNA to silence the target gene and observe whether breast cancer cells can sensitive to tamoxifen. This is a simple and low cost experiment, but it is important to support the conclusions of this study and should be presented by the authors.
6. “DEG Gene Ontology and pathway enrichment analysis” Line 228-231. Some genes should measure protein levels. In particular, two genes were tested for IHC.
7. “HSPD1 and HSPH1 levels correlate with tamoxifen resistance” Line 238-250. In tamoxifen-resistant patients, the comparison of expression differences between ANT and tumor tissues is not an important data supporting the conclusions of this study. The authors should compare the expression between tumor tissues of patient’s sensitive to endocrine therapy and those insensitive to endocrine therapy, or compare the expression between tumor samples sensitive to endocrine therapy and tumor samples resistant to endocrine therapy of the same patient. Since the author has collected 50 samples of endocrine therapy resistance from 2001 to 2016, I believe that the author is sure to get more samples of patient’s sensitive to endocrine therapy.

Minor concerns:
1. Table 1. Just keep 3 decimal places. List the probe number, gene full name, and gene ID number.
2. Figure 1. Flowcharts should not only show the process of bioinformatics analysis, but should present the entire research process.
3. Figure 2. Please make sure the text in the picture is large and clear for the author to read. The title of the axes should be clear.
4. Figure 3. The nodes in the diagram need to distinguish significantly between the DEGs and other genes. And add color legends.
5. Figure 5. Please make sure the text in the picture is large and clear for the author to read. To avoid direct screenshots, use vector images.
6. Figure 6. The control tissue in Figure 1B does not look like normal breast tissue. The author should check it with a pathologist.


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

Input:
Title: IDENTIFICATION AND CHARACTERIZATION OF CRITICAL GENES ASSOCIATED WITH TAMOXIFEN RESISTANCE IN BREAST CANCER
Review round: 1
Reviewer: 3

Output:
Basic reporting: Figure 2: Why do A and C lack the detailed of the number of genes in the graphs, whereas they are shown in B?
Figure 3: the interaction graph should be improved, spread out further to be able to appreciate all the interactions.
Table 4: the layout should be improved, it is confusing and not intuitive.
Figure 6: more IHC should be shown (at least 3 patients for each protein). Also, in the case of Patient D the normal tissue does not look like normal mammary gland. Is it stroma? Could a more representative section be chosen?
Experimental design: The experimental design is adequate.
Validity of the findings: The paper is valid. The authors should explain why they chose a threshold of 1.5 for the DEG. The conclusions are adequate.
Additional comments: The paper entitled: Identification and characterization of crucial genes 3 associated with tamoxifen resistance in breast cancer, by Kai Zhang, Kuikui Jiang, Ruoxi Hong, Fei Xu, Wen Xia, Ge Qin, Kaping Lee, Qiufan Zheng, Qianyi Lu, Qinglian Zhai, Shusen Wang compares gene expression profiles of MCF7 and MCF7/TR cells  using a GEO dataset. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses using  Database for Annotation, Visualization and Integrated Discovery (DAVID). A protein-protein interaction (PPI) network is generated, hub genes were analyzed. Finally, the authors conducted the immunohistochemical analysis of samples from breast cancer patients.
The paper is clearly written. The following issues should be considered:
1) The authors should explain why they chose a threshold of 1.5 for the DEG.
2) Figure 2: Why do A and C lack the detail of the number of genes in the graphs, whereas they are shown in B?
3) Figure 3: the interaction graph should be improved, spread out further to be able to appreciate all the interactions.
4) Table 4: the layout should be improved, it is confusing and not intuitive.
5) Figure 6: more IHCs should be shown (at least 3 patients for each protein). Also, in the case of Patient D the normal tissue does not look like normal mammary gland. Is it stroma? Could a more representative section/area be chosen?


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

Input:
Title: IDENTIFICATION AND CHARACTERIZATION OF CRITICAL GENES ASSOCIATED WITH TAMOXIFEN RESISTANCE IN BREAST CANCER
Review round: 2
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: I think the authors have improved the manuscript significantly and answered my criticisms satisfactorily.


