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: NOBLELLA THIUNI SP. N., A NEW (SINGLETON) SPECIES OF MINUTE TERRESTRIAL-BREEDING FROG (AMPHIBIA, ANURA, STRABOMANTIDAE) FROM THE MONTANE FOREST OF THE AMAZONIAN ANDES OF PUNO, PERU
Review round: 1
Reviewer: 2

Output:
Basic reporting: The authors describe a new species of Noblella from Southern Peru. The paper is well written and conforms to the “standard sections”. The authors used well referenced, actual and fundamental literature related to the subject. They present important new data on a very interesting and difficult group of direct developing frogs, however important information needed for the species description is limited or missing. These are discussed in the next section.
Experimental design: While the manuscript has the potential to be published, I think that there are a number of serious concerns that preclude its consideration for publication in its current state.

1. My main concern is that the description of the new species is based on only one specimen. While there are many cases when new species were described based on only one specimen it is important to address the problems associated to singleton descriptions (see Castroviejo-Fisher et al. 2011; Lim et al. 2012 and more recently Guayasamin et al. 2018). The authors fail to do this in the current manuscript. Almost no information concerning their field work is provided (when and how many times was the study site visited, methodology used etc.) and no details provided why they were not able to collect additional specimens (they just mention that the specimen was obtained during a “short survey” of the type locality - L 310). From personal experience I know how difficult it is to find the small frogs of the genus Noblella, but with regular visits (ideally in different seasons and weather conditions) is possible to collect a decent number of individuals needed for the description.

This problem is especially severe as the new species seems very similar to the problematic type species of Noblella, N. peruviana, for which we lack any new data (molecular and morphological) as no additional specimens were found since the original description. The morphological differences found between the new species and N. peruviana don’t seem to be sufficient to separate them, considering the scarce information: old specimens on one hand (possibly even from different species; see De La Riva et al. 2008) and only one specimen on the other. By the way, how far from the putative type locality of N. peruviana (the vicinity of the Inca Mine, in Santo Domingo de Carabaya, De La Riva et al. 2008) was collected the N. thiuni specimen? I strongly recommend that the authors address these issues in their discussion.

2. A second problem that I could identify is with the phylogenetic analysis. I understand that the authors didn’t try to resolve the phylogenetic relationships of this difficult group and their analysis is preliminary, but I still think that a robust analysis should be performed. I think that the used 16S sequences are not sufficient (especially for this group), and ideally inclusion of at least one nuclear gene would be desirable. Also, I would recommend the use of the primers 16SC and 16SD for the 16S gene as these are producing better quality sequences (more than 830 bp), in the case of Noblella, than the used 16Sar - 16Sbr pair. Additionally, I would recommend the use of a more versatile phylogenetic analysis tool like RAxML or Garli for the ML analysis (and with 1000 bootstrap replicates) and ideally coupled with a Bayesian inference in order to present a better resolved phylogenetic tree (as many groups had very low support in the authors Mega analysis). And finally and more importantly the authors don’t provide any information on the genetic distances of the closely related species. I recommend the inclusion of this data in the manuscript.
Validity of the findings: I’m afraid that the authors don’t provide sufficient data to support the validity of the new species in the manuscripts current form, especially because the analyzed specimen is so similar to a controversial species. I would recommend the inclusion of additional specimens in their description and the rerun of the molecular analysis.
Additional comments: Minor comments:

Figure 5 - Please correct the scale bar to mm: cannot be 2 cm!

Appendix 1 - Please add the legend for the collections abbreviations for the examined specimens

16S rRNA sequences (FASTA format) for the holotype of Noblella thiuni sp. n. - I don’t think this is useful as the sequence will be available from GenBank. It would be more useful (but not mandatory) to attach the whole alignment matrix.

Appendix 2 - is not properly presented as there is no legend and the listed species don’t match with the ones from the tree.


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: NOBLELLA THIUNI SP. N., A NEW (SINGLETON) SPECIES OF MINUTE TERRESTRIAL-BREEDING FROG (AMPHIBIA, ANURA, STRABOMANTIDAE) FROM THE MONTANE FOREST OF THE AMAZONIAN ANDES OF PUNO, PERU
Review round: 2
Reviewer: 1

Output:
Basic reporting: No comment
Experimental design: No comment
Validity of the findings: No comment
Additional comments: In my view, the authors have fully addressed the comments raised by myself and the other reviewers. I think that the description is solid and should be published.


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: NOBLELLA THIUNI SP. N., A NEW (SINGLETON) SPECIES OF MINUTE TERRESTRIAL-BREEDING FROG (AMPHIBIA, ANURA, STRABOMANTIDAE) FROM THE MONTANE FOREST OF THE AMAZONIAN ANDES OF PUNO, PERU
Review round: 2
Reviewer: 2

Output:
Basic reporting: no comment
Experimental design: The authors added a detailed description of the problems related to singletons species and important data that was missing in the original MS. They also significantly improved the discussion section.
Validity of the findings: The authors managed to add sufficient information to support the validity of the new species. I think that the MS is publishable in its current form after correcting some very small errors presented in the next section.
Additional comments: Minor comments:

- I still would add in the Materials & methods section a short description of the field work: when and how the short survey was implemented and other details of the field work (how many persons, survey methods etc.). I would remove these details from the “Distribution, natural history and threats” and add them to the Materials & methods. Sorry, but I didn’t find sufficient information related to the field work not even in the article naming Psychrophrynella glauca. I think that it’s very important that you present the details of how your field work was carried out.

356 - “Santo Domingo is a cloud forest at 1690 m asl is in a small tributary of the upper Inambari River, ~150 km SE of the type locality of N. thiuni.” Something is missing from this sentence; please correct it.

Appendix 2 - Replace “Celio F. B. Haddad Field Series” with “CFBHT – Celio F. B. Haddad Field Series” (add CFBHT).


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: A NETWORK APPROACH TO IDENTIFY BIOREGIONS IN THE DISTRIBUTION OF MEDITERRANEAN AMPHIPODS ASSOCIATED WITH POSIDONIA OCEANICA MEADOWS
Review round: 1
Reviewer: 1

Output:
Basic reporting: English overall good, but a few passages are hard to read or grammatically flawed:

line 335
line 396
line 413
line 372: Relationships with Posidonia oceanica history - relationship of amphipods with...?
Experimental design: no comment
Validity of the findings: no comment
Additional comments: It is a brave and interesting effort, to bring so much data together and the scope of the paper is clear and indeed fills a gap in the knowledge in biographic classes in amphipods. However, in some respects the outcome remains rather abstract. About the hypothesis referred to in line 413 – this hypothesis or scenario is so broad in its predictive value that it makes almost every conceivable mode of distribution plausible.

