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: RESTRICTION DIGEST SCREENING FACILITATES EFFICIENT DETECTION OF SITE-DIRECTED MUTATIONS INTRODUCED BY CRISPR IN C. ALBICANS UME6
Review round: 1
Reviewer: 1

Output:
Basic reporting: The authors incorporation of restriction enzymes into CRISPR homologous replacement mutagenesis screens is related to previous work using restriction screening for site directed mutagenesis. They should cite Neff et al., Plant J 1998 (which used a similar approach to identify site directed mutations) and Hodgens et al., 2017 (which extended Neff's approach to identify CRISPR indels with their indCAPS tool).
Experimental design: The manuscript reports a clever incorporation of restriction enzymes into a CRISPR mutagenesis screening regime, as well as results of site-directed mutagenesis of the C. albicans UME6 transcription factor.  One point of confusion arises from the C. albicans strain used for the study. Was it SC5314 "wildtype"?  Also, were the mutations homozygous or heterozygous, as C. albicans is an obligate diploid?  Heterozygous mutations would have left 50% cut / 50% uncut on the gel. Were such mutations recovered?  Did the authors do anything to remove the pv1093 plasmid after CRISPR mutagenesis?  If not, heterozygotes would repair randomly to homozygous wt or homozygous mutant, right?
Validity of the findings: While the experiments were well done and appropriate controls and statistical tests were performed, the authors overstate the results in the discussion line 223. Their results don't show that UME6 is "essential for filamentation", as they don't show any defect in hyphal growth in figure 2. There may be such a defect (visible under the microscope, e.g.). If so, please show it. In addition, there wasn't a "dramatic loss of filamentation and virulence". Virulence was reduced, not lost.  This is just overstated. Probably "led to decreased filamentation and virulence" would be fine, assuming they show some change in filamentation beyond altered colony morphology.
Additional comments: Generally nice paper. Adding references to previous restriction enzyme screening approaches and lightening the interpretation of UME6 mutants a bit would make this stronger. Also, the authors should revise the manuscript to clarify the distinction between homozygous and heterozygous mutants.


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: RESTRICTION DIGEST SCREENING FACILITATES EFFICIENT DETECTION OF SITE-DIRECTED MUTATIONS INTRODUCED BY CRISPR IN C. ALBICANS UME6
Review round: 1
Reviewer: 2

Output:
Basic reporting: No comment.
Experimental design: No comment.
Validity of the findings: No comment.
Additional comments: The work "Restriction digest screening facilitates efficient detection of site-directed mutations by CRISPR in Candida albicans" by Evans and colleagues describes a useful mutagenesis and screening strategy for the important fungal pathogen Candida albicans. While mutagenesis at some loci is very efficient, others are more difficult, requiring extensive screening to identify successful mutants. The approach presented can significantly reduce the cost of identification of such mutants. Though not emphasized, the authors have additionally identified mutants which impact filamentation and pathogenesis.

A few minor suggestions for the authors, in no particular order:
1. The authors discuss the importance of codon usage in designing a species specific mutagenesis strategy (in paragraph starting at line 200), but do not discuss the codon usage for the codons they chose. Perhaps worth noting if the codon frequency was/was not limiting for the E codons they chose.

2. C. albicans is a diploid, therefore successful CRISPR mutagenesis requires mutation of both copies of a gene - even more for guides targeting multiple genes. A key advantage of the restriction strategy proposed is that it can sensitively/reliably detect mixtures, which would result in incomplete digestion. Such mixtures are not always reliably detected by sequencing, where mixed base calls rely on more ideal templates. This additional benefit of the authors' strategy might be worth highlighting in the discussion.

3. The rationale for mutagenesis of the N-terminal domain of UME6 at codons 754 and 755 was not given. Is this a conserved region of the protein? Is there a predicted function? Have other studies been done to suggest this is important, or is there any clear reason why the mutants chosen would have the hypomorphic phenotype shown?

4. As this article is intended to provide some guidance for mutagenesis strategies, perhaps it would be worthwhile to depict the guide in Figure 1C-E, and how mutagenesis destroys the recognition motif. If so may be worth showing Figure 1E as "WT" sequence, rather than "failed mutant," and simply note the failed mutant had WT sequence. If so, it should be shown before mutant sequences.

There were a few minor errors in the text noted below:
Line 67: "reviewed in" should be inside the reference parenthesis
Line 117: C. albicans instead of Candida albicans
Line 146: The statement refers to data shown at 2A, not 2B.
Table 2 (last line,  last column): should be I-SceI not 1-SceI


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: RESTRICTION DIGEST SCREENING FACILITATES EFFICIENT DETECTION OF SITE-DIRECTED MUTATIONS INTRODUCED BY CRISPR IN C. ALBICANS UME6
Review round: 1
Reviewer: 3

Output:
Basic reporting: Figure are not relevant in light of the title. No figure shows any restriction digest, and one figure relates to the function of a gene, again not the topic of the title. I would suggest changing the title.

Otherwise, the basic reporting is fine.
Experimental design: No Comment.
Validity of the findings: Any conclusion about the importance of KK to the function of ume6 is premature without verification of expression by something like western blot. If the KKíEE mutation causes a loss of soluble expression, no conclusion about the functionality of amino acids is possible.

Introduction of EE mutation appears to introduce multiple PAM sites. Sequencing of many clones is needed to analyze the non-RE digested samples, that might have incorporated the mutations but later mutated the site again by Cas9 activity.
Additional comments: I don't really understand what type of scanning mutatgenesis library is proposed? I am not familiar with applications where two adjacent alanines are needed. A reference would help.

Can you add the CRISPR target site to your Figure 1A?

How does this work to make scanning libraries, as suggested?


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: GASTROINTESTINAL TRANSIT TIME AND HEART RATE VARIABILITY IN PATIENTS WITH MILD ACQUIRED BRAIN INJURY
Review round: 1
Reviewer: 1

Output:
Basic reporting: This is an interesting convenience sampling study that looked at the gastrointestinal transit time and heart rate variability in TBI patients.

In general, the sentences were good with only minimal grammar errors.

Abstract
- Good abstract with precise sentences.

Intro.
1) The statistics data used fo the ABI incidence was quite old data, nearly a decade. I am sure there is a newer data to support this study.
2) The research gap is well explained.
3) Good discussion and referring to other previous papers but maybe emphasize can also be made on how the study will impact the  medical and general community.
Experimental design: Methodology.
1) Good description of inclusion & exclusion criteria
2) However, for TBI, the classification used is quite broad (ie based on GCS at admission to ICU which sometimes is masked by some drugs). Do you have any other classification for usage of TBI classification (ie GCS on first clinic apppointment, Rancho Los Amigos cognitive scale etc?)
3) Any specific classification for Subarachnoid hemorrhage patients included in this study?
4) For exclusion criteria, do you rule out peripheral nerve problem ie pre-existing diabetic neuropathy which is unrelated to the central nervous system?
5) How “blinding” was performed in selecting control subjects?
6) For the neurogenic Bowel dysfunction scoring and FIM scoring, are you using the same person to ask the questionaires? How do you standardised this part of your study?
Validity of the findings: Discussion.
- Good discussion with good explanation on the findings.
- Limitation of the study was explained well.
- Interprets the findings of this study well and good comparison with previous studies in this topic
Additional comments: - This is a good paper, and introduce new finding in the brain injury rehabilitation field.


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

Input:
Title: GASTROINTESTINAL TRANSIT TIME AND HEART RATE VARIABILITY IN PATIENTS WITH MILD ACQUIRED BRAIN INJURY
Review round: 1
Reviewer: 2

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: I have reviewed the manuscript “Gastrointestinal transit time and heart rate variability in patients with acquired brain injury”. This is an interesting study evaluating the gastrointestinal transit time after brain injury. I have several queries as listed below.

1.	Methods: The authors did not mention whether the patients stopped any laxatives they were on at least 3 days before GITT. However we understand from Results Section that 12 (48%) of patients received laxatives. This is a pitfall.
2.	Methods: Please move Neurogenic bowel dysfunction score subtitle after GITT as this will be flowing with the results.
3.	Discussion: It may be better to discuss the GITT results first (to the 3rd paragraph), before discussing GITT-correlation.
4.	Discussion: Since some patients continued to use laxatives, this probably affected GITT results. May this be one reason for no correlation between HRV and GITT other that low statistical power?
5.	Discussion: Authors need to discuss more the correlation with other studies.
6.	Discussion: There are several limitations of this study that are already given in the Discussion section. Actually limitations are much widely given than other discussion.
7.	Conclusion: Authors need to state her that no correlation was detected between GITT and HRV.


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: GASTROINTESTINAL TRANSIT TIME AND HEART RATE VARIABILITY IN PATIENTS WITH MILD ACQUIRED BRAIN INJURY
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 responded my queries made changes on manuscript accordingly.