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

Input:
Title: IDENTIFICATION AND CHARACTERIZATION OF CRITICAL GENES ASSOCIATED WITH TAMOXIFEN RESISTANCE IN BREAST CANCER
Review round: 2
Reviewer: 2

Output:
Basic reporting: No comment.
Experimental design: No comment.
Validity of the findings: No comment.
Additional comments: Breast cancer is one of the predominant types of tumors in women worldwide. Despite great progress in the early diagnosis and treatment recently, drug resistance and distal metastasis remain major causes of cancer-related mortality. In this manuscript, Kai Zhang and colleagues investigated the potential mechanisms of tamoxifen resistance through bioinformatics analysis. The study revealed a number of critical hub genes that might serve as therapeutic targets in breast cancer resistant to tamoxifen and provided potential directions for uncovering the mechanisms of tamoxifen resistance. The paper is improved and most concerned raised by the reviewer have been addressed. I think it is might suitable for publication at this version of revised manuscript.


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

Input:
Title: PATTERNS OF TELOMERE LENGTH WITH AGE IN AFRICAN MOLE-RATS: NEW INSIGHTS FROM QUANTITATIVE FLUORESCENCE IN SITU HYBRIDISATION (QFISH)
Review round: 1
Reviewer: 1

Output:
Basic reporting: Figure 1D: It is very hard to see the interstitial telomere signal. Please provide another example.

A more detailed description of the FISH TL method used will be helpful to critically review the results. For example, how were the samples batched? Was there a quality control sample? Were there technical replicates?  If so, what was the assay coefficient variation of the replicates? What criteria were used to choose the 50 cells analyzed in each animal?
Experimental design: Molar tooth wear and eruption patterns were used to place animals into different age groups for DMR and MMR because these animals were caught in wild and there was no other way of knowing their age. But NMR is bred in-house with age records. So, it would make sense to analyze TL with known age in the NMR group. Relatedly, as the exact age of DMR and MMR is not known, the authors should discuss this as a limitation of this study.

An independent telomere length method, such as qPCR, should be considered to verify the FISH results. It is understandable that the quantity of specimens is challenging especially with bone marrow samples. It will be helpful even if it is done in only a subset of samples.
Validity of the findings: It is possible that the lack of age correlation in NMR and MMR is caused by the high intra-specific differences in the telomere fluorescence signals and small sample size. This point needs to be discussed.

Figure 1: It appears that the telomere signals vary significantly among DMR, MMR, and NMR. Can you comment on whether this reflected different telomere lengths in these three species or technical variations?
Additional comments: Overall, the manuscript provided new sight on the telomere length in these three African mole-rats.
Addressing the small sample issue and the method details will be extremely helpful to bolster the conclusions.


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: PATTERNS OF TELOMERE LENGTH WITH AGE IN AFRICAN MOLE-RATS: NEW INSIGHTS FROM QUANTITATIVE FLUORESCENCE IN SITU HYBRIDISATION (QFISH)
Review round: 1
Reviewer: 2

Output:
Basic reporting: no comment
Experimental design: Calculation of total telomere fluorescence in qFISH experiment for telomere length estimation should cite a proper published research paper.
Validity of the findings: Telomere length measurement by qFISH only might not be enough to support the conclusions of the manuscript. Therefore, telomere length comparisons between individuals of the same species with 3 different ages must be performed using the “gold standard” telomere length measurement method—Southern blotting with a radioactive 32P or DIG-or Biotin-labelled TG-rich probe. Alternatively, could be more challenging, the PCR-based method to measure the telomere length difference between different age groups, consistent results will be more supportive.
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: PATTERNS OF TELOMERE LENGTH WITH AGE IN AFRICAN MOLE-RATS: NEW INSIGHTS FROM QUANTITATIVE FLUORESCENCE IN SITU HYBRIDISATION (QFISH)
Review round: 1
Reviewer: 3

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: The study by Leonida et al analyze telomere dynamics with age in three species on mole rats. They show that telomeres shorten in DMRs, but don not change or slightly lengthen in MMR and NMR (the longest-lived species). The study provides novel information on diversity and evolution of telomere maintenance mechanisms in these unique species of rodents.
The study is clearly written and a pleasure to read. I enthusiastically recommend publication.

Minor suggestions:
1.	NMR shows dimmer telomere fluorescence compared to DMR. This is consistent with the earlier observations that NMRs have human-sized telomeres (10-12 kb), while other short-lived rodents tend to have longer telomeres (30+ kb). The authors may want to comment on it.
2.	Figure 2B has chromosomes that appear not separated from each other. A better image or an explanation in the legend would be helpful.