I would like to see more reference to certain groups of amphipods referred to in line 413. Which amphipods dependent on Posidonia would be possible candidates in a follow up research to verify the hypothesis and why. It would certainly help strengthening the paper if more attention is given to this in the conclusions.


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

Input:
Title: A NETWORK APPROACH TO IDENTIFY BIOREGIONS IN THE DISTRIBUTION OF MEDITERRANEAN AMPHIPODS ASSOCIATED WITH POSIDONIA OCEANICA MEADOWS
Review round: 1
Reviewer: 2

Output:
Basic reporting: I feel that this manuscript needs major revision.

In my opinion, the entire manuscript is poorly structured, with long unclear statements. I feel that words are used incorrectly, such as "rate of endemisms" in the introduction. The authors keep referring to a "network approach based on modularity." I feel that this should be explained in the introduction as it is essentially jargon to anyone who is unfamiliar with these techniques. As it is, this article is relevant to a small group of researchers.

The figures are too busy and not explained well. There is no description of the labels.

Finally, the article title is "Biogeography of Mediterranean amphipods associated with Posidonia oceanica meadows", yet there is no mention of a single amphipod species in the article. I feel that this is crucial to the article and should not be supplementary information.
Experimental design: The research question is vaguely defined with jargon, and needs clarification. I am still unsure of exactly where the data came from.
Validity of the findings: I am sure the findings are valid and may be useful, but I feel that they should absolutely be presented in terms of what amphipod species are where. It is impossible to describe the biogeography of amphipods without doing this.
Additional comments: I think this manuscript is interesting, but needs to be presented differently, with more of a focus on the amphipods and less of a focus on your "network approach based on modularity."


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: A NETWORK APPROACH TO IDENTIFY BIOREGIONS IN THE DISTRIBUTION OF MEDITERRANEAN AMPHIPODS ASSOCIATED WITH POSIDONIA OCEANICA MEADOWS
Review round: 2
Reviewer: 1

Output:
Basic reporting: The readability has much improved. I added or removed an 's' here and there on the track changes version which staff will forward to the authors.
Experimental design: no comment
Validity of the findings: The conclusions gained focus by pointing out relevant amhipod goups.
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: A NETWORK APPROACH TO IDENTIFY BIOREGIONS IN THE DISTRIBUTION OF MEDITERRANEAN AMPHIPODS ASSOCIATED WITH POSIDONIA OCEANICA MEADOWS
Review round: 2
Reviewer: 2

Output:
Basic reporting: This version of the manuscript is much better than the first, but I still have several suggestions for improvement (see comments in PDF file). The authors need to add in text taxonomic authorities for all species mentioned and these also need to be added to the reference list. I feel that the title is misleading and perhaps should be changed to reflect that you are actually reporting on your model rather than the biogeography of amphipods. In my opinion you still do not discuss the biogeography of amphipods, but instead are reporting on your modeling using amphipods as a model group.
Experimental design: I think that the experimental design is sound, except for the literature used for data mining.
Validity of the findings: I have a concern as to the validity of your data as it is based on literature that is apparently not current (although I still feel that the sources of data are unclear). For example, you mention Leucothoe spinicarpa as a cosmopolitan species, yet it is not cosmopolitan and is not believed to be present in the Mediterranean Sea (see Krapp and Menioui, 2005). My concern for your results is that they are based on literature that may or may not be currently relevant.
Additional comments: There are still several grammatical and reference errors with the manuscript.


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

Input:
Title: GASTROCNEMIUS FASCICLES ARE SHORTER AND MORE PENNATE THROUGHOUT THE FIRST MONTH FOLLOWING ACUTE ACHILLES TENDON RUPTURE
Review round: 1
Reviewer: 1

Output:
Basic reporting: Thank you for letting me review this interesting and novel paper. The manuscript ”Gastrocnemius fascicles are shorter and more pennate throughout the first month following acute Achilles tendon rupture” is well written and may add some new, interesting facts in this field. The purpose of this study was to quantify the immediate structural changes to the medial gastrocnemius the first 4 weeks after an Achilles tendon rupture.
However, there is a need for some revisions, especially in the Conclusion and Discussion section in order to avoid too much speculation.
Experimental design: The design of this study is adequate and described in a proper way. A power analyze has been performed that showed that 10 subjects were needed for detect a 10% decrease in fascicle length. The coefficient of variation was calculated to be less than 10%. There is a lack in explanation how the subjects in this particular study were recruited from the mentioned 1-year long prospective study. This needs to be clarified.

An advantage in this paper is that the same person performed all the ultrasound measurements. The coefficients of variation between the measurements on the healthy limb have been calculated and they were found to be less than 10%. However, this says nothing about the normal variation between the fascicle length between the healthy and the normal side. There is a need to clarify the facts in this matter.
Validity of the findings: The data in this study is well presented and there is also supplementary material to clarify all the measurements.
However, the conclusion in the abstract and in the Conclusion section are not expressed in the same way. In my opinion, the authors can not draw the conclusion that “careful immobilization paradigms may prove to be effective for preserving muscle structure and quality in patients following tendon injuries” from the results in this fairly small study performed during 4 weeks. It is important for the authors to keep in mind what variables you used for power analysis before drawing any far-reaching conclusions. A suggestion is to use the last sentence in the abstract for Conclusion.
Additional comments: Sometime you use the word “pennation” and sometimes “pennation angle” in the paper. Please, use the same word throughout the manuscript.

Abstract
Line 21-25: Please clarify that the measurements refer to the injured limb and (I suppose) in comparison with the healthy limb.
Introduction
Line 69-80: This paragraph is the purpose and the hypothesis. This section needs to be re-written since there are some difficulties to follow what is the purpose/hypothesis and what is more background. A suggestion is to put line 69-73 at the very end.

Methods
Line 86: You have listed the inclusion criteria. Were there any exclusion criteria? I miss the inclusion criteria in to this study from the “1-year long prospective study” (line 101).