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: COMPARATIVE DESCRIPTION AND OSSIFICATION PATTERNS OF DENDROPSOPHUS LABIALIS (PETERS, 1863) AND SCINAX RUBER (LAURENTI, 1758) (ANURA: HYLIDAE)
Review round: 1
Reviewer: 1

Output:
Basic reporting: A enormous and important bibliographic references are absent. In this sense, data  about the condrochranium and ossification pattern in Scinaxinae could be found for:  Scinax acuminatus  (Fabrezi & Lavilla 1992; Alcalde et al., 2011); S. boulengeri (Vera Candioti 2007);  S. fuscovarius (Fabrezi & Vera 1997); S. granulatus (Alcalde & Rosset 2003);  S. nasicus (Fabrezi & Vera 1997; Vera Candioti et al. 2004; Vera Candioti 2007), S. squalirostris (Alcalde & Rosset 2003), S. skuki (Rodrigues et al., 2017); Julianus. aff. pinimus, and J. uruguayus (Alcalde et al. 2011); Ololygon aromothyella and O. berthae (Alcalde et al. 2011). In the same way, exist data about condrochranium  in Dendropsophinae (i.e. Dendropsophus nanus; Fabrezi & Lavilla 1992; Vera Candioti et al., 2004). Despite most of these references absent in the manuscript, being crucial to provide a proper discussion, the authors should perform an exhaustive reading of the bibliography available.
Experimental design: Please describe the methodology in more detail. for example:
“tadpoles of Dendropsophus labials (N= 32), and Scinax ruber (N=114), and one adult of each species were double stained for bone and cartilage following Dangerous and Uhler (1977). Voucher are stored in …….”
In addition in the entire manuscript the authors should perform an update in the taxonomy of Hylidae following Duerman et al., 2016 or use the taxonomy of Faivovich et al., 2005, but not a mixture of both.
Validity of the findings: The discussion section should be focused to compare related species. In this sense, S. ruber should be compared with the information available for Scinax species (i.e. Alcalde and Rosset, 2003; Alcalde et al., 2011), and inferences (with caution by the absence of data in most taxa) with julianus and other Scinaxinae (like Ololygon), in the framework of Hylidae. In Dendropsophus, the authors omitted references useful to make comparisons (see above) and the result obtained should be discussed considering the complete framework of Hylidae. The similitudes observed between between taxa not closely related: Dendropsophus labials (Dendropsophinae) and Scinax ruber (Scinaxinae) could be consequence of common origin, but also could be consequence of homoplasy with independent origin. So the absence of data not allow a clear comparison, and the authors should be caution with such issue.
Additional comments: Some other minors correction are provide to the authors:

Introduction Section
Line 36: deleted Frost, 2006. add Frost 2017 and/or Duellman et al., 2016
Line 52: delete Colombian. The fact that these taxa are distributed in Colombia is not really important for the study of the phylogenetic relationship of the group.
Line 36-38: review Hylidae taxonomy, now is considered Phyllomedusidae as a full family, so Hylidae (sensu Duellman et al., 2016) now is composed by 705 species (Frost, 2017).
Materials and methods Section
Line 45: add potential informative characters
Line 74: replace & by and
Line 240: Add Duellman et al., 2016
Line 244: replace Hyla by Boana,
Line 245: delete Scinax species:. Replace Scinax uruguayus and S. aff. pinima by the actual genera Julianus (J. uruguayus, J. aff. pinimus). Scinax aromothyella and Scinax berthae by Ololygon aromothyella and O. berthae.
Line 247: Gastrotheca riobambae is not a Hylid!, belong to Hemiphractidae. replace Hypsiboas pulchellus by Boana pulchella.
Discussion section:
Line 235-236: Is not true!. The 125 species of hylids frogs present in Colombia is less than half of the number of species assigned to Hylidae for Brazil.
Line 235-262: Is not a real discussion, only repeat the results obtained and none discussion is provided. Please re write these paragraph with a proper discussion (or delete or move the paragraph to introduction and results).
Line 288: Check this reference Hass, 1996.
Line 292: H. microcephala???? replace by D. nanus.

Tables and figures.
FIGURE 1. Could the authors provide photograph of the condrochranium?, as was performed in 1D?.  the drawings are really poorly detailed, and multiples structures could not be proper identified with those pictures (mainly in 1A, and 1B).

TABLE 4. Phyllomedusa valianti belongs to Phyllomedusidae not Hylidae!. Please check the table in order to update the taxonomy.


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: COMPARATIVE DESCRIPTION AND OSSIFICATION PATTERNS OF DENDROPSOPHUS LABIALIS (PETERS, 1863) AND SCINAX RUBER (LAURENTI, 1758) (ANURA: HYLIDAE)
Review round: 1
Reviewer: 2

Output:
Basic reporting: The work "Comparative description and ossification patterns of Dendropsophus labialis (Peters, 1863) and Scinax ruber (Laurenti, 1758)(Anura: Hylidae)" propose a comparative description of the skeletal development of two species of anurans, Scinax ruber and Dendropsophus labialis. Although it presents valuable unpublished data, in my opinion the work has problems of organization and presentation.
1) The authors should contextualize or justify why they study these species? What is the hypothesis? What is the importance / contribution of this work? That they are not studied species of anura from Colombia would not be enough for their publication in an international journal with a wide audience.
2) I suggest to present the results in a comparative way, and that the idea of the development of the described structures be clearly reflected:
Results:
The data of the tables 1 and 2 would be useful to see in a comparative way
-	The authors study the development of the skeletal system of two species of anurans, but in the description is not recorded in which stages / moments of development the structures, ossifications, etc., appear. This is a serious problem since the main objective of the work is to describe the development, and such as the results are presented, this is not reflected. In the section "ossification sequence" there is a summary on this subject, but it is not useful to transmit to the reader what are the elements (each one) that appear before or later, and what would be the difference between both species studied in this aspect.

3)	Discussion
Since the authors says: “The species of the family Hylidae show variation in the taxonomic arrangements, as it was showed by the variety of phylogenetic hypotheses based on molecular, chromosomal, and morphological data from both larvae and adults”. It would be very useful to organize your ms so that it is clear what would be the support that the development characters would potentially give to the composition of the phylogeny, taxonomy and evolution of the group.
There are other problems of closing the paragraphs and discussing the results with what was previously published that are marked in the pdf.
Experimental design: no comment
Validity of the findings: no comment
Additional comments: I recommend to re-organize yours data because that they are more profitable in multiple aspects


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: COMPARATIVE DESCRIPTION AND OSSIFICATION PATTERNS OF DENDROPSOPHUS LABIALIS (PETERS, 1863) AND SCINAX RUBER (LAURENTI, 1758) (ANURA: HYLIDAE)
Review round: 1
Reviewer: 3

Output:
Basic reporting: This manuscript provides a thorough description of the skeletal development of two hylid frog species. It is clearly organized, and the anatomical terminology is appropriate.
For anatomical terms that have alternative uses in the literature, references have been provided. The English could be improved. In addition to several typos and grammatical mistakes, there are some instances of awkward phrasing that make it a bit difficult to understand.

The first paragraph of the introduction does not seem to fit with the rest of the manuscript---the link between ossification sequence and potential phylogenetic characters is not well developed. A discussion about sequence variation or heterochrony might make a more compelling argument.

The figures and tables, in general, are appropriate for the type of study and the data have been presented in a way that allows for the observations to be repeated. I would like to see a full table of specimens examined (so that I can see how many specimens from each stage were examined). Some of this information can be gleaned from the table(s) and figures, but not easily. This is particularly important when discussing degree of chondrification---i.e., could a lack of stain really be a lack of cartilage or just one or two poorly stained specimens? Is it hard to tell if the number of specimens is not listed.

The figures could be much more detailed—for example, are the craniopalatine and carotid foramina in D. labialis visible? They aren’t shown in Figure 1.  You describe them in text but do not show them in the illustration.  Also, Figure 5. Really doesn’t provide much information and should probably be deleted (or given more treatment in text).

There are some papers listed in text that are not cited in the reference section (e.g., Shearman and Maglia, 2014)
Experimental design: This is a descriptive paper, so the experimental design is minimal. For the type of study, the methods and approach are appropriate.
It is not entirely clear to me why this paper was written as a comparison of the two species. If the main goal (as described in the introduction and conclusions) is to provide baseline information to be used later in comparisons of heterochrony within phylogenetic context and/or for phylogenetic character, why not just describe each species individually? What new information does comparing the two species provide? Do the developmental differences relate to adult morphological differences? Providing more justification for the comparative approach in the introduction would make the paper more interesting, and the significance and new knowledge gained by comparing them should be discussed in the conclusions.

The authors present a clearly defined research question, namely how does the skeletal development of these two species differ from one another. While in a larger context, the question is relevant and meaningful, the authors do not provide much in the way of a larger comparative context. That being said, publication of these data will enable other to use them for broader studies.
Validity of the findings: The anatomical descriptions seem straightforward and thorough without being redundant. The sample size for the D. labialis is very small, and it is not clear how much influence this might have over perceptions of interspecific variation. It would be good to address this.

Line 243: First paragraph of the discussion---this list is not fully consistent with the list in the introduction. You’ve listed papers in the intro that include chondrocranial descriptions, but then do not include them on this list.

Line 317: Regarding the ossification sequence, the first bones ossified in the cranium were the exoccipital, the frontoparietal and the parasphenoid at GS 36.  I’m a bit confused---this doesn’t seem consistent with the tables. In fact, in Table 2, shouldn’t exocippital be listed at GS38?

There is little in terms of conclusion beyond the fact that the data might be useful in future studies.  I think the paper might be more interesting if you used the conclusions to comment/speculate on why you think there might be differences among these two species. Just a thought.
Additional comments: In general, this paper provides a detailed anatomical comparison between two species, and the ossification sequence information may provide some additional data that could be useful for larger comparative studies conducted in the future (although a more robust sample of D. labialis would have been preferred).  The morphological descriptions are well done, but the illustrations could be a bit more detailed.