Vera Gorbunova


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: PATTERNS OF TELOMERE LENGTH WITH AGE IN AFRICAN MOLE-RATS: NEW INSIGHTS FROM QUANTITATIVE FLUORESCENCE IN SITU HYBRIDISATION (QFISH)
Review round: 2
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: I am not convinced to support the publication of this manuscript. At least another independent telomere length method must be performed to obtain consistent experimental results as shown in this version. Current data are basically one-dimensional, which can lead to ambigous conclusions.
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: PATTERNS OF TELOMERE LENGTH WITH AGE IN AFRICAN MOLE-RATS: NEW INSIGHTS FROM QUANTITATIVE FLUORESCENCE IN SITU HYBRIDISATION (QFISH)
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 my concerns


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

Input:
Title: COMPREHENSIVE ANALYSIS OF CIRCRNA EXPRESSION PROFILES AND CIRCRNA-ASSOCIATED COMPETING ENDOGENOUS RNA NETWORKS IN IGA NEPHROPATHY
Review round: 1
Reviewer: 1

Output:
Basic reporting: The manuscript ''Comprehensive analysis of circRNA expression profiles and circRNA associated competing endogenous RNA networks in IgA nephropathy'' is interesting, well-structured (conforms to PeerJ standards), and professional English language is used throughout. However, the whole manuscript could use some editing to make it more clear and unambiguous. Some specific changes are suggested below:

Line 85 suggest -> suggesting
Line 98 have providing evidence -> provide evidence
Line 102 procession -> processing
Line 104 remove: therefore the circRNAs been produced
Line 104 are -> is
Line 107 regulating -> regulate
Line 108 please check: thus been discrible involed
Line 109 have -> has
Line 110 remove: ''many''
Line 112/116 please check: ''; novel_circ-113 0004153/rno-miR-1443p/Gpnmb ceRNA relationship have been reported involved in acute kidney injury of rat model (Cheng et al., 2019); in another way, human antiviral circRNAs are activated for responding to Kaposi's sarcoma herpesvirus infection, which been proved work as an antiviral mechanism by suppressing crucial viral genes''
Line 122 the change of -> changes in
Line 123 please check: ''Because PBMCs are full of lymphocytes (with 70-90% T cells, B cells, and NK cells) could partially represent the change of the immune system, it had been broadly investigated in IgAN''
Line 127 remove: ''underlying''
Line 135/136 patients were excluded from any other systemic diseases or secondary IgAN -> patients with any other systemic diseases or secondary IgAN were excluded
Line 141 of -> using
Line 143 ugs -> g
Line 144 left -> remaining
Line 146 synthesis -> synthesized
Line 164 denovo -> de novo
Line 165 ''; '' -> ''. ''
Line 166 remove: ''were''
Line 187 are -> were
Line 188/189 please check: ''Normalize Between Arrays in the Limma package was used to normalize''
Line 190 Significantly differential -> Significance of differential
Line 201 which expressed -> which are expressed
Line 207 found -> find
Line 211 screened -> screen
Line 226 have -> that
Line 227 validation the expression -> validation of the expression
Line 229 with the following: -> with the following conditions
Line 232 and listed -> and are listed
Line 239 differential -> differentially
Line 241 is listed -> are listed
Line 250 remove: ''briefly''
Line 256 Fc gamma R- -> Fc gamma Receptor-
Line 258 upregulate -> upregulated
Line 262 function -> functions
Line 265 ''; '' -> ''. ''
Line 265 upregulate -> upregulated
Line 274 remove: ''have''
Line 275 overlapping -> overlapped
Line 278 identified -> identify
Line 285 and 286 remove: '', ''
Line 287 most -> mostly
Line 296 remove: ''And''
Line 299 was -> were
Line 368 transformer -> differentiation?
Line 369 prepossessing -> preprocessing
Line 381 remove: ''using''
Line 382 has -> have
Line 383 please check: ''adaptions''
Line 401 These Vivo and Vitro phenotypes -> These in vivo and in vitro phenotypes
Line 432 remove: ''of''

The authors explain the context of their study and support their data with well referenced and relevant literature.

The figures are relevant and high quality. However, there are some errors in some of the titles:

Table 1- Title:  Most -> most
Table 1- Column 2: please check ''CricBase ID'', is it CircBase?
Table 1- Columns are too close to each other. A suggestion is to reduce the unnecessary decimals in the last two columns (log2(FC) and p-value).

Table 2- Title: deferentially -> differentially
Table 2- Column 4- Title: Different -> Differential

Additionally, there is a lack of cohesion throughout the manuscript when referencing Figures. Sometimes the full word appears. Other times they are named as ''Fig.''. I suggest homogenizing.