Line 96-99: What are normal differences between limbs (in healthy subjects) in fascicle length, pennation angle, muscle thickness and echogenicity?

Line 109-124 For proper intra-reliability calculations between measurement, there may be a need for ICC-values for the measurements with ultrasound.

Line 114: How did you confirm that you measured in the middle of the muscle belly? Please clarify.

Line 124: The coefficient of variation is often expressed in percent. This is just a suggestion and not necessary to change.

Line 134: How did you define “poor quality”? Please, clarify.

Results
The results are presented in a clear and consistent way that make it easy for the reader to follow.
I know that you present your measurements in percent, but what unit has echogenicity?

Discussion
In this section, I miss a discussion of your results in the aspects of:
- Treatment with or without surgery
- Gender differences (you have only evaluated men)
- How is the fascicle length in the beginning of the injury since the mean time before the first evaluation was 4,5 days? This is linked to the question about how much is the normal difference in fascicles length between limbs in healthy subjects.

Line 163-171: This paragraph is more like a continuing of the Introduction. A suggestion is that you start with your major findings (like you write at line 172-174).

Line 174: How do you know that the changes is a mechanical response to a sudden loss of intra-muscular tension? This sentence is more of a speculation than a conclusion of your results.

Line 179: I agree that your results are an important advancement in your study, but not (at least yet) in the treatment of tendon injuries.

Line 182: Please clarify that you are referring to the injured gastrocnemius.

Line 202: You do not need to refer to your own figure in the Discussion section.

Conclusion
The conclusion in the abstract and in the Conclusion section are not expressed in the same way. In my opinion, the authors can not draw the conclusion that “careful immobilization paradigms may prove to be effective for preserving muscle structure and quality in patients following tendon injuries” from the results in this fairly small study. A suggestion is to use the last sentence in the abstract for Conclusion.

Please, keep in mind what variables you used for power analysis before drawing any far-reaching conclusions.

Figures
Figure 2: If possible, it would be valuable to present a bigger picture. About the figure legend: Here, there is no need to write that the same investigator performed all evaluations. It would be valuable to know where (on the medial gastrocnemius muscle) the imaging is taken.


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: GASTROCNEMIUS FASCICLES ARE SHORTER AND MORE PENNATE THROUGHOUT THE FIRST MONTH FOLLOWING ACUTE ACHILLES TENDON RUPTURE
Review round: 1
Reviewer: 2

Output:
Basic reporting: The language is clear and professional. Literature references and background/context are provided for the relevance of Achilles tendon injuries and plantar flexor function, whereas information is missing to explain why the chosen time period of the first four weeks after injury is relevant.
Experimental design: The design of the experiment is solid but I believe originality and relevance of the study could be greatly improved by reporting the follow-up of the patients over a longer time period. Other variables, for example soleus muscle architecture, would also strengthen the paper since soleus is a primary contributor to ankle joint torque during locomotion (e.g. Hamner 2018 J Biomech). Although research questions are well defined by the authors, they do not convincingly convey the relevance of the work.
Validity of the findings: The conclusion that muscle architecture changed because of the tendon rupture is not directly supported by the data and that should be acknowledged by the authors. To conclude this, it would have been necessary to perform the same measurements before injury. The present study-design allows only to conclude that there are differences between the injured and the healthy leg. Whether these differences resulted from the tendon rupture or whether they existed before and possibly have contributed to a higher injury risk, is unclear from the present data.
Additional comments: The authors explain that no functional tests can be performed within this time frame, so I am wondering why the later follow-up measurements of the patients were not reported to relate changes in muscle architecture to function and to see whether the short term changes persist. The authors could not convince me why changes in muscle architecture are important in the first four weeks after injury when the injured side cannot be loaded. I recommend to add measurements over a longer period and to test functional outcomes as early as possible before resubmitting.


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: GASTROCNEMIUS FASCICLES ARE SHORTER AND MORE PENNATE THROUGHOUT THE FIRST MONTH FOLLOWING ACUTE ACHILLES TENDON RUPTURE
Review round: 1
Reviewer: 3

Output:
Basic reporting: No comment
Experimental design: In this study, the authors determine the effects of Achilles tendon rupture on medial gastrocnemius muscle architecture. They find that an acute AT rupture elicits immediate changes in muscle fascicle length and pennation angle, that persist for up to 4 weeks post injury. The authors hypothesize that these structural changes occur to maintain MTU tension following injury. The manuscript is well-written, however as discussed below, you will see that I have a few concerns related to the experimental protocols and methodologies that I hope can be addressed by the authors.

Major Comments:

Methods – Did you, and if so how, ensure that ankle angle was consistent between the injured and uninjured limbs when acquiring ultrasound images? This is critical to ensuring that structural changes are due to mechanical changes rather than kinematics. Also, did you measure this angle at each testing session and ensure that it was the same at week 0, 2, and 4 for each participant. For these comparisons to be made, ankle angle must be controlled. The authors state ‘with their feet and ankles supported by the edge of the table in PF and kept in the same position for all imaging sessions’. Please report what this plantarflexion angle was and also how you ensured it was in the same position for each participant across testing sessions.

Comparisons to control limb – it is unclear why the authors used the uninjured limb at week 0 as the control for all comparisons. Given that it is likely for de-training effects to have also affected muscle structure on the uninjured limb since participants would not be exercising between weeks 0 and 4, the authors should compare structural changes between the injured and uninjured limb at EACH time point, in an attempt to remove the confounding effects of de-training/exercise. I believe the authors should have these data given that they confirmed probe placement repeatability measuring the control limb at each visit.

Reliability of ultrasound measurements – the authors state that many of the ultrasound images show to be a poor quality and should conduct and report intra or inter-rater reliability measures on these data.

Figures 1 and 2 are poor quality and not informative. I suggest changing these. See methods section.
Validity of the findings: No comment
Additional comments: Minor Comments:

Abstract

Line 14 – ‘on medial gastrocnemius’ should be ‘on medial gastrocnemius structure’

Line 16 – move B-mode from Line 18 to Line 16 when ultrasound is first discussed

Line 19 – was ‘initial clinical treatment’ always the first image acquisition? Or did patients receive clinical treatment before this first time point of data?