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: COMPARATIVE DESCRIPTION AND OSSIFICATION PATTERNS OF DENDROPSOPHUS LABIALIS (PETERS, 1863) AND SCINAX RUBER (LAURENTI, 1758) (ANURA: HYLIDAE)
Review round: 2
Reviewer: 1

Output:
Basic reporting: After reviewing the manuscript entitled "Comparative description and ossification patterns of Dendropsophus labialis (Peters, 1863) and Scinax ruber (Laurenti, 1758)(Anura: Hylidae)", I have observed that it has been considerably improved. However, many issues must be addressed in more detail. The list of changes proposed are detailed below.
Experimental design: No comment
Validity of the findings: The validity of the data obtained is of great importance for the study of anuran development patterns. However, these data are not used in the best way, giving a poor discussion regarding the available literature about development.
Additional comments: ABSTRACT
Delete: of neotropical frogs (the lack of data of ossification pattern is common in all anurans groups, being not restricted to the neotropical region).
Is not clear if the differences described are between both taxa studied, between related species or in fact correspond to intraspecific differences. Please re-write the sentence.
INTRODUCTION
LINE 28: Add reference
LINE 31: Add reference
Line 34-40: i really don’t understand this paragraph. The authors propose that the morphology in the hylids has contributed to the group's systematics because they are many species?. I suggest delete the paragraph or re-write these.
Line 47-51: Please include in each species with ossification data the specific name with which it was described. For example: Boana pulchella (as Hypsiboas pulchellus, Hoyos et al., 2012).

MATHERIALS AND METHODS
LINE 64: replace “These specimens belong to the” by “Specimens examined are stored at”.

RESULTS
LINE 101: replace “in the two species” by “both studies species”.

DISCUSSION
The discussion section is not properly organized, still multiple paragraphs are only repetition of the results obtained and the comparison and discussion with the data available in the literature is poor, and need more work.

LINE 251: Add de Sá, 1988
LINE 251-252: delete Alcalde and Rosset 2003 from D. nanus reference.
LINE 253: delete “species: J.”
LINE 255: delete “Ololygon species”
LINE 257: delete “Scinax species”
LINE 262-271: are results, nothing is discussed…delete.
LINE 265-277: discuss the data obtained in relation to the data available in literature.
LINE 275-279: Move this paragraph to the end of the discussion section, is a conclusion more than a discussion.
LINE 283: replace Hyla by Boana
LINE 284: compare first the palatoquadrado between D. labialis and the others Dendropsophus with data available (i.e. D. nanus), the same structure of S. ruber with the others Scinax species, and later could perform some inference in the Hylidae context.
LINE 299: But Alcalde and Blotto (2006) report this characteristic in Limnomedusa macroglossa, is not an hylid frogs, so the differences could not be explained so lightly.
Line 318: add while in Boana raniceps….
Line 327-330: multiples hylids frogs (specially studied in Scinax by Faivovich, 2005) present a suprascapular process, review the literature.
Line 339: detailed the species transferred to Dendropsophus adding the proper reference.
Line 343: replace “species of Hylidae” by hylids.
Line 351: the taxa assigned to Litoria nannotis by Hass (2003) belongs to Ranoidea (Pelodryadidae). Again the differences or similitudes founded could not be homologues.

FIGURES
Despite the explanation given by the authors, I still consider that chondrocranium drawing is not the best technique to observe the anatomical characteristic of this structure. As the drawings were not updated, I leave the decision on the images to consideration of the editorial in chief.


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: COMPARATIVE DESCRIPTION AND OSSIFICATION PATTERNS OF DENDROPSOPHUS LABIALIS (PETERS, 1863) AND SCINAX RUBER (LAURENTI, 1758) (ANURA: HYLIDAE)
Review round: 2
Reviewer: 2

Output:
Basic reporting: improved from previous review; see editorial suggestions in attached document
Experimental design: improved from last version
Validity of the findings: appropriate, although some of the discussion paragraphs are a bit confusing and I was not really sure what points they were trying to make (see in-text comments).
Additional comments: In general, the manuscript is much improved. I have made editorial and content suggestions in the attachment.


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: AN INEXPENSIVE, CUSTOMIZABLE MICROSCOPY SYSTEM FOR THE AUTOMATED QUANTIFICATION AND CHARACTERIZATION OF MULTIPLE ADHERENT CELL TYPES
Review round: 1
Reviewer: 1

Output:
Basic reporting: This paper is well written and easy to read. I have some minor points that I would like to see corrected or addressed.

Minor Points:

-The acronym MTT isn’t defined (line 52).

-Please make the axes label font size larger in Figure 3 and 7

-I’d remove a few of the stronger modifier adjectives (see “extraordinary” line 234, “incredible” line 310, “remarkable” line 384/409/411)

-I’m not sure what you mean by binary counts on line 409, how does that contribute to the system resolution? You might just want to get rid of that sentence.
Experimental design: The overall design of testing the accuracy of the imaging/segmentation based on serial dilutions is sound. Adding in the option to deal with co-culture situations with secondary stains is a logical extension. I see no significant problems with the experimental design.

The research niche the pipeline fills is correctly identified and appears to be a relevant problem in the sub-field of cell counting in difficult cell types.
Validity of the findings: Overall, I think the paper supports the claims of the software’s performance adequately. I have a few clarifying points that should be fixed.

I don’t think Figure S1 accurately demonstrates whether the automated nuclei segmentation is completely working at very high confluency levels. In order to assess the quality of the segmentation, an overlay of the segmented nuclei and the original image would need to be produced. I do agree with the authors that even on this difficult segmentation task that most nuclei are identified. I would change the wording on line 228 or attempt to measure the accuracy of the confluent segmentations with manual counting.

I’m finding Figure 4 to be a bit confusing. Did the dilution series start at 100,000 or 200,000 cells/well? If the dilution started at 100,000, the first reading at Dilution Series value 1 of about 180,000 is far from the expected count of 100,000. I’m also having trouble visualizing how confluent the wells are at these various dilution series, can you add sample DAPI and segmentation images for some of the points along the series?

Can you also report the slope of the best fit line in Figure 5?
Additional comments: Have you tested your pipelines with CellProfiler 3.0.0 on Mac? I was able to load both pipelines, but received this error when loading S1:

Error while loading SaveImages: Unsupported image type: Objects. Use <i>ConvertObjectsToImage</i> to create an image.
Do you want to stop processing?

And this error when loading S2:

Error while loading ClassifyPixels: Could not find the ClassifyPixels module
Do you want to stop processing?

It looks like ClassifyPixels is provided by Ilastik, which is supposed to be provided by CellProfiler, is it not packaged in CellProfiler 3.0.0? Does Ilastik need to be installed as well on Mac? I’m not sure if either of these errors are show-stoppers, but I thought you might want to know about them.


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

Input:
Title: AN INEXPENSIVE, CUSTOMIZABLE MICROSCOPY SYSTEM FOR THE AUTOMATED QUANTIFICATION AND CHARACTERIZATION OF MULTIPLE ADHERENT CELL TYPES
Review round: 1
Reviewer: 2

Output:
Basic reporting: In general, this technique article complies with all standards envisioned for basic reporting of the journal but needs to be supplemented with some more literature data analysis to outline originality of the work. As for the figures, the axes of some graphs are finely labeled, so enlargement is required.
Experimental design: All experiments are well designed and performed in compliance with the journal’s requirements, but some issues may need to be addressed (see general comments) to further consider the validity of the findings.
Validity of the findings: Validity of the findings is supported by series of replicate experiments. The data are generally robust, statistically sound, and controlled.
Additional comments: In the manuscript entitled “A customizable microscopy system for the automated quantification and characterization of multiple adherent cell types: an alternative to flow cytometry” (#24707) the authors propose an inexpensive microscopy-based automatic system for counting cells with chosen parameters in adherent mono- or co-cultures. In general, ideology of the work is well understood, and the results of are quite interesting and well described. However, the manuscript needs some revision including improvements in editing.

The authors reasonably state that trypsinization or scraping of some types of cells is not suitable for flow cytometric measurements since such treatments may alter viability of cells and some other important cell properties. It should also be stated that in suspension some cells can stick each other thus forming aggregates, a serious disadvantage in flow cytometry- or Coulter-based absolute cell counting. Indeed, at first sight the microscopy-based image analysis technique for counting of adherent cells seems superior. However, this technique may not be ideal either particularly if cells attach to the inner surface of walls of the culture plates or multiwell plates. Usually, in small volume cell cultures using standard multiwell plates  (≥ 24 wells), ratios of heights of culture media to diameters of wells are considerably higher than those in large volume cell cultures, assuming that more cells can probably attach to the walls. Obviously, without these cells the absolute cell counting per well would not be complete. This issue needs to be addressed. Otherwise, cell density values (cells/mm2) should be used instead of absolute cell counts to monitor effects.

It is unclear whether similar techniques have been applied by other researchers if standard fluorescent microscope, motorized x-y-z stage, and image analysis software are only needed for cell counting. It should be stated in Introduction. Fluorescence image analysis technique designed for cell quantification in co-cultures was proposed a while ago by Krtolica A. et al. Cytometry 49:73-82 (2002). This work should be referred and compared with the current work in terms of delineating the novelty and possible advantages.

By the way, there are multi-color laser scanning cytometers (Pozarowski P. et al. Methods Mol Biol 931:187-212 (2013)) equipped with a computer controlled stage and autofocus. Although these commercial instruments are not cheap, are they principally different from the current one in terms of image collection, processing and analysis? Notably, in addition to cell quantification, laser scanning cytometry (LSC) like flow cytometry is capable of acquiring cell information sufficient to generate histograms (e.g., DNA content histograms, etc.). More demonstrative and illustrative information on LSC can be found in http://research.uthscsa.edu/facs/PDF%20Files/BCI_Webinar_Telford_2010.pdf

There is also another technique published by Spink B.C. et al. Cell Biol Intl 30:227-238 (2006). They proposed to measure cell numbers in black 96-well microtiter plates with flat and clear bottoms using a fluorescence microplate analyzer. This work should be taken into account and referred as well.