The authors supplied raw data for circRNA (available at GEO accession number GSE154046) and RT-PCR (Excel file).
Experimental design: The original primary research of this manuscript is within the scope of the journal.

IgA nephropathy remains a significant cause of renal failure that results from the formation of IgA1-containing immune complexes. Our current understanding of its pathogenesis is mostly based on the abnormal increase of circulating O-galactosylated IgA1 and the production of O-glycan-specific antibodies. However, it has become evident that other genetic, epigenetic, and immunological mechanisms are responsible for mesangial IgA1 deposition and renal injury.  Hence, the authors´ research question is relevant and meaningful in furthering our understanding of IgA nephropathy's pathogenesis. The authors state that their research fills an identified knowledge gap (understanding the molecular pathogenesis of IgA nephropathy) that could lead to improved targeted therapies to ameliorate disease progression.

Rigorous investigation is performed to a high technical and ethical standard. Overall,
the methods are described with sufficient detail and information to replicate. Some minor comments:

Methods section- A description of RT-PCR data normalization and analysis is missing. Looking at the Excel file containing the raw data it is evident that they used actin as the house-keeping gene for normalization. However, this is not stated in the manuscript and the primers used for actin amplification are also missing in Table S1. Also, the full names of ASB16, HLA-B, TRIM21, and SEC24C are not provided.
Validity of the findings: All underlying data have been provided, they are robust and statistically sound. I found the results to be supported by the data presented.

Conclusions are well stated. However, I was left wondering how their findings compare to other studies of non-coding RNAs in PMBCs from patients diagnosed with glomerular diseases other than IgA nephropathy. Could any of their circRNAs, miRNAs, and mRNAs have the potential to be used as diagnostic or prognostic markers? Additionally, I found the number of patients in which they performed the validation of their findings through RT-PCR rather small (IgA nephropathy n=4 and healthy controls n=4). I suggest elaborating on this issue as a limitation of their study.
Additional comments: The GEO accession number of the circRNA data is not written in the manuscript. Is this intentional?


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

Input:
Title: COMPREHENSIVE ANALYSIS OF CIRCRNA EXPRESSION PROFILES AND CIRCRNA-ASSOCIATED COMPETING ENDOGENOUS RNA NETWORKS IN IGA NEPHROPATHY
Review round: 1
Reviewer: 2

Output:
Basic reporting: 1.	The title of this manuscript is “Comprehensive analysis of circRNA expression profiles and circRNA associated competing endogenous RNA networks in IgA nephropathy”. It should be better using “circRNA-associated” instead of “circRNA associated”.
2.	The English language should be improved to ensure that an international audience can clearly understand your text. Some examples where the language and grammar could be corrected or improved include lines 48, 75, 85, 90, 107, 108, 122, 358 and so on – the current phrasing makes comprehension difficult.
3.	The introduction needs to be more concise, and highlights the background of circRNA-mediated ceRNA networks in IgA nephropathy. Therefore, line 88 – line 99 elucidating that miRNA has universal role in the pathophysiology of IgAN could be redundant.
4.	Line 124, the last sentence of the introduction described that “PBMCs from IgAN and healthy controls were collected and sequenced with next-generation technology”. It’s necessary to indicate clearly PBMCs were collected from IgA nephropathy patients instead of animal model.
5.	Figure 2A: The spelling of “cricRNA” was wrong. Please correct it.
Experimental design: no comment.
Validity of the findings: 6.	The author didn’t use qRT-PCR to confirm the differential expressed circRNAs identified in the circRNA-sequencing data. It will improve the accuracy of this manuscript if the qRT-PCR confirmation experiment could be supplemented.
7.	In Conclusions (line 439), the manuscript elucidated that “4 circRNAs competed with miR765 to regulate the expression of GALNT2 in IgAN”. However, the experiment design of this article didn’t validate this point. Therefore, the author couldn’t draw this definite conclusion. Suggest the author could modify the content of “Conclusions”.
Additional comments: 8.	In the Discussion, line 338 – line 343 described that “the hsa_circ_007056、hsa_circ_006671、hsa_circ_0073237 and circRNA3302 were significantly upregulated in our study, and all of them may play roles as ceRNAs of miR765, therefore participating in the manipulation the expression of target GALNT2. However, all the 3 circRNAs were up-regulated, therefore didn’t follow the classic circRNA(down in IgAN)-miRNA(up in IgAN)-mRNA(down in IgAN) interaction regulation”. This finding was interesting, but the explanation of this part was not sufficient. Hope the author could discuss deeply about this finding.