Line 27 – I suggest beginning this sentence with ‘Our results suggest…’ or a similar statement and please replace ‘configuration’ structure or architecture to be consistent in your language throughout.

Introduction:
Line 44 – Please add in whether decreased resting ankle angle refers to more PF or DF, as sign conventions are often different across the literature.

Line 45 – what is calf raise ‘function’ referring to? Force, kinematics, number of reps?

Line 49 – Highlighting that these results are from animal models would be useful to the reader

Line 54 – ‘PF function is governed by …. and mechanical properties’.  Mechanical properties of what? (Muscle, tendon?)

Line 66 – modeling should be remodeling

Line 68 – have should be has

Line 72 – ‘and would persist’ should be ‘and these changes would persist’

Methods

See above major concerns regarding limb configuration during image acquisition, comparison to control image and reliability of US measurements.

Line 99 – requires a reference.

Methods – states that ultrasound video sequences were acquired but single images were analyzed. How did you choose which image to analyze?

Line 142 – did you also run the t-test of the echogenicity data? This is not included.

Figure 1 is not a very informative figure (and the image quality of the leg traces is quite poor). I suggest replacing Fig 1 with a more powerful methods figure that has either a cartoon or camera image of a person in the position which you acquired images. You can still include the details within your current Figure 1, but this is already within the text more than once (and can remain in the figure caption as is).

Figure 2 – I would like to see the authors choose one representative subject and present the control and the injured ultrasound images together (A and B) with the representative fascicle, pennation and thickness outlined on each of the images. Further, in this figure the line drawn is not on the deep aponeurosis.

Discussion

Line 176 – biologic should be biological

Line 195 – compares should be compare

Line 244 – the authors mention that they are prospectively following these patients for 1 year to link changes in muscle structure with tendon elongation and functional deficits. Why was tendon length during these time 0, 2, and 4 week time points not measured and reported. See reference below for ultrasound – tape measure method.

Barber, L., Barrett, R., & Lichtwark, G. (2011). Validity and reliability of a simple ultrasound approach to measure medial gastrocnemius muscle length. Journal of anatomy, 218(6), 637-642.

From Fig 4 it is apparent that muscle structure, especially pennation angle, changes from week 2 to week 4. Can you authors include some discussion regarding this change. What changes from a cast to a boot may lead to alterations in muscle structure? (Weight bearing, activity level?)


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: GASTROCNEMIUS FASCICLES ARE SHORTER AND MORE PENNATE THROUGHOUT THE FIRST MONTH FOLLOWING ACUTE ACHILLES TENDON RUPTURE
Review round: 2
Reviewer: 1

Output:
Basic reporting: Thank you for letting me re-review the manuscript “Gastrocnemius fascicles are shorter and more pennate throughout the first month following acute Achilles tendon rupture”.

The manuscript has improved significantly. I have only one major concern; I think it already in the Method section (Study design) should be clarified that the findings in the present study is a preliminary data set from a larger clinical cohort. Now, the reader is informed in the last paragraph in the Discussion section which may be confusing (line 280-284). In my opinion, there is also a need for clarification how these 10 patients were chosen from the larger dataset? Consecutively?
Experimental design: No comment
Validity of the findings: No comment
Additional comments: No comment


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

Input:
Title: GASTROCNEMIUS FASCICLES ARE SHORTER AND MORE PENNATE THROUGHOUT THE FIRST MONTH FOLLOWING ACUTE ACHILLES TENDON RUPTURE
Review round: 2
Reviewer: 2

Output:
Basic reporting: I understand the authors position that they don’t have the relevant data available yet and that is regrettable because of the limited information brought by the current data.
Experimental design: no comment
Validity of the findings: no comment
Additional comments: No additional comments


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

Input:
Title: GASTROCNEMIUS FASCICLES ARE SHORTER AND MORE PENNATE THROUGHOUT THE FIRST MONTH FOLLOWING ACUTE ACHILLES TENDON RUPTURE
Review round: 2
Reviewer: 3

Output:
Basic reporting: The authors have done a nice job in revising their manuscript in response to the minor comments and suggestions, however, I still have 3 major concerns that should be addressed.

Major Comments:

(1)
Reviewer: Methods – Did you, and if so how, ensure that ankle angle was consistent between the injured and uninjured limbs when acquiring ultrasound images? This is critical to ensuring that structural changes are due to mechanical changes rather than kinematics. Also, did you measure this angle at each testing session and ensure that it was the same at week 0, 2, and 4 for each participant. For these comparisons to be made, ankle angle must be controlled. The authors state ‘with their feet and ankles supported by the edge of the table in PF and kept in the same position for all imaging sessions’. Please report what this plantarflexion angle was and also how you ensured it was in the same position for each participant across testing sessions.

Author: We thank the Reviewer for this comment and agree that ankle angle during imaging is indeed an important consideration. During the start of each image acquisition, patients lay on a treatment table with their feet freely hanging off the edge of the table. Next, the patients were carefully shifted onto the table while preserving the resting ankle angle of the uninjured leg. We have used a similar approach for longitudinally studying Achilles tendon properties with ultrasound (Kagan et al). Due to time constraints, we decided against measuring this ankle angle but did confirm that muscle structure measurements had low coefficients of variation and intra class correlations between the three scanning sessions for the uninjured side (< 10%). This has been described in our methods section (Lines 97-102, 128-129).

Reviewer: This needs to be added to limitations – as visual inspection is not the standard in literature measuring plantarflexor resting muscle architecture. Further, while this may allow for ‘preserving’ ankle angle during one testing session, it does not allow for a constant angle ankle across testing sessions, which is of critical importance for the comparisons being made in this study. I urge the authors to carefully control this (or measure using a goniometer) in future experiments.

(2)
Reviewer: Comparisons to control limb – it is unclear why the authors used the uninjured limb at week 0 as the control for all comparisons. Given that it is likely for de-training effects to have also affected muscle structure on the uninjured limb since participants would not be exercising between weeks 0 and 4, the authors should compare structural changes between the injured and uninjured limb at EACH time point, in an attempt to remove the confounding effects of de-training/exercise. I believe the authors should have these data given that they confirmed probe placement repeatability measuring the control limb at each visit.