Line 72-73: Intercellular physical contact was shown to be also important for the induction of proliferative responses of bystander cells co-cultured with irradiated cells (Gerashchenko B.I. and Howell R.W. Cytometry Part A 56A:71-80 (2003) can be added to the references).

The term “optical cell cytometer” throughout the text is suggested to be replaced with the term like “microscopy-based cytometer” since in the former term (a) cytometry already implies cell measurements; (b) flow cytometry is an optical technique as well.

In the title of the article the phrase “… an alternative to flow cytometry” is not necessarily.
Can the title sound like “An inexpensive customizable microscopy system for the automated quantification and characterization of multiple adherent cell types”?

The judgment of killing of tumor cells with 51Cr together with intention to recreate this experiment using fluorescent tracers should be moved from Results to Discussion.

How much time does it take to obtain the final results from a single well of 24- or 48-well plate since starting collection of images?
-------------------------------------
Specific comments:

The word “unique” or “uniquely” is often used throughout the text.

Line 50: In the phrase “unique limitations” the word “unique” can probably be replaced by the word “apparent” or “obvious”.

Line 51: Can “metabolic” be replaced with “physiologic”?

Lines 52-53: Abbreviation MTT should be uncovered. This assay along with alamarBlue assay should be briefly described.

In Fig. 1, 6-well plates are demonstrated, but experiments were conducted in 24- and 48-well plates.

In the legend of Fig. 2 it is unclear what the type of cells is used.

Fig. 3: Is the average number of cells per image estimated in 24-h cultures? If so, it should be mentioned in the legend.

Fig. 4: Is the total number of cells per is estimated in 24-h cultures? If so, it should be mentioned in the legend.

Lines 286-288: This statement should be placed in Discussion.

Statistical analysis section should be added to Materials and Methods. Do deviations represent SD or SEM of replicate experiments? In Fig. 7, indication of statistical significance (where appropriate) may be helpful to dissect the effect.


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: AN INEXPENSIVE, CUSTOMIZABLE MICROSCOPY SYSTEM FOR THE AUTOMATED QUANTIFICATION AND CHARACTERIZATION OF MULTIPLE ADHERENT CELL TYPES
Review round: 2
Reviewer: 1

Output:
Basic reporting: The paper is well written and the minor language modifications have improved the text. I see no problems with in this area.
Experimental design: I see no major problems remaining with the experimental design.
Validity of the findings: My previous concerns have been addressed, I see no problems in this area.
Additional comments: I think you should add a short note about the analysis pipeline requiring CellProfiler 2.2.0. I apologize if I missed this addition in your revisions, but a quick search didn’t yield any changes in this respect. I see the largest impact of your paper coming from people looking to use the provided analysis pipeline, but current users of CellProfiler are likely to try to load your pipeline in CellProfiler 3.0.0, see the same errors I did and move on to some other method. A section before “Explanation of CellProfiler Codes and Associated Functions” should fulfill this need.


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: AN INEXPENSIVE, CUSTOMIZABLE MICROSCOPY SYSTEM FOR THE AUTOMATED QUANTIFICATION AND CHARACTERIZATION OF MULTIPLE ADHERENT CELL TYPES
Review round: 2
Reviewer: 2

Output:
Basic reporting: In general, this technique article complies with all standards envisioned for basic reporting of the journal and now it is supplemented with some more additional literature data. As for the figures, the plots in Fig. 3 are too small, so some enlargement is required.
Experimental design: All experiments are well designed and performed in compliance with the journal’s requirements, however, an issue may need to be addressed (see general comments) to further consider the validity of the findings.
Validity of the findings: Validity of the findings is supported by series of replicate experiments. The data are generally robust, statistically sound, and controlled. The conclusions are appropriately stated and connected to the original question investigated. Also, they are limited to those supported by the results.
Additional comments: There are some improvements in the revised manuscript. However, it still unclear whether this technique is capable of performing precise absolute cell counts per well. In previous commentaries, the possibility was mentioned that some cells may attach to the inner surface of the well’s wall, which is perpendicularly positioned to the well’s bottom (this is not about well’s edge). If they do attach, without them the absolute cell counting per well would not be complete, however. How to clarify this situation? I would probably suggest conducting a simple experiment as follows. Plate an equal numbers of cells into 2 separate wells and culture them for 24 h. Then quantify cells in the entire well by microscopy-based cytometry and compare cell counts with those of another well in which cells were trypsinized and collected for hemocytometry (an old and quite reliable technique). I hope that there will not be any significant differences in cell counts, but this has to be proven. The results of this experiment can be presented as a supplementary material.


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: THE MITOCHONDRIAL GENOMES OF TWO WALNUT PESTS, GASTROLINA DEPRESSA DEPRESSA AND G. DEPRESSA THORACICA (COLEOPTERA: CHRYSOMELIDAE), AND PHYLOGENETIC ANALYSES
Review round: 1
Reviewer: 1

Output:
Basic reporting: I thought the paper is meet the Peerj published standards. The English is clear and can be understand. Two mt genomes of Coleoptera were sequenced and compared.
But the paper need some revisions.
I thought the all sequenced download from GenBan should be referenced. So the authors should added the references of 36 mt genomes of Coleoptera from GenBank. The other literature references is no problem.
The figs 6 and 7 should be added all bootstrap and BPP number above the branch not only one value.
Experimental design: The experimental desing and analysis method is no problem.
If the authors can obtain the complete mt genomes of Gastrolina depressa depressa, the paper can deeply discuss the difference within two subspecies. I just find one pair of primers you designed in Table 1. If you failed to sequence the AT region you can sequence this region by colon method. I also suggest the authors compare the genetic divergence in all genes and whole genomes, which can give us the more different informations between two subspecies.
Validity of the findings: Two mt genomes were sequenced.
Some different gene charachters were found in two mt genomes.
Conclusion is well.
Additional comments: Two mt genomes of Coleoptera were sequenced and compared in the paper. The paper is  meet the Peerj published standards. The English is clear and can be understand.
1. But the paper need some revisions. I thought the all sequenced download from GenBan should be referenced. So the authors should added the references of 36 mt genomes of Coleoptera from GenBank. The other literature references is no problem. The figs 6 and 7 should be added all bootstrap and BPP number above the branch not only one value.
2. If the authors can obtain the complete mt genomes of Gastrolina depressa depressa, the paper can deeply discuss the difference within two subspecies. I just find one pair of primers you designed in Table. If you failed to sequence the AT region you can sequence this region by colon method or design the other primers. I also suggest the authors compare the genetic divergence in all genes and whole genomes, which can give us the more different informations between two subspecies.

Other minor revsions is revised in PDF. Please see the PDF.


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: THE MITOCHONDRIAL GENOMES OF TWO WALNUT PESTS, GASTROLINA DEPRESSA DEPRESSA AND G. DEPRESSA THORACICA (COLEOPTERA: CHRYSOMELIDAE), AND PHYLOGENETIC ANALYSES
Review round: 1
Reviewer: 2

Output:
Basic reporting: The English writing is generally professional, only with some minor errors in a few  word ore sentence expression. All the errors was listed or labelled in the revised manuscript with detail and the authors was suggested to checked them carefully and correct them.
Experimental design: no comment.
Validity of the findings: no comment.
Additional comments: 1. The studies firstly reported the mitochondrial genomes of two walnut pests,Gastrolina depressa depressa and G. depressa thoracica (Coleoptera: Chrysomelidae), and conducted the relevant phylogeny within Chrysomeloidea. This report is valuable for enriching the mitogenomic data of beetles and related future colepteran phylogenetic studies.
2. Some minor errors of word and sentence expressions in the English writing were all listed or labelled in the revised manuscript, and the authors was suggested to check and correct them carefully.
3. The two phylogenetic trees shown in the Figure 6 and Figure 7 of the manuscirpt were suggested to be incoporated into one consesus tree and shown in one figure.


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: THE MITOCHONDRIAL GENOMES OF TWO WALNUT PESTS, GASTROLINA DEPRESSA DEPRESSA AND G. DEPRESSA THORACICA (COLEOPTERA: CHRYSOMELIDAE), AND PHYLOGENETIC ANALYSES
Review round: 1
Reviewer: 3

Output:
Basic reporting: The mitochondrial genomes of two walnut pests, Gastrolina depressa depressa and G. depressa thoracica (Coleoptera: Chrysomelidae), and phylogenetics within Chrysomeloidea (#21999) by Qiqi Wang, and Guanghui Tang

The authors describe the mitochondrial genomes of two walnut leaf pests, Gastrolina depressa depressa and Gastrolina depressa thoracicia, illustrating similarities in gene content and gene arrangement across these two insects, as well as conserved elements found in other coleopteran taxa.  In addition, they use nucleotide and amino acid sequences from the 13 protein-encoding genes from these two taxa, along with 34 other species within the Chrysomeloidea (and outgroups), to infer the pattern of relationships within this large superfamily of beetles using ML and Bayesian analyses.  Their phylogenetic analyses provided consistent support for the monophyly of Eumolpinae + Cryptocephalinae + Cassidinae, but little resolution for basal relationships within the Chrysomeloidea.

This study is a little unusual in that two central taxa at the focus of the study are presented as subspecies of a single species Gastrolina depressa.  If the authors were interested in generating two new mtDNA genomes, in part to help better understand mt genome dynamics and phylogenetic relationships within the Chrysomeloidea, why choose two subspecies?  This fact alone made the central rationale for this study seem a little tenuous.  Despite their subspecies designation, however, the authors present these two taxa as if they were separate species, noting differences in morphology, and referring to past work on their systematics, occurrence and spatial distribution.  Yet they never comment on the species vs. subspecies status specifically.  This is odd, particularly when Ge et al. 2003 indicate that these two subspecies should be considered two separate species: Gastrolina depressa and Gastrolina thoracica.  A discussion of this paper would seem to be important for this study. Why was this recommendation not followed?