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

Input:
Title: COMPREHENSIVE ANALYSIS OF CIRCRNA EXPRESSION PROFILES AND CIRCRNA-ASSOCIATED COMPETING ENDOGENOUS RNA NETWORKS IN IGA NEPHROPATHY
Review round: 2
Reviewer: 1

Output:
Basic reporting: No comment
Experimental design: No comment
Validity of the findings: No comment
Additional comments: The authors have modified their manuscript to address all my previous concerns.


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

Input:
Title: COMPREHENSIVE ANALYSIS OF CIRCRNA EXPRESSION PROFILES AND CIRCRNA-ASSOCIATED COMPETING ENDOGENOUS RNA NETWORKS IN IGA NEPHROPATHY
Review round: 2
Reviewer: 2

Output:
Basic reporting: no comment.
Experimental design: no comment.
Validity of the findings: no comment.
Additional comments: no extra comment. The author has answered my questions quite well. The work is good.


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: BACTERIAL EXPOSURE LEADS TO VARIABLE MORTALITY BUT NOT A MEASURABLE INCREASE IN SURFACE ANTIMICROBIALS ACROSS ANT SPECIES
Review round: 1
Reviewer: 1

Output:
Basic reporting: Dear editor and author,
Thank you very much for the invitation; it was a pleasure to do the manuscript review. The study titled “Lethal and antimicrobial responses to bacterial exposure across ant species” it aims to compare four ant species’ responses to non-pathogenic bacterial exposure in order to broaden the understanding of microbial health-threats to ants and their ability to defend against them. The results are interesting and after some modifications it could be considered for publication.

Here is my main concern:

-Authors should consider restructuring the entire introduction. The introduction is long and has no objectivity. In addition, the four species used in the study were not highlighted in the introduction and it would be of great help to the reader if in the introduction the defense strategies against pathogens of each species were presented.

-Adding figures with experimental design of the two experiments would be of great help to the reader.

- Authors should consider presenting a mortality curve for each species and treatment to illustrate and highlight the main findings of the study.

Specific comments:
L1: Title is not very informative. I suggest focusing on the main results.

L22: It would be interesting for the reader to know here what these four species are.

L44: Please provide references.

L48-50: Please provide references and examples.

L51-53: It seems strange to me: using ant antibiotics to understand which microbes are pathogenic. How is this possible? I believe that a study showing the function of these bacteria would be more efficient to discover this. Could you provide more details?

L68: It would be of great to give credit to the taxonomist for all the ant species you mention in the text. Check this out on antcat.org

L106: What are the species? Restructuring the introduction would be of great help to the reader. Provide information on each of the species and strategies that each would have the greatest impact on the study.

L129: You need to assume that readers are not American and do not know what this location. I suggest adding the country and the geographic coordinates here.

L129: Finally, the 4 species! Authors should reveal these species in the abstract, in the introduction, and provide data on the strategies for defending these species in the introduction.

L129-135: How were these species identified? Morphology? Which taxonomic key? Were these ants deposited in an entomological collection? Do you have a voucher?

L136-138: This information should be moved for introduction and not in MM.

L139-149: it would be very helpful for the reader to explain why this.

L142: How long did these ants stay in the lab until the experiment was carried out. I wonder if stress is a factor to be considered as responsible for the results of the study.

L155: How many ants were added in each treatment? Were they together?

L157: How many ants died in the control group? This will tell me if the ants were under stress.

L175: Why only those two? Do the other species not produce inhibitors? A restructured introduction should address these issues.

L175-177: This information should be moved and explored in the introduction.

L183: Same thing about the experiment 1. For the manuscript to gain international appeal, the authors should also offer the country and the geographic coordinates.
How were these species identified? Was it deposited in an entomological collection? Please provide vouchers.

L185: 200 in total, right? and how many were added in each treatment? It is still difficult to visualize and understand the experiments. I believe that figures with experimental designs could offer more support to the reader.

L194-195: Do you have data that provide a basis for this choice? These data would give more credibility to this choice and to the study in general. Please provide this data.

L234-237: Excellent! It would be interesting to put the n of dead ants in control. In addition, providing a mortality curve for each treatment and each species would be very helpful and would help to highlight the findings of the present study.

L242-243: Where is this result? Quote the Figure or Table here.

L285: Please, remove that square.

L350-352: Please provide references.