Author: We appreciate this comment and would like to clarify our decision to compare the injured scans to the week 0 injured scans. First, our hypothesis was that Achilles tendon ruptures result in differences in muscle structure from baseline data. While it is possible that the patients underwent some de-training effects, our high ICC and low coefficients of variation in the control muscle measured at each clinical visit suggest that de-training did not occur. We have provided additional metrics of measurement variability between clinical visits for the control muscle to highlight the consistency in this measurement (Lines 128-130).

Reviewer: I would like to see a supplemental figure of Figure 3 where the measures are compared to the control limb at each time point --given that the authors have collected this data. Although the coefficient of variation is low (less than 10%), the change in fascicle length from control to week 0 is 15 %.

(3)
Reviewer: Reliability of ultrasound measurements – the authors state that many of the ultrasound images show to be a poor quality and should conduct and report intra or inter-rater reliability measures on these data.

Author: We apologize for the confusion created by this previous wording. We noticed that muscle quality on the injured side appeared to be ‘poor quality’. This was evident by a lack of contrast between the fascicles and interstitial space. To preserve consistency between scan sessions and subjects, we kept all scan parameters constant. A previous report by Fukumoto et al (2012) that found echo intensity correlates with muscle strength and suggested that this was caused by increased fibrous and adipose tissues within the muscle belly. This agreed with our initial intuition of our observations so we decided to include this ultrasound analysis as a means of quantifying ‘muscle quality’. We have clarified this in the methods section (Lines 141-142).

Reviewer: I can see from figure 2 that the image quality, as evident by the contrast between fascicles and connective tissues to be very poor. Ensuring scan parameters were consistent is useful for the echo intensity analysis but not for the fascicle architecture analysis, as presumably you could have optimized these parameters for each participant (ie: changed power, gain, focus depth, etc). My concern is that the quality of the images make it challenging to track fascicles, and I would like to see an inter-rater reliability testing conducted to ensure that fascicle tracking is consistent between raters.
Experimental design: no comment
Validity of the findings: no comment
Additional comments: No additional comments


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

Input:
Title: GASTROCNEMIUS FASCICLES ARE SHORTER AND MORE PENNATE THROUGHOUT THE FIRST MONTH FOLLOWING ACUTE ACHILLES TENDON RUPTURE
Review round: 3
Reviewer: 1

Output:
Basic reporting: No comment
Experimental design: No comment
Validity of the findings: No comment
Additional comments: Thank you for letting me re-re-review your manuscript “Gastrocnemius fascicles are shorter and more pennate throughout the first month following acute Achilles tendon rupture”.
Thank you also for your clarifying answer to my concern about the inclusion criteria in this study. I no longer have any comments about your study.


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: GASTROCNEMIUS FASCICLES ARE SHORTER AND MORE PENNATE THROUGHOUT THE FIRST MONTH FOLLOWING ACUTE ACHILLES TENDON RUPTURE
Review round: 3
Reviewer: 2

Output:
Basic reporting: I have two remaining comment that I would like to see addressed.

(1)	As the authors acknowledge, the lack of controlling for ankle angle is a valid concern. However, this has not been included within the limitations section of the manuscript as suggested in the previous review. Please include these details, along with the potential effects on results and interpretation of results, within your discussion

(2)	The new figure 3 caption is incorrectly matched with the figure labels. (A) is echogenicity in the figure but in the caption is refers to (A) as fascicle length. It appears that A and D need to be swapped.
Experimental design: NA
Validity of the findings: NA
Additional comments: NA


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: CYP2C11 PLAYED A SIGNIFICANT ROLE IN DOWN-REGULATING RAT BLOOD PRESSURE UNDER THE CHALLENGE OF A HIGH-SALT DIET
Review round: 1
Reviewer: 1

Output:
Basic reporting: The manuscript entitled "CYP2C11 played a significant role in down regulating rat blood pressure under the challenge of a high-salt diet" by Wei Liu et al. is a good observational manuscript. The rat model for CYP is crucial to decipher the salt diet and blood pressure relation. However, the mechanistic insight is lacking, which may be acceptable since it is first correlational study. Language of the manuscript is clear and satisfactory with very few grammatical errors. I do recommend it for publication only if authors address the following concern list below.
Experimental design: I have few concerns with experimental design, which I had listed below. qPCR should have been performed for all four genes. Explanation of author for not performing qPCR for two other genes is not acceptable.
Validity of the findings: Findings may have a broader impact.
Additional comments: Line 29: Our not our
Line 30: remove ‘a’

In result section: Authors are advised to compare each subject sequentially throughout the manuscript. For eg. If you are comparing male and female data as in line 150 and 151, keep the sequence same in line 153 and 154 and not jumble it like female and male.
In feeding duration of 2 months, did authors have checked the change in weight pattern of mice? Also, what was the weight of mice at the time/day of measuring blood pressure?

In the western blot result:
1. There is no loading control in figure 2, although author explained that common control proteins like β-actin and GAPDH are absent from renal microsomes, Ponceau staining of blot can be used instead for the same.

2. Without quantification of western blot signal, it is difficult to conclude anything from the data. Quantification of blot signal is required for comparison and can be done by freely available NIH ImageJ software.

3. Also mention the molecular weights in figure 2 themselves, even it is mentioned in legend. Describe each lane individually since it is unclear whether the samples were loaded in duplicate in adjacent lanes or samples were from two individual?

4. Please explain in detail the microsome isolation in the methods section, although reference was cited for the same.

5. Did author have used any quality control for the purity of microsomal fractions? Since, β-actin and GAPDH are not supposed to be present in microsomal fractions, western blot with these antibodies could serve as an excellent control for the purity of microsomes.

Line 163: CTP2C11 or CYP2C11?

In qPCR:
Author chose to quantify the renal mRNA expression level of CYP2C23 and CYP2C24 genes only, since antibodies of these two proteins were not present. It would be more comprehensive if mRNA expression level of all the four genes i.e, CYP2C23, CYP2C24, CYP2J, and CYP4A were analyzed and western blot of CYP2J and CYP4A can complement the findings.

Immunohistochemistry section needs more attention. Author should explain their result and observation in more detail. Authors have labeled each figure but didn’t explain any one of them in result section. For which parameters, images were analyzed by image-pro plus version 6, explain them in detail in result section. Also, convert table 2 in bar diagram and merge with figure 4.