In addition, the authors also claim on lines 71/72 that this study was undertaken to understand the taxonomic status of these two insects, and the phylogenetic relationships within the “Chrysomeloidae” (which I assume is a typo – it should be Chrysomeloidea or Chrysomelidae).  Yet, in the Results/Discussion, the authors never address how their data have any bearing on the taxonomic status of Gastrolina depressa depressa and Gastrolina depressa thoracica, other than that they group together in their phylogenetic analysis.  Hence, this study appears to be somewhat muddled in terms of its focus, and the choice of the focal species used.  A stronger introduction to the species and their current taxonomic status would have helped to resolve some of these issues.

The conclusions that can be drawn from their phylogenetic analyses based on the 13 protein-encoding genes of 36 taxa are also somewhat limited given the superficial coverage given to their results and discussion. There also appear to be some important outcomes of their analyses that are not discussed at all, but need to be.  For instance, they state on line 209-211 that Galerucinae + Chrysomelinae were monophyletic (albeit only in the tree derived from the nucleotide dataset), but don’t mention that the difference in analyses is because Paleosepharia falls outside the Chrysomeloidea clade entirely and groups with the outgroup taxon in the amino acid dataset.  Surely some explanation is warranted here.  Likewise, there is a focus on the monophyly of Galerucinae + Chrysomelinae (despite the weird results of the amino acid dataset) but no mention of the fact that neither analysis shows either subfamily as monophyletic.  Despite this, latter on in the Conclusions (line 224-226), Galerucinae + Chrysomelinae are referred to as having high nodal support as sister taxa.

There are other issues too, including recent (2016, 2017) references that would appear to be relevant to this study that were not cited in this paper, including:
•	Ming-Long Yuan, Qi-Lin Zhang, Li Zhang, Zhong-Long Guo, Yong-Jian Liu, Yu-Ying Shen, Renfu Shao. 2016. High-level phylogeny of the Coleoptera inferred with mitochondrial genome sequences. Molecular Phylogenetics and Evolution 104: 99-111, ISSN 1055-7903, https://doi.org/10.1016/j.ympev.2016.08.002.
•	Hongli Zhang, Nian Liu, Zhiping Han, Jianxia Liu. 2016.  Phylogenetic analyses and evolutionary timescale of Coleoptera based on mitochondrial sequence. Biochemical Systematics and Ecology 66: 229-238. ISSN 0305-1978, https://doi.org/10.1016/j.bse.2016.04.014.
•	Haddad, S. and D. McKenna.  2016. Phylogeny and evolution of the superfamily Chrysomeloidea (Coleoptera: Cucujiformia). Systematic Entomology 41: 697–716 DOI: 10.1111/syen.12179
•	Nan Song, Xinming Yin, Xincheng Zhao, Junhua Chen & Jian Yin.  2017. Reconstruction of mitogenomes by NGS and phylogenetic implications for leaf beetles. Mitochondrial DNA Part A. [Epub ahead of print].
The most recent of these may not have been seen by the authors because it is a 2017 publication, only currently available as an epub.  Nevertheless, it is clearly overlapping with the present study in terms of focus on mtDNA genomes and chrysomelids.

In addition, this study is also undermined by grammatical problems and spelling mistakes, which can lead to challenges in interpretation in several places. Although this is understandable if the authors’ first language is not English, these errors must be corrected before publication.

In terms of figures:
Figure 1: The region that was not sequenced should be clearly noted for G. depressa depressa.  It was also unclear to me why the genome size could not be estimated for this species based on the size of amplification products.  If this was a sequencing issue (lines 136 – 137), then amplification products could be used to get an estimate of the total genome length.

Figures 2 and 3 could be included as supplementary information.

Figures 6 and 7.  It is unclear where the phylogram came from that was used to map the BP and PP values.  Is this from the ML or Bayesian analysis?  Why are different rules used for the two trees in terms of BBP and PP reporting?  For instance, PP>60 and BBP>0.7 are reported in Figure 6, and PP>75 and BBP>0.6 reported in Figure 7.  What are the standard metrics for reporting PP and BBPs?
Experimental design: As mentioned above, the central rationale for this study is unclear, in part because the authors undertook a detailed study of the mtDNA genomes of two closely related taxa in one genus (Gastrolina), and then attempted to use these genomes to better understand phylogenetic relationships within the diverse superfamily Chrysomeloidea.  In doing so, they have given short shrift to the (apparent) initial focus of this study – the taxonomic status of of two walnut leaf pests, Gastrolina depressa depressa and Gastrolina depressa thoracicia – and an extremely superficial overview of their analysis/results pertaining to phylogenetic relationships within the family. Similarities/differences between the analyses based on nucleotide vs amino acid datasets are poorly summarized, and glossed over very quickly.  Odd results such as that pertaining to the placement of Paleosepharia in the amino acid analysis are not addressed.

On lines 92-93, the authors report that the PCR products were 1.1 to 1.4kb in length, but then provide a list of primer pairs in Table 1 that are associated with amplification products ranging in size from 1.1 – 4.1 kb. How can this be so?

It would have been interesting to know too, why the authors didn’t elect to try longer amplifications using LA-PCR protocols, and attempt to size the mtDNA genome of Gastrolina depressa depressa even though they couldn’t sequence through the AT-rich region.

Giving the size of the partial genome in the Abstract (for comparison to G.depressa thoracica) isn't very meaningful without some explanation as to why this was not completely sequenced, and that the sequenced region included all the standard 37 genes.

The presentation of a Results/Discussion section as a combined unit rather than as separate entities obscures the fact somewhat that this study really lacks a comprehensive  discussion of the results, including an attempt to interpret the relevance of the results to the taxonomic status of the two focal taxa (an objective of the study), or the higher level phylogenetic relationships within the Chrysomeloidea (a second objective).
Validity of the findings: The presentation and reporting of the two mitochondrial genomes appears to have been done well, with the standard features reported and unique and conserved elements of these genomes highlighted, with a limited comparison to some other coleopteran mtgenomes.  The authors purport to have provided a detailed comparative investigation into the composition of mitogenomes for Chrysomelinae (line 70-71).  I’m not sure that I saw this here, but certainly some comparisons were made to other taxa.

Bigger concerns associated with this study relate to the phylogenetic analyses presented in Figure 6 & 7, which are reported and discussed somewhat superficially, with minimal attempts to discuss strange outcomes, and with errors in reporting mentioned above.  The statement on line 205 – “Regardless sequencing or database errors in some taxa” also lead one to question the quality of some of the data included in this study, and really requires the authors to qualify what they mean by this statement, and to ensure that they winnowed out data that were suspect prior to undertaking their analyses.

Also, frequent issues with typographical and grammatical errors lead to difficulties in understanding what the authors are trying to say in places, as well as in interpreting the data.  These should be resolvable, however, with editorial assistance.
Additional comments: I felt that this paper would be a stronger contribution, as written, without the phylogenetic component.  This seems to have distracted the authors from providing more of a focus on the mitochondrial genomes of two focal species, particularly with respect to providing a more comprehensive comparison of the structure and composition of mtDNA genomes across the Chrysomeloidea.  If the authors want to include the phylogenetic component, then I think that it is important to interpret their results more fully, explore the odd placement of taxa (as noted above), and incorporate more of the recent literature into their 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: THE MITOCHONDRIAL GENOMES OF TWO WALNUT PESTS, GASTROLINA DEPRESSA DEPRESSA AND G. DEPRESSA THORACICA (COLEOPTERA: CHRYSOMELIDAE), AND PHYLOGENETIC ANALYSES
Review round: 2
Reviewer: 1

Output:
Basic reporting: One minor suggestion is that those different topology of nodals in Figure 6 can be used as 0.54/- ..... not just 0.54, which maybe give reader clear to understand.
Experimental design: The experimental designing and analysis method is no problem.
Validity of the findings: The data is robust and conclusion are well stated.
Additional comments: I thought the paper is meet the Peerj published standards now. It can be accepted.


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: THE MITOCHONDRIAL GENOMES OF TWO WALNUT PESTS, GASTROLINA DEPRESSA DEPRESSA AND G. DEPRESSA THORACICA (COLEOPTERA: CHRYSOMELIDAE), AND PHYLOGENETIC ANALYSES
Review round: 2
Reviewer: 2

Output:
Basic reporting: I appreciate the fact that the authors have put considerable effort into responding to criticisms of the reviewers, but I still find the manuscript unacceptable for publication in its current state.

The writing still needs to be improved substantially. There are sentences, even in the Abstract, that are difficult to interpret. I am sympathetic to the challenges the authors are facing here, but it is in the authors’ best interests to ensure that their writing is clear and understandable.  I would encourage the authors to seek editorial support from a native English speaker -  some of the same problems in version 1 of the manuscript remain in this version. Spelling mistakes also abound - e.g. Celeoptera (L. 42)and Chrysommeloidea (L49) - which suggest a lack of attention to detail.