Figure 1: Very small and poor-quality image.
It would be interesting if the authors showed the mortality curve of this first experiment with respect to the control group.
If I managed to visualize the graph well, A rudis and B. chinensis was no different from the control group in terms of mortality, is that it? Can we say that these ants were under stress and can we not consider these results, whether positive or negative?

Figure 2 and 3: It would gain more impact if the authors added the statistic to that image (p-value).
Experimental design: It has already been commented above.
Validity of the findings: It has already been commented above.
Additional comments: It has already been commented above.


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: BACTERIAL EXPOSURE LEADS TO VARIABLE MORTALITY BUT NOT A MEASURABLE INCREASE IN SURFACE ANTIMICROBIALS ACROSS ANT SPECIES
Review round: 1
Reviewer: 2

Output:
Basic reporting: In the manuscript titled „Lethal and antimicrobial responses to bacterial exposure across ant species” the authors investigate a whether surface substances ants influence the ant’s ability to survive exposure to two bacteria that likely did not coevolve with the ants and whether the antimicrobial activity of the ant’s surface substances changes upon exposure to these bacteria. Unfortunately, although I liked the basic premise of their two experiments, I cannot recommend the study in the present format as it contains some lines of argumentation that I think the authors should reconsider, methods that might be inappropriate to answer the questions raised by the authors and at places a poor choice of references.
Experimental design: See 3. Validity of the findings
Validity of the findings: 1. Argumentation.
I like the authors idea to use bacterial species that likely did not coevolve with the ants, as I agree with the authors statement that harmfulness of bacteria or other microorganisms is often only tested with likely pathogens. However, exactly this point is also problematic and I think the authors need to reconsider their lines of argumentation for the following points.
1.1 The authors may find one effect with the bacteria they used but potentially an opposite effect with other bacteria. As there are literally tons of different bacteria in the environment of ants, choice of representative bacteria becomes crucial. As far as I understand this choice was mainly based on a previous study by Penick et al (2018) and I wonder why the authors did not choose for example widespread bacteria species that these ants are likely to encounter in their environment. Moreover, in line 82-84 the authors write: “we do not know whether exposure to these non-entomopathogenic bacteria represents an actual health threat that ants would benefit from having evolved defences against.” This implies that part of the aim of the authors study was to investigate evolved defences, i.e. antimicrobial surface substances. Altogether, this leaves the reader wondering what exactly the expectation of the authors was investigating evolved defences against non-coevolved bacteria. That susceptibility to bacteria varies under these premises (main finding reported in line 289) is hardly surprising.
1.2 In line 309-310 the authors state that their aim was to broaden our understanding of how microbes might influence antimicrobial traits of ants. Have the authors considered that antimicrobial traits might also influence microbes, especially those in the environment of ants?
1.3 At the end of the discussion the authors raise the hypothesis (line 332-333) that ants that are susceptible to environmental bacteria are more likely to employ antimicrobials. This brings me to the part of the study that I consider most problematic: In contrast to for example fungal entomopathogens that enter their host via penetration, bacterial entomopathogens and likely also the non-entomopathogenic bacteria used in this study usually represent a health threat to insects when orally ingested. However, once ingested surface substances are unlikely to play a role in the fight against bacteria. How this might influence the results presented in the study or the hypothesis raised in line 332-333 is unfortunately completely ignored.
1.4 Another argumentation line that should be reconsidered is in line 310-319: here the authors criticize the use of bacteria that show a coevolutionary association with insects. However while the authors used in their experiment free-living bacteria, examples given here concern the intracellular bacteria Wolbachia and Spiroplasma which, apart from several other differences, cannot be compared as they are most often maternally transmitted and will likely never encounter surface substances of ants.