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: CYP2C11 PLAYED A SIGNIFICANT ROLE IN DOWN-REGULATING RAT BLOOD PRESSURE UNDER THE CHALLENGE OF A HIGH-SALT DIET
Review round: 1
Reviewer: 2

Output:
Basic reporting: Liu el al, investigated the in vivo effect of CYP2C11 function in epoxyeicosatrienoic acids (EET)-mediated blood pressure regulation and showed that high-salt diets only elevate rat blood pressure in CYP2C11 knockout rats. Authors conclude that high salt alone is insufficient for blood-pressure increase and rats without CYP2C11 are more vulnerable to the challenge of high-salt diet. Authors investigated the expression of various P450s involved in renal Arachidonic acid (AA) metabolism, including CYP2C23, CYP2C24, CYP2J, and CYP4A. Overall authors try to understand the important of cytochrome P450s  in regulating blood samples. Manuscript is written clearly with sufficient background, however I have concern for the conclusions drawn from the current data.
Experimental design: Methods are described with sufficient detail and information.
Validity of the findings: Authors should add sufficient detail in figure legends and also add information about the controls used in each experiment.
Additional comments: Comments :
(1). "Under a normal lab diet, CYP2C11-null and WT rats did not differ in blood pressure. However, a high-salt diet significantly increased blood pressure among CYP2C11-null rats compared with a normal diet. The increase in CYP2C11-null females was significantly higher than in CYP2C11-null males and WT females". Can authors  describe/speculate the reason for this variability ?

(2). Figure 2, under high-salt diet, renal CYP2J2 expression increased both in male and female CYP2C11-null rats compared with WT rats. Authors suggest no significant effect on CYP4A renal expression, however in CYP4A lanes, I can see two very undifferentiated bands both in male and female samples and these bands intensity increased under high-salt condition.
My concerns are following :
a) Authors should show loading control for each sample
(b) Quantify the data by normalizing to loading control
(c) Gel should be run longer to see whether there is single or two bands for CYP4A. These could be two isoforms of  CYP4A. After quantifying data, authors can reach to conclusion that there is no change in CYP4A level under high salt condition.
(d) What about the protein expression for CYP2C23 and CYP2C24 ?

(3).  In figure 3, authors suggest that after knocking out the CTP2C11 gene, renal CYP2C23 and CYP2C24 mRNA did not differ between CYP2C11-null and WT rats and diet had no effect on mRNA expression. However, I observed that authors have different scales for y-axis in each sample, hence it is difficult to compare the expression level by looking at current graphs. By looking at data in current graphs, I see that expression level of CYP2C23 is lower under high salt diet whereas expression level of CYP2C24 is high in both null mutants.  To make right conclusion about the expression level and significance change, authors should have similar Y-axes and show the mean value/or other parameter for each sample.  How the mRNA levels correlate for CYP4A and CYP2J2?

(4). It is difficult to follow the variability in figure 4. Authors should add arrows to indicate variability and also add description in the figure legend.


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

Input:
Title: CYP2C11 PLAYED A SIGNIFICANT ROLE IN DOWN-REGULATING RAT BLOOD PRESSURE UNDER THE CHALLENGE OF A HIGH-SALT DIET
Review round: 2
Reviewer: 1

Output:
Basic reporting: The manuscript has improved significantly, but still need few improvement:
Result:
Results subheading should be conclusive as in line 160, so make changes accordingly in all further result subheadings.
Experimental design: Not applicable
Validity of the findings: Already provided.
Additional comments: The explanation for the comment mentioned below is still not substantial to consider for the purity of microsome fractions. Both the articles recommended by author doesn't provide the protocol for microsomes isolation and their purity and above that referred paper cite the other paper for the protocol. I would suggest the author to refer the correct paper and methodology and proper controls and accordingly mention them all in method section.

5. Did author have used any quality control for the purity of microsomal fractions? Since, β-actin and GAPDH are not supposed to be present in microsomal fractions, western blot with these antibodies could serve as an excellent control for the purity of microsomes.
We didn’t use any quality control for the purity of the microsomes. This method was learned from my Ph.D. advisor Dr. Xinxin Ding (Department of Pharmacology and Toxicology, University of Arizona) and had been used for many years. This method worked well in our previous studies. (For example: Wei et al., 2010; Wei et al., 2018; etc.)


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: CYP2C11 PLAYED A SIGNIFICANT ROLE IN DOWN-REGULATING RAT BLOOD PRESSURE UNDER THE CHALLENGE OF A HIGH-SALT DIET
Review round: 2
Reviewer: 2

Output:
Basic reporting: Manuscript is clearly written.
Experimental design: In revised manuscript, authors have added relevant detail in method section
Validity of the findings: Data is robust and conclusions are well stated.
Additional comments: Relevant changes have been included in revised manuscript.


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

Input:
Title: NEXT-GENERATION SEQUENCING OF BRCA1 AND BRCA2 GENES FOR RAPID DETECTION OF GERMLINE MUTATIONS IN HEREDITARY BREAST/OVARIAN CANCER
Review round: 1
Reviewer: 1

Output:
Basic reporting: See below
Experimental design: See below
Validity of the findings: See below
Additional comments: Thank you for asking me to review the manuscript by Nicolussi et al. entitled: “Next-Generation Sequencing of BRCA1 and BRCA2 genes for rapid 2 detection of germline mutations in Hereditary Breast/Ovarian Cancer”

The study describes the use of Ion Torrent (semiconductor sequencing) technology to sequence germline DNA for BRCA1/2 variants. This providers of this technology claim that it provides a cheaper and faster method of detecting variants.
Nicolussi et al. show that the technology can be used to detect germline BRCA1/2 variants in the training set (n=11) and then validate the technology on a large series (n=136 probands), with additional Sanger sequencing of 60 samples as a gold-standard sequencing techniques.
The authors show for SNV/indels detection concordance with Sanger sequencing of 100% (excluding LGRs). The authors also detect 30 BRCA1/2 pathogenic variants in the VS, and confirmed these variants using Sanger sequencing.