The authors continue to state that their results will help to understand the taxonomic status of these two insect taxa, but then once they have their results, shy away from trying to comment on this other than to say that their data are insufficient to do so.  This still is problematic for me.  Perhaps a solution to this is to avoid making this statement entirely, and place this study firmly within the context of understanding where their two taxa fit within the Chrysomeloidea.
Experimental design: In my last review, I criticized the paper because the central rationale for this study seemed a little tenuous – because the authors claimed to be using mtDNA genomes of two subspecies to “help to examine the detailed classification status of both insects”  but yet never addressed  what their results had to say about the relationship of these two taxa.  This seemed odd, particularly when Ge et al. 2003 indicated that these two subspecies should be considered two separate species: Gastrolina depressa and Gastrolina thoracica.  In the current revision the authors do address this.  They state that “the existing molecular data especially mitogenomes of species in Chrysomelinae, are not sufficient enough to detailedly classified the two newly sequenced insects” or variations thereof throughout the manuscript.  I am not sure what they mean by this or exactly what data they feel that they would need to make this sort of determination.  Barcoding approaches using COI gene regions are routinely used as coarse metrics of species-level status within animal taxa, as so could be referred to if nothing else, and presumably there are lots of associated studies to compare their genetic divergences to.  If they don’t feel that they have enough data to lend their support to species or subspecies status, what data do they need?

In the abstract the authors draw attention to several results, two of which I am puzzled by.  They give a fair amount of space to the presentation of repetitive elements in three regions of the genome, but fail to demonstrate why this is important or significant.  Secondly, they draw attention to the fact that G. depressa thoracica and G. depressa depressa were found to be strongly supported as sister taxa in their analyses.  This seems hardly a surprise unless the classification of these two species/subspecies was really off.  These were the only two members of the genus examined in their phylogenetic analysis, and two of only three species in the subfamily Chrysomelinae included in their analyses.  Hence, I wonder if there aren’t other aspects of their results which are worth highlighting in the Abstract instead of these two points above.
Validity of the findings: The authors have done a lot of work to generate and annotate the mtDNA genomes of their two focal taxa, and to undertake the phylogenetic analyses to place these taxa within a broader phylogenetic setting.  My major challenge as a reviewer of this manuscript is with the context of this study.  I don’t think that there is a good match with what the study proposes to do, and what it actually does.  As explained above, this needs to be addressed by either changing the overarching goals, or trying to examine the data so that some commentary can be made towards satisfying those objectives.  A second issue is with the writing. As good as the writing is considering that the authors are (I assume) not native English speakers, improvements are necessary to  ensure that the meaning of the authors is fully understood by the reader.  I think that this can be resolved by having the manuscript read/revised by a native English speaker.
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: SHIFT IN VEGFA ISOFORM BALANCE TOWARDS MORE ANGIOGENIC VARIANTS IS ASSOCIATED WITH TUMOR STAGE AND DIFFERENTIATION OF HUMAN HEPATOCELLULAR CARCINOMA
Review round: 1
Reviewer: 1

Output:
Basic reporting: Article has clear structure, provided with literature references, conclusions are supported with the data.
Most of the raw data shared with the exception of clinicopathological data for each patient - instead, authors summarize those feature in Table 1.
Experimental design: Research questions are clearly defined: authors sought to analyze distribution of different isoforms of VEGFA in HCC and adjacent non-tumor tissues. Elaborated methods were used to quantify the transcripts, those methods are described with sufficient details.
Validity of the findings: Findings are explained and supported by the data. Statistical analysis could have more details (see comments below).
Additional comments: I only have certain minor comments regarding the presentation of the results and methods:
1) Description of primers in Table S1: it may be a bit confusing for the reader that the same primers are shown under different names (for example - VEGFA-total, VEGFA-intron5, VEGFA-189, VEGFA-165, VEGFA-121- is all the same primer).
2) Figure 1A - dashed lines continuing exon 8b might imply presence of other exons which is not the case
3) Figure S1 - for easier understanding, might be useful to mark the heteroduplex band as well
4) Figure 2B - on the left plot, maybe worth showing VEGFA-xxxb distribution for both tissues (like on the right plot)
5) For Table S2, authors should give better explanation for what exactly is shown in the "Unique sequence region" of the table. The sequence provided there is shorter than the length indicated in "PCR product" column (for example, for the row marked "VEGFA-iso (523 bp)" the sequence is 228 bp only, etc.) - I guess it's probably limitation of sequencing - but this should be explained
6) Methods and data of Table 2 should be better explained. For example, what measure was used for the "change" - fold change, log2, etc? Which variables were used as continuous, categorical, or discreet? As authors show Spearman's correlation, I assume all the data was ranked - but this should be explained explicitly.
7) To visualize correlation with clinicopathological properties, it could be helpful to add corresponding color bands on the heatmaps (Fig. 2D, Fig. 3C)
8) Right barplots at Fig. 3B might be confusing (one would assume to sum up values within one bar which is not the case). As one alternative, authors could use a scatterplot instead: each case would be represented by a dot with each axis corresponding to a tissue type.
9) Authors performed valuable work to determine absolute number of copies for each transcript. It would be interesting to estimate approximate number of copies per cell or at least per mg of tissue: this would give reference potentially helpful for future researchers.
10) In contrast to literature data about other tissues, authors did not find significant VEGFA-xxxb expression in both normal and tumor liver tissues - it's probably worth to mention in the abstract.


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: SHIFT IN VEGFA ISOFORM BALANCE TOWARDS MORE ANGIOGENIC VARIANTS IS ASSOCIATED WITH TUMOR STAGE AND DIFFERENTIATION OF HUMAN HEPATOCELLULAR CARCINOMA
Review round: 1
Reviewer: 2

Output:
Basic reporting: The manuscript "Shift in VEGFA isoform balance towards more angiogenic variants is associated with tumor stage and differentiation of human hepatocellular carcinoma" is well written in plain and professional English. It provides a short, but sufficient overview of the background, with relevant references.
The figures are of sufficient quality, mostly quantification of the raw data (which is also provided).
The hypothesis is clearly formulated and addresses an important issue of different roles of splice variants in regulation of VEGFA role in HC progression. In addressing this issue, clear questions are answered with robust results.
Experimental design: While the semi-quantitative and quantitative analysis of the VEGFA isoforms is well done, the analysis is limited to the RNA level. This is problematic for 2 reasons:
1) There is no corroboration of the data with independent methods. This may result in biasing due to the chosen methodology.
2) It is well known that VEGFA in particular is regulated very extensively at all levels of expression: splicing, transcription AND translation (for review see Nucleic Acids Res. 2013 Sep; 41(17): 7997–8010). Moreover, VEGFA is active at a protein level rather than at the mRNA level.
Therefore, I suggest the authors confirm the results they obtained with a simple semi-quantitative Western blot analyis. They may use total VEGFA antibodies and distinguish isoforms with SDS-PAGE electrophoresis by size, which is a trivial task. It is important to confirm close correlation between the qPCR and the Western blot data, as well as the correlation with the tumor characteristics (see below).
Otherwise, the obtained results are quantified and analyzed well.
Validity of the findings: The most important finding of the article is shown in table 2. It gives validity to the data obtained, confirms the original hypothesis and even provides some predictability power to the obtained results on VEGFA isoforms.
It is important to notice, however, that the best obtained correlation coefficients and the p-values are not very strong. They are within the 0.05 standard (for the p-values), but in all but one cases higher than 0.01, for instance. This is especially critical because the authors are analyzing their data for correlation against 16 tumor sample characteristics, so a possibility of finding a low statistically significant correlation by chance is high.
The data can be strengthened if the authors, again, provide a similar analysis using quantification on a protein level. This would be an independent analysis, which, together with the already produced data, could make it more robust and biologically relevant.
Additional comments: In general, I like the article for its logical structure, sound data and clinical relevance. However, in my opinion the data needs to be corroborated with an independent method and on another level of gene expression. Therefore, I suggest to use A Western blot analysis to semi-quantitatively analyze VEGFA isoforms and correlate with the HCC properties.


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: SHIFT IN VEGFA ISOFORM BALANCE TOWARDS MORE ANGIOGENIC VARIANTS IS ASSOCIATED WITH TUMOR STAGE AND DIFFERENTIATION OF HUMAN HEPATOCELLULAR CARCINOMA
Review round: 1
Reviewer: 3

Output:
Basic reporting: The language is clear and professional throughout the manuscript, litterature references of relevance to the topic are provided, and the background information presented is sufficient to provide context. The structures is professional and the article is self-contained.
Experimental design: The research question is well defined, and the statistical Foundation for the study is solid. It would have been easier to interpret the expression levels of VEGF-A isoforms if a better description of the samples had been given. How did the authors know that the samples were in fact tumor or non-tumor samples (this can be very difficult to judge from simply looking macroscopically at the tissue). What extent of tumor cells versus non-tumor cells were present in the tumor tissue? What extent of necrosis/viable cells was present in the tumor tissue? The selection, characterization and validation of the tumor tissue should be described in more detail in the materials and methods section.
We are asked to judge whether the research fills an identified knowledge gap. It is known that VEGF-A is deregulated in many tumors, and the authors also make this clear in both the introduction and discussion. Is it a knowledge gap how VEGF-A189 specifically is regulated? Unless the authors can demonstrate that the down-regulation of VEGF-A189 without the up-regulation of other VEGF-isoforms, as what the authors are demonstrating in their work, leads to a difference in angiogenesis, it is hard to see how the results fill a knowledge gap. That does not mean, however, that the results could not be proven to do so in the future.
Validity of the findings: The impact of the findings are not clear at present, as the functional consequence of reduced VEGF-A189 levels are not known. The data, however, seem robust and statistically sound, and does seem to impact tumor progression (or advanced progression may impact VEGF-A189 production). The conclusions and discussions are however done at a reasonable level, without attempts to overreach.
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: DISCOVERY OF DIGESTIVE ENZYMES IN CARNIVOROUS PLANTS WITH FOCUS ON PROTEASES
Review round: 1
Reviewer: 1

Output:
Basic reporting: This review provides a good overview of research so far in the discovery of enzymes from carnivorous plants. Overall, this is a solid contribution to the literature and puts in context recent work from several groups focusing on different carnivorous plants.