2. Methods.
2.1 In their first experiment on the mortality of ants upon exposure to bacteria on an agar block the authors did not control exposure. Although they write in line 156 that they ensured direct contact between ants and bacteria at least once and in line 157-158 that observations of ants tunnelling into the agar were recorded, I wonder whether the finding of variable susceptibility to bacteria is an intended/unintended consequence of avoidance or attraction of the ants to the agar block that has nothing to do with the existence of antimicrobial surface substances. I wonder whether the authors have considered that some ants will use the agar block as a food source and therefore tunnel into it and through this orally acquire an infection with the bacteria present on the agar block. Unfortunately, the observations on tunnelling are not reported or discussed.
2.2 In their second experiment the authors measured the antimicrobial activity of surface extracts of the ants. While this or similar methods have been used in previous studies to investigate a relationship between antimicrobial strength and sociality (Turnbull et al. 2011) or sociality and group size (Penick et al. 2018) I am highly sceptical that this method to assess the antimicrobial activity of surface substances will give valid insights for the following reasons. 1) This method likely extracts substances from exocrine glands all over the body of the ants and not just substances that are present on the cuticle all the time. Most of these glands will contain compounds that at some concentrations will show antimicrobial activity, especially defensive glands such as the poison gland. An antimicrobial activity of a substances at certain concentrations is hardly surprising, as the dose makes the poison (alleged statement of Paracelsius). What is however completely ignored with this method is whether ants use antimicrobial compounds from these glands for their antimicrobial activity, i.e. actively use them to fight microbes. Without knowing this the antimicrobial activity of compounds, gland or surface extracts is unfortunately only of pharmacological but not really of biological interest. 2) Depending on the solvent used for extraction the method will give varying results as it will extract different compounds from the same gland or compounds form different glands. While the authors acknowledge this partly in line 306-307, this is and reason 1) mentioned above make it impossible to connect the results of the method to create surface extracts with the biology of the species under question.

3. References:
3.1 I also like the authors aim to test for a possible increase in antimicrobial ability in response to exposure to bacteria. As far as I am aware there is only one study that has investigated this with antimicrobial metapleural gland secretions: Yek et al. (2012; https://doi.org/10.1098/rspb.2012.1458). Yet this reference is unfortunately missing in the reference list.
3.2 Line 48: The reference to Cremer et al. (2007) is placed behind part of the sentences talking about antibiotics, suggesting that this reference mainly deals with antibiotics, which is clearly not the case.
3.3 Line 140-142: Here the authors write that reduced carbohydrate levels have been shown to reduce antimicrobial expression and that this was the reason ants were fed sugar water to avoid weakening possible antimicrobial activity. However, with their method of extracting surface substances it is highly unlikely that antimicrobials in the form of antimicrobial peptides were investigated. Instead the authors likely extracted antimicrobials from exocrine secretions with their method.
3.4. Line 328-330: The study of Lucas et al. (2019) although an interesting study itself, is certainly not appropriate to reference strategies used by some ant species in controlling and mitigating the risk of fungal pathogens
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: BACTERIAL EXPOSURE LEADS TO VARIABLE MORTALITY BUT NOT A MEASURABLE INCREASE IN SURFACE ANTIMICROBIALS ACROSS ANT SPECIES
Review round: 1
Reviewer: 3

Output:
Basic reporting: This paper introduces many fascinating topics, but it left me scratching my head. I think there could be improvements on clarity, relating the study to the literature, and expanding on relevant details.

I found the conclusions to be a bit vague and confusing. The authors aim to make a connection between the ability to produce antimicrobial compounds and susceptibility to bacteria. There were two ant species (S. invicta and B. chinensis) that were used in both experiments. B. chinensis extracts had relatively low inhibitory effects on E. coli, yet they enjoyed relatively high survivorship when exposed to it (the opposite of what the authors hypothesize). Additionally, the other ant species, S. invicta, had relatively strong inhibitory effects against E. coli, yet suffered higher mortality when exposed (also opposite of hypothesis). This suggests that the antibacterial activity of extracts do not predict protection against the bacteria in the setting they tested. If these results are valid, this is perplexing, but the authors don’t discuss what could be explaining this pattern. The authors still say in the discussion “our results could be an indication of a linkage between an ant species’ susceptibility to bacterial exposure and antimicrobial ability” (lines 290-291) and end the discussion saying “We hypothesize that ants that are susceptible to environmental bacteria are more likely to employ antimicrobials (and perhaps less likely to rely on immune responses)” (lines 332-334). Seems odd to end with a hypothesis that was not supported by the study. (Also, it would be helpful to have this hypothesis more clearly stated in the introduction.)

The authors call the experimental bacteria “non-pathogenic” but go on to state that the goal of the experiment was to test if those bacteria had harmful effects. I would argue that it isn’t accurate to call something non-pathogenic if its pathogenic properties haven’t yet been tested. I suggest the authors change this descriptor to environmental bacteria, to indicate that they are not ant-specific. I don't think a microbe has to be coevolved with a specific host species to cause disease.