Major points:
•	Introduction: The introduction needs more explanation of the Ion Torrent PGM beyond… “The Ion Torrent PGM is a new sequencing platform that substantially differs from other sequencing technologies by measuring pH rather than light to detect polymerization events.”
•	Results: Could the authors clarify if they detected any pathogenic BRCA1/2 variants using Sanger sequencing that were not detected using the Ion Torrent technology please? I.e. False-negative rate.
•	Results: Are the authors able to provide the sensitivity, specificity, PPV and NPV for SNV/indels in a table format with Sanger Sequencing as the gold standard?
•	Results: There was no information/discussion regarding time the assay took and costs, until it is briefly mentioned in the conclusion.
•	Discussion: The discussion needs more emphasis on the broader implications of using the technology within a laboratory e.g. why should we change our current platforms to use this technology?
•	Discussion: The general conclusion of the paper is that Ion can be used to detect SNV, indels with excellent sensitivity, but fails to detect LGR. Therefore MLPA should be continued to be used as a method for detecting LGRs. This conclusion needs clearly stating.

Minor points:
1.	The following reference should be included regarding the risk of breast/ovarian cancer in BRCA1/2 carriers – Kuchenbaecker et al. JAMA 2017
2.	The introduction requires a sentence regarding the clinical utility of germline BRCA1/2 testing for breast and ovarian cancer therapies (please see following clinical trials: SOLO1, SOLO2, ARIEL3, NOVA, EMBRACA, OLYMPIAD – all phase 3 PARPi trials).
3.	Is studio the correct terminology in this statement? “The BRCA1 and BRCA2 panel used in this studio generates 167 amplicons that cover all targeted coding exons and exon-intron boundaries”
4.	A flowchart for the methodology would be nice as the description is very detailed
5.	In the discussion, could the authors make a comment on whether they feel this technology can be applied to DNA extracted from breast/ovarian cancer tumour tissue please?
6.	In Table 1, please change the LGR DNA level to describe the Exon deleted instead (e.g. Exon 13 duplication).
7.	In Table 1 and 4, the “reference ID according to NCBI” can be removed as this is irrelevant
8.	In Tables 1 and 4, please define what the column “quality” means?
9.	In Table 4, a number of the variants are defined as indels, but they are actually small deletions or duplications, not insertion-deletions
10.	In Table 4, the variant c.4131_4132insTGAGGA is not “complex” – it is an “insertion”
11.	In Figure 4, where any other family members tested for this variant in germline/tumour DNA as part of segregation analysis?


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: NEXT-GENERATION SEQUENCING OF BRCA1 AND BRCA2 GENES FOR RAPID DETECTION OF GERMLINE MUTATIONS IN HEREDITARY BREAST/OVARIAN CANCER
Review round: 1
Reviewer: 2

Output:
Basic reporting: The manuscript includes a relevant topic and introduces the rationale for the project. Overall the manuscript reads well and follows the standard manuscript structure. A more detailed legend for Figure 1 D en F would be helpful, especially as the have the appearance of a boxplot while they depict other type of information with a SD. A minor detail is the varying use of the terms mutations and (pathogenic) variants, which may be confusing.
Experimental design: -The aim of the study could be clarified. The aim stated in abstract and in the introduction seems to differ slightly. The final sentences of the introduction read as a conclusion rather than a study aim.
-The method section provides structured information of the different analyses steps. The outcomes measurements of the comparison of both methods are not included. Please include a short statements on this.
-The distinction between the training set and validation set is clear. Though it would be useful to clearify the rationale for the different number of patients included in various analyses (e.g. subset of 11 TS + 60 VS samples for Sanger sequencing [methods]; a critical analyses of coverage in 73 VS sample [results]).
Validity of the findings: -The detection of all types of variant is evaluated between the NGS method and the conventional methods (sanger/MLPA), and therefore the training set includes SNVs, indels and large genomic regions. However, the methods and conclusion of the comparison are based on the SNVs or indels detected by NGS method versus Sanger sequencing. This should be clarified in the aim and methods of the manuscript, and the impact of missing the LGRs should be addressed in the results and discussion section.
-The conclusion of the study includes statements on the performance regarding costs and turn around times, which has not been addressed in the main text as outcome measurements and data.
-The validation set consists of 136 samples, of which 30 with a pathogenic variant, 2 VUS, and 60 no pathogenic variant. What were the findings for the other 44 samples? In case these where cases without pathogenic variants and a less extensive personal and family history of cancer, please discuss why for this group no additional Sanger sequencing has been performed as BRCA1/2 variants are present in patients with less suspect phenotypes.
Additional comments: The authors address an important topic. Given the increasing demand for genetic diagnostics, improvement in the efficient use of means and time, meanwhile ensuring quality, is a pressing issue.


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: NEXT-GENERATION SEQUENCING OF BRCA1 AND BRCA2 GENES FOR RAPID DETECTION OF GERMLINE MUTATIONS IN HEREDITARY BREAST/OVARIAN CANCER
Review round: 1
Reviewer: 3

Output:
Basic reporting: 1. Well structured paper with clearly written methods, results and discussion.

2. Whilst the novel variant you described (c.72delA) is an interesting finding to report, I did not find that it was completely necessary to include the pedigree (Fig 4) in your main manuscript. Could this be included in supplementary material? I would also be interested to know if the variant segregated with the affected family members in Family BR1270.

3. Could you include criteria used to enrol the probands for BRCA1 or BRCA2 sequencing in either the main manuscript or supplementary material.

4. Last paragraph of Results section, lines 243 to 252: Some elements of this paragraph may be better suited in the Discussion section where you are interpreting the significance of your findings in light of your research. You could therefore make the last section of your results more concise and fact-based.

5. Your discussion states that the sequencing technology has 100% sensitivity if large genomic rearrangements are counted as false negatives. Are there any other methods you could undertake or suggestions that you have to improve the detection of LGRs?

6. Please check references for spelling errors.
Experimental design: 1. You have mentioned the guidelines for variant interpretation from Plon et al, 2008 in the analysis of the novel variant. We use American College of Medical Genetics guidelines for variant interpretation (2015) in our local centre and I therefore wondered why you were not using more updated guidelines for your variant analysis?
Validity of the findings: No comment
Additional comments: No additional comments


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

Input:
Title: NEXT-GENERATION SEQUENCING OF BRCA1 AND BRCA2 GENES FOR RAPID DETECTION OF GERMLINE MUTATIONS IN HEREDITARY BREAST/OVARIAN CANCER
Review round: 2
Reviewer: 1

Output:
Basic reporting: Please see below.
Experimental design: Please see below.
Validity of the findings: Please see below.
Additional comments: The authors have addressed all points raised, thank you.