In practice, the focus is mainly on proteases. It may make sense to either change the title to reflect this emphasis, or if not, references to some of the work on chitinases from carnivorous plants should be added, for example:

I. Matusíková, J. Salaj, J. Moravcíková, L. Mlynárová, J. Nap, J. Libantová, Tentacles
of in vitro-grown round-leaf sundew (Drosera rotundifolia L.) show
induction of chitinase activity upon mimicking the presence of prey, Planta 222
(2005) 1020–1027.

H. Eilenberg, S. Pnini-Cohen, S. Schuster, A. Movtchan, A. Zilberstein, Isolation
and characterization of chitinase genes from pitchers of the carnivorous plant
Nepenthes khasiana, J. Exp. Bot. 57 (2006) 2775–2784.

S. Rottloff, R. Stieber, H. Maischak, F.G. Turini, G. Heubl, A. Mithöfer, Functional
characterization of a class III acid endochitinase from the traps of the
carnivorous pitcher plant genus, Nepenthes, J. Exp. Bot. 62 (2011) 4639–4647.

K. Ishisaki, Y. Honda, H. Taniguchi, N. Hatano, T. Hamada, Heterogonous expression
and characterization of a plant class IV chitinase from the pitcher of the
carnivorous plant Nepenthes alata, Glycobiology 22 (2012) 345–351.

T. Renner, C.D. Specht, Molecular and functional evolution of class I chitinases
for plant carnivory in the caryophyllales, Mol. Biol. Evol. 29 (10) (2012)
2971–2985.

P. Paszota, M. Escalante-Perez, L.R. Thomsen, M.W. Risør, A. Dembski, L. Sanglas,
T.A. Nielsen, H. Karring, I.B. Thøgersen, R. Hedrich, J.J. Enghild, I. Kreuzer, K.W.
Sanggaard, Secreted major Venus flytrap chitinase enables digestion of arthropod
prey, Biochim. Biophys. Acta. - Proteins and Proteomics 1844 (2) (2014)
374–383.

M. Unhelkar, V. Duong, K. Enendu, J. Kelly, S. Tahir, C. Butts, R. Martin, Structure prediction and network analysis of chitinases from the Cape
sundew, Drosera capensis, Biochim. Biophys. Acta - General Subjects 1861 (2017) 636–643.
Experimental design: Not applicable - review paper.
Validity of the findings: Not applicable - review paper.
Additional comments: If possible, light editing for English usage is recommended. This concern is stylistic rather than essential for understanding the paper, however.


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: DISCOVERY OF DIGESTIVE ENZYMES IN CARNIVOROUS PLANTS WITH FOCUS ON PROTEASES
Review round: 1
Reviewer: 2

Output:
Basic reporting: Authors have discussed the classification of carnivorous plants by trapping methods, they also summarized how digestive enzyme discovered and characterized as well as the potential application. Although authors did a complex review on this topic, there are several logical issues related to the review.
1.	There is no need to discuss the classification of the carnivorous plants by trapping methods. Authors talked about the enzyme discovery by their families and species, not trapping methods, so the first part has no relationship with the second part. I would suggest either remove it or write the classification by families.
2.	The key point of this review is to discuss the enzyme discover, authors states all discovered enzyme with their common names such as protease or phosphatase, does the structure of each enzyme discovered from different species the same? Can you find the same enzyme in other places like in animals? Authors should provide the structures of several enzymes.
3.	In table 3, authors should list the name of each protease from different species, the enzyme should have difference because the conditions listed are different. Same requirement applies to Chitinase and lipase as well phosphatase.
4.	The main purpose of this review is to discuss the potential application of digestive enzyme from carnivorous plants, however, in table 4, authors also listed the protease from other source such as animal and microbial, this doesn’t make any sense.
5.	The language need to improved, a lot of worlds are missing.
Experimental design: not applied
Validity of the findings: not applied
Additional comments: not applied


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: BIOCHEMICAL AND GENETIC ANALYSES OF THE OOMYCETE PYTHIUM INSIDIOSUM PROVIDE NEW INSIGHTS INTO CLINICAL IDENTIFICATION AND UREASE-BASED EVOLUTION OF METABOLISM-RELATED TRAITS
Review round: 1
Reviewer: 1

Output:
Basic reporting: 1.	Basic Reporting
The article was written in a clear way, and sound technically correct. The manuscript is well structured and the references are in agreement with the objective of this study.
Experimental design: 2.	Experimental design
The research question is clearly postulated at the end of the introduction. This issue is relevant since the mechanisms of pathogenicity and metabolic pathways of this microorganism are not yet completely known.
The investigation was conducted rigorously and the methods used are in line with those prevalent in the field. The methods were well described and could be replicated by other researchers.
Validity of the findings: 3.	Validity of the Findings
The data presented in this manuscript highlighted the need of a more comprehensive knowledge of the biochemical features of this oomycete before to propose these features for identification and differentiation from others pathogens. The results of the biochemical profile analysis are similar to data already published by other authors, but the analysis of the urease and urease accessory protein sequences are new data and can be useful for future studies.
The data are robust, well explained on discussion and appropriately concluded.
Additional comments: I have only minor correction to improve the manuscript:
Line 65: the authors cited Chaiprasert et al., 1990 to say that isolation of the pathogen from infected tissues is difficult. This statement is not very correct, since the isolation of the pathogen from infected tissue is easily done, at least from infected horses and experimental animals. Please, rephrase this sentence.
Line 75: “ in the clinical identification…”
Line 83: “Although the use…”
Line 114: “ ten, instead of 10”
Line 466, 468: Figure 1 legend – arrowhead, instead arrow head


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: BIOCHEMICAL AND GENETIC ANALYSES OF THE OOMYCETE PYTHIUM INSIDIOSUM PROVIDE NEW INSIGHTS INTO CLINICAL IDENTIFICATION AND UREASE-BASED EVOLUTION OF METABOLISM-RELATED TRAITS
Review round: 1
Reviewer: 2

Output:
Basic reporting: The authors reported the phylogenetic tree and evolution of urease enzyme and provide the new information for the study of Pythium insidiosum.

1.	Title:  The experiment mainly worked on the urease in terms of phylogenetic tree and evolution. Thus, the appropriate of this tiltle should focus on more specific to include the key word of  “Urease”.  Also very less pathogenesis was mentioned in the discussion part thus the  title should be considered.  Other biochemical tests were like adding without much association of urease story.  It might be better to consider for process another manuscript.

2.	Keywords:  No keywords couldnot be found.
Experimental design: 3.  Materials & Methods :
        a.  Microorganism & Growth : P 3 :
              i.   L. 109: One-week-old, actively growing colonies of P. insidiosum : It seems reasonable to prepare the inoculum like that for growth rate experiment. It is not clear that how the authors prepared the cultures which stocked before. These cultures require, at least, subculture 2-3 times to revive. This point will be very critical influence to the growth rate experiment.
             ii.   L. 112: It will be better to add reference for preparing the inoculum size : 5 mm.
Validity of the findings: 4.	Result :
        a.   L. 168: Sample: Please state whether the sample size was statistic calculation or …….. .
        b.   L. 174:  The authors recruited strains from 3 Clades of Pythium insidiosum, from this study, what the reader can learn from the clade point of view.

5.	Discussion :
        a.   L. 233: Failure to utilize the sugars was markedly associated with slow growth : What is the theory supported this result. Or in other hand, how do you prove that it is not the "co-incidence"?
        b.   L. 238: Utilization of urea not associated with growth rate :  any mechanism or Similar as above comment. How do you know that it is not the co-incidence between the hydrolysis of sugar and growth rate? : Urea breakdown can be observed in slow growing strains : Normally, the newly isolates from host show more rapid growth that the old ones. How do know that the slow growing is not the effect from reviving the stock before study?

6.	 Conclusion : The main story should be very interesting if the manuscript is focused on urease story. Another notice is that the conclusion of "several biochemical characteristics, and provided new insights into pathogenicity and metabolism-related evolution of P. insidiosum.“ , isn’t it too broad as comments above.
Additional comments: The authors presented and analysed of urease gene including phylogenetic tree and evolution using Pythium insidiosum strains from Thailand and from other regions.  The manuscript will be more focused if the authors presented the story of urease.


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: THE ENERGETIC COST OF MOUNTING AN IMMUNE RESPONSE FOR PALLAS’S LONG-TONGUED BAT (GLOSSOPHAGA SORICINA)
Review round: 1
Reviewer: 1

Output:
Basic reporting: No comment
Experimental design: No comment
Validity of the findings: In abstract, it is difficult to compare APRs with other species without controlling for dose of LPS.  This should be made clear in the abstract.
Additional comments: This study examines the effect of immune challenge (using bacterial lipopolysaccharide) upon energy expenditure and resting metabolic rates of the Pallas's long-tongued bat.  The study is fairly straightforward and well-designed. The authors acknowledge several drawbacks of the study in the discussion: (1) Interspecific comparisons of APRs is somewhat misleading when different mass-corrected doses of LPS are used, and (2) body temperature was not measured. It would have been useful to know whether bats become hypo-or-hyperthermic from the LPS treatment vs. saline-treated bats.  In small birds, LPS treatment can induce short-term hypothermia in some cases.