I found there to be little background information about the bacteria, especially Staphylococcus epidermidis. It would be helpful to explain whether or not this is a bacterial species that ants are likely to encounter in nature. In Penick et al. 2018, they say: “Testing inhibition of Staphylococcus bacteria is the standard technique to measure antimicrobial activity of insect compounds, and it is the same approach used for the comparative studies in insect groups described above [11–13,26,27]”. This justification, and a similar one for E. coli, would be helpful to have in the text.

The authors do not explain the reasons for picking these four species, how exactly they differed in antimicrobial activity from Penick et al. 2018, or how they are related. The authors also discuss potential medical applications but don’t relate it to the results, the bacteria used in the study, or the compounds the ants produce. It seems like there is a lot of interesting ideas in there, but none are really flushed out and tied to the study.

Much of the paper is based off a previous paper (Penick et al. 2018). The authors report findings from that paper inaccurately. 40% of the species in that study were ineffective at inhibiting growth (line 76 says 40% were effective). In the Discussion, the authors bring up the fact that they found a result that contradicts this previous study (lines 304-307), which they speculate may be due to different extraction solvent. This could be discussed more and could make a call for repeating Penick’s experiment since it appears it’s possible some of the 40% of species that did not show responses to S. epidermidis may need isopropanol to be extracted.

There were also numerous language and grammatical errors, which I did not comment on here.
Experimental design: 200 workers were used for S. invicta instead of 50 like the other species. If they did not use larger petri dishes for S. invicta, density of ants would impact their results by altering likelihood of ingesting the bacteria or experiencing allogrooming. It seems to me this should be discussed, and the results for S. invicta should be plotted and interpreted separately from the other species.

I found it to be problematic that there was no estimation of the number of bacterial cells on each piece of agar that the ants were exposed to. And was the bacterial density or abundance increased in S. invicta’s dishes? I think this information would be important for interpreting the results.

I don’t quite understand the explanation of the unbalanced design in lines 224-227. Are nests different than colonies?

“Control group responses were subtracted from treatment group responses to directly compare treatment effects” (lines 234-235). I'm not sure I understand why the authors did this. How could they compare the treatments to the controls if one was subtracted from the other? The figures are unintuitive – I think it would be easier to just plot the control values alongside the treatments instead of subtracting.

The authors say that the ants in Experiment 2 were “subjected to a sub-lethal period of bacterial exposure” (lines 293-294) but that “dead individuals were removed” (lines 192-193; and they don’t state how many individuals died). The authors then used “the remaining living workers for extraction”. Assuming a different number of individuals died from each species (especially since their first experiment showed differences in mortality), wouldn’t that result in different numbers of workers being used for the extractions? It would be helpful if they included how many workers died in each group if they believe that difference in workers didn’t affect their results.
Validity of the findings: The data and analyses for the second experiment are not provided.

There are areas where confounding variables might be present, which I bring up in the Experimental Design comments (i.e. the number of individuals used for the extractions, the number of bacterial cells administered), which may compromise the validity of the findings if present or unaccounted for.
Additional comments: The authors conducted two studies about ant susceptibility to bacteria. The authors first tested the lethality of two species of bacteria for four ant species. They administered bacteria or control agar to ants in petri dishes and counted the number of dead ants after 24 and 48 hours. They found that two of the four species had higher mortality when exposed to bacteria compared to the control ants. One of these species was only susceptible to one of the bacteria; the other was susceptible to both. This experiment was nice and simple and showed that there is variation in susceptibility to different bacteria in ants. I think this is a great result and opens up many other questions.

The authors then sought to test whether these differences in mortality were reflected by inhibitory cuticular antimicrobial compounds and whether this antimicrobial activity was induced by non-lethal exposure (vs. constitutive). This is a very interesting question and nice to pair with the first experiment and a previous study (Penick et al. 2018). They used two of the four species used in experiment one and exposed them to bacteria or control agar for 6 hours before extracting their cuticular compounds and running antibacterial assays on the same bacteria the ants were exposed to.

However, this paper left me feeling confused. There were lots of ideas brought up in the introduction and discussion, but they were not connected to the goal or findings of this study. Additionally, some of the terminology was confusing (e.g. using “non-pathogenic” to describe a bacterial species that has not yet been tested for pathogenic effects). Lastly, I found the methods to be problematic in a few ways; please see the Experimental Design 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: BACTERIAL EXPOSURE LEADS TO VARIABLE MORTALITY BUT NOT A MEASURABLE INCREASE IN SURFACE ANTIMICROBIALS ACROSS ANT SPECIES
Review round: 2
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: The authors accepted almost all of my previous suggestions. I am satisfied with the changes and believe that the manuscript can be published.