In regards to body mass changes from LPS, the authors compare their findings to other bat and birds.  The authors only report house sparrows (Passer domesticus) in the discussion, but a number of other species have been examined (see my review paper on LPS-responses in birds. Ashley, N. T. and J. C. Wingfield.  2012. Sickness Behavior in Vertebrates: Allostasis, Life History Modulation, and Hormonal Regulation. In: G. E. Demas and R. J. Nelson, eds. EcoImmunology. Pp. 45-91. Oxford University Press).
.  Also, there is pretty strong evidence that body mass loss is tied to initial body condition. Is there a method to assess body condition in bats?, and if so, this could help explain some of the variation in metabolic and body mass responses.

- The mean ambient temp was below the lower crit level of the animal… meaning all animals were thermogenic prior to injection and was wondering how much this would affect the estimates of RMR. Just something to think about...

Lines 276-LIne 280. This is a very long and confusing sentence. Please rephrase and break up.

LInes 286-288. What the same mass-corrected LPS dose in your study used in the fish-eating Myotis study? If not, differences in daily energy expenditure could be due to dose....

Line 293. ", then the results reveal"


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: THE ENERGETIC COST OF MOUNTING AN IMMUNE RESPONSE FOR PALLAS’S LONG-TONGUED BAT (GLOSSOPHAGA SORICINA)
Review round: 1
Reviewer: 2

Output:
Basic reporting: Major Comments:
(1)	The writing throughout the manuscript needs refining – as is, the writing presents some issues with readability due to basic, repeated grammatical errors. For example, the first sentence of the abstract:

“Activation of immune response has been long assumed to be an energy-costly process but direct measures of changes in metabolic rate after eliciting immune response disputes the universality of this assertion.”

This sentence should, as a first pass, be revised as follows:

“Activation of the immune response has long been assumed to be an energetically-costly process, but direct measures of changes in metabolic rate after eliciting an immune response disputes the universality of this assertion.”

However, further work to streamline the writing would really improve the manuscript:

“Mounting an immune response is thought to be energetically costly, however direct measures of metabolic rate during immune challenges contradict this assumption.”

These issues should be addressed throughout by careful editing and attention to grammar/sentence structure. I also found the logical order of sentences throughout difficult to follow. Again, in the abstract, the authors introduce the immune response and the assumptions about immune response activation in the first sentence (Lines 2-4). In the second sentence, authors define the immune response they’re addressing (the APR) and again state that it is energetically costly (lines 4-5). The connection between energetic cost and metabolic rate from the first sentence is not made explicit (only implicit), and the second sentence essentially repeats the same information. Authors can greatly improve the quality of the manuscript by carefully editing and revising the writing to address logical consistency and redundancies throughout.

Minor Comments:
1.	It is not clear from the methods when body mass measurements were taken relative to treatment. Please clarify. Also, in discussing the changes in mass (line 222-223). The published report on the species of particular interest (M. vivesi) to the authors has issues in how their calculations were done overall – they suggest that bats lost weight in response to LPS injection, however their “pre-injection” mass measurements suggest that the change they are seeing is primarily explained by time of day and not the LPS injection (see Fig. 1 in Otalora et al 2016). So I don’t see this as a legitimate comparison. The Stockmaier paper also looked at 24 h rather than 10 h after injection. These differences in timing etc. should be made explicit because the LPS APR and recovery is so rapid (<48 h)

2.	Throughout the results, authors are inconsistent on defining abbreviations when introduced in new sections (e.g., PI; line 202).

3.	It’s unclear from the methods when animals were sampled. Were these data collected across a 30-day period, or were all individuals sampled on the same day? How did animals cope with captivity (body mass throughout this period)?

4.	I find the Y axis label in Figure 1B confusing – “% Variation in MR after injection”. At first, I thought this meant, “how variable was the MR within an individual” – similar to heart rate variability – however it was later made clear to me that the authors meant “% increase in MR over baseline following injection”. This should be clarified.

5.	In Table 1, I don’t understand the inclusion of the Mean body mass (Mean Mb) column. What does this metric tell us about? Mbi and Mbf seems sufficient.
Experimental design: No comments
Validity of the findings: The authors frame their work as an important comparative step, and contrast their data with data from small mammals, birds, etc. in an attempt to make broad generalizations about how different taxa respond to immune challenges. First, the authors need to be careful throughout to avoid over-generalizing. For example, the abstract, intro, and discussion state that:

“…large increase[sic] in metabolic rate after APR activation measured in piscivorous bat species … suggests that immune response is unusually costly for bats.” (Line 7-9)

I don’t think one species should be thought to represent the response of an entire (extremely diverse!) taxa, even if it’s the only published data out there, and the authors should not attempt to generalize to this degree.

More importantly though, there are some major problems with contrasting the manuscript under review with published literature (and therefore the presented premise of this project). First, different doses (and lots) of LPS are used across all these studies. The immune response to an LPS dose varies among lots, across different doses, parasite lode and exposure?, and also with life history stage/social context, etc. Authors acknowledge this fact briefly in their discussion (Line 264), however they do not address the fact that this variability largely undermines or at least limits comparative use of their study. I think this fact needs explicit attention throughout the manuscript. Due to the marked variation in response to LPS injection across these various contexts and among experiments, it is inappropriate and misleading to attempt to directly and quantitatively compare the data presented here with data from other studies. While qualitative comparisons that evaluate differences between the sexes, energetic states, or life history stages may be appropriate, the authors do not present analyses or discussion that could be used in such a context. The authors also state that their data collection and analytical methods for measuring metabolic rate differ from those in other studies. These difference further underscore that premising the entire paper on such comparisons is invalid. Finally, I would add that because doses are usually chosen by the ability to elicit a response (rather than some other external item), it's difficult to contrast among systems and the justification easily becomes circular.

Again, the authors introduce the idea that the differences in metabolic rate/cost of the immune response may be explained by “ecological factors” (Line 309-317). This is a really interesting idea, but the authors throw it into their conclusions without addressing it anywhere else, and they seem to suggest that this explanation makes the most sense without presenting any data from their own system to support this. It makes sense to me to bring this up as a contributing factor, but it remains unsubstantiated as a conclusion.

When these comparisons are appropriately limited and/or removed, these data are really only anecdotal. It is extremely difficult to assess whether these data provide any information to contrast this species with other studies, and there is too little data and the samples sizes are too small to address basic questions about the life history of this species or the evolutionary/taxonomic variation in the immune response.
Additional comments: As stated elsewhere, the data are interesting but stand largely as anecdotal. The scope of the article, as framed by the abstract, introduction, and discussion, needs to be substantially altered. Comparison of this work to others should remain qualitative, limited, and marked as speculative. Limitations of comparisons should be clearly discussed.


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: THE ENERGETIC COST OF MOUNTING AN IMMUNE RESPONSE FOR PALLAS’S LONG-TONGUED BAT (GLOSSOPHAGA SORICINA)
Review round: 1
Reviewer: 3

Output:
Basic reporting: The authors have done an adequate job framing their study with relation to the literature. It is professionally written in relatively straightforward language (with a few exceptions) and the figures are sufficient and well-constructed. There are small typos and grammatical mistakes throughout that need attention.

There are some major style shifts throughout the manuscript that could be smoothed out. For example, the Introduction is really well constructed and is pleasant to read. The Abstract and Discussion, however, are much more disjointed and need the same kind of attention paid in setting up the manuscript in the Introduction. In the revisions please consider paying more attention to the overall flow and style of the manuscript.
Experimental design: Overall the experimental design is good. But there are several points that should be addressed, including a re-analyses of the entire data set.

1) the lack of inclusion of Ta into the analysis is a fundamental flaw to the data set. There was a large range of Ta measured during the study (at least 4 degrees -- but the minimum is never specified), including temperatures outside of the bat's reported TNZ. I suggest  re-analysis of the data that tests for effects of Ta on MR.

2) The definition of the 'net RMR' is convoluted and needs to be clarified.

3) Instead of using Mb as a covariate, why not analyze mass-specific metabolic rates?

4) It is not clear how or in which software the data were analyzed.
Validity of the findings: The data appear to be robust, but see my above comments on analysis.

Hanging this around the 'large increase in RMR in fish-eating Myotis' seems a bit disingenuous, since in that study it is not clear if the bats were normothermic or thermoconforming at high temperatures. I think it it more interesting to rephrase this around the diversity of immunological adaptations that are emerging in bats and the strong selection on a reduced immunological tool kit.

The paragraph on L 245-280 in the Discussion meanders and could use substantial editing down to find the really important points.
Additional comments: I have made comments in the attached pdf.


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: THE ENERGETIC COST OF MOUNTING AN IMMUNE RESPONSE FOR PALLAS’S LONG-TONGUED BAT (GLOSSOPHAGA SORICINA)
Review round: 2
Reviewer: 1

Output:
Basic reporting: The manuscript has improved editorially, but there are still minor editorial issues that need to be addressed. Perhaps having a colleague read who can assist with English language corrections would benefit the MS.
Experimental design: No comment
Validity of the findings: No comment
Additional comments: Overall, the authors have done a good job in responding to previous criticisms and toning down conclusions based upon previous studies.


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: THE ENERGETIC COST OF MOUNTING AN IMMUNE RESPONSE FOR PALLAS’S LONG-TONGUED BAT (GLOSSOPHAGA SORICINA)
Review round: 2
Reviewer: 2

Output:
Basic reporting: The authors made effective changes to the writing in the manuscript.
Experimental design: The experimental design results in some important caveats to the interpretation, but these are now made clear by the authors in the methods and discussion.
Validity of the findings: The authors have made changes to the introduction and discussion that improves the validity of their conclusions.
Additional comments: Instead of placing the Ta analyses in the methods, where it can be easily missed, the authors shoudl move the analyses to the results section. They should also add information on the analyses run to their "statistical analyses" section.

There are a few typos throughout the corrections the authors made:

Line 52: “whithin” should be “within”

Line 64: add a space between “;” and “increase”