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: NEXT-GENERATION SEQUENCING OF BRCA1 AND BRCA2 GENES FOR RAPID DETECTION OF GERMLINE MUTATIONS IN HEREDITARY BREAST/OVARIAN CANCER
Review round: 2
Reviewer: 2

Output:
Basic reporting: The authors have addressed the previous remarks.
The supplemental files do not include any table/figure number, title and description. Please add these.
Experimental design: No comment
Validity of the findings: No comment
Additional comments: No additional comments


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

Input:
Title: EXTINCTION AND THE U.S. ENDANGERED SPECIES ACT
Review round: 1
Reviewer: 1

Output:
Basic reporting: Manuscript is written in a clear, and concise manner using professional language. Text is very direct ,easily understood and answers the questions posed by authors.. References cited are sufficient but more detaile discussions of P of survival of Hawaiian birds are available.

An easy read.
Experimental design: Suffient to the task and well defined.
Validity of the findings: Findings are defensible but I believe a bit conservative regarding status of Hawaiian birds e.g. Kamao Ou and Kauai Oo.Conclusions are well linked to original  research question. They set up a more detailed follow up paper in which effort to locate "extinct species was quantified.
Additional comments: Greetings

Please consider the following comments
1) Lines 58-61 REcognizing that there will be wide variation in the answer discuss briefly what are expected time to recover after listing. For a quick assessment of  the question you could use previously delistedy species as your data base.2) Page 2 lines 139 -142 provide reference


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: EXTINCTION AND THE U.S. ENDANGERED SPECIES ACT
Review round: 1
Reviewer: 2

Output:
Basic reporting: The basic reporting is good.
Experimental design: I have questions regarding the methodology, which are discussed in my review.
Validity of the findings: Due to my concerns about the methodology, I am not convinced of the findings.  See details in my review.
Additional comments: This is a very important topic, and the authors are to be commended for tackling it in a quantitative fashion.  However, contrary to the assertion on lines 72-73 that such a study has not been done previously, there was a paper addressing this same issue that reached some of the same conclusions (see: McMillan and Wilcove. 1994. Gone but not forgotten: why have species protected by the Endangered Species Act become extinct? Endangered Species UPDATE 11(11): 5-6).  But it was published in an obscure journal, so it’s understandable why the writers have overlooked it.  Moreover, this manuscript by Greenwald et al. is more comprehensive and also attempts to calculate the number of species whose extinctions have been prevented by the ESA.

With respect to the first part of the manuscript – determining how many and which species have gone extinct before ESA listing and after ESA listing – I have the following questions:

1.	 The authors state that 71 of 97 extinct (EX) or possible extinct (PE) species were last observed before they were listed and thus “are not relevant” to determining the Act’s success in preventing extinctions.  These 71 species were last seen anywhere from 1-80 years prior to listing.  It strikes me that unless there were fairly intensive searches of the habitat immediately prior to or immediately after listing, it is not reasonable to exclude these species from an assessment of the Act’s success.  That a species was last seen 1, 2, or 10 years prior to listing is, in itself, uninformative, unless we know that serious efforts were made to find it right before or after listing.  The authors may well have factored this into their determinations but it is unclear from the text and SI that they did so.
2.	Of the 26 species that disappeared after listing, the authors note that 8 had total populations <10 individuals at time of listing that their survival was always questionable.  This could be fleshed out more: what was the distribution of population sizes at time of listing for these 26 species?

With respect to the second part of the manuscript – calculating how many species extinctions have been averted by ESA listing – I have several concerns regarding the methodology:

1.	An underlying assumption of the methodology is that the ESA designations of Endangered and Threatened can be equated to the IUCN Red List designation of endangered, with the associated probability of persistence.  This cannot be assumed; it must be tested.  The Red List criteria are based on quantitative criteria related to population size and number, rate of decline, and or of habitat loss, which (allegedly) are they linked to quantitative assessments of probability of extinction as assessed via PVAs.  However, the statutory definitions in the ESA of “Endangered” and “Threatened” are notably vague; nor, to the best of my knowledge, has FWS set about establishing quantitative guidelines as what puts a species in one status versus the other.  In the absence of such guidance, the authors need to look at the population sizes or rates of habitat loss of T&E species and see if those values more or less align with the IUCN criteria.
2.	The equation they use for determining the null model – how many species would have disappeared in the absence of ESA protection – is as follows:

Expected extinctions = (Spp. X 100 year Extinction Risk X Portion of a century with protection)

Are they using 45 years as the numerator in determining the proportion of 67 years that a species has enjoyed ESA protection?  If so, this puzzles me because species only get that protection once they are listed, and they have been added to the list at varying times.  Thus, should not the null model use this equation and make a separate calculation for each species based on how long it has enjoyed ESA protection and then sum the results to derive the expected figure for how many species should have perished in the absence of ESA protection?  Perhaps this is what they did, although I cannot find confirmation of this in the material sent to me.  (And, of course, this does not negate my belief that the foundation of the calculation may be invalid for the reason outlined in the previous point).

Finally, the authors allude (lines 186-7) to steps FWS can take to streamline the listing process.  If space permits, it would be great to provide at least a summary of these proposed changes.


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: EHEALTH ADOPTION AND USE AMONG HEALTHCARE PROFESSIONALS IN A TERTIARY HOSPITAL IN SUB-SAHARAN AFRICA: A QMETHODOLOGY STUDY
Review round: 1
Reviewer: 1

Output:
Basic reporting: The overall reporting was good. The authors should consider the following:

Abstract
To ease readability and comprension, it would be great to structure the abstract. Authors should consider PeerJ’s example format of (Background, Methods, Results and Discussion) to structure the abstract.

Introduction
Spell out the abbreviation HCP (see line 60) at first use
Space in beginning of line 64; please edit it
Line 66. Should show or cite literature that suggest that eHealth promotes "efficiencies in the provision of quality of healthcare gain of credibility among patients"

Line 71, sentence seem incomplete

Should consider the citation of appropriate literature to highlight eHealth adoption in SSA, including its barriers and challenges?

Revise the sentence in line 95…This they said…a bit awkward
Same as 97…awkward sentence…same as line 99
Line 115...mentioning the management of the area of study at this part of the narrative is a bit confusion. First study setting must be described. Second, it sounds more of anecdotal instead of a generalized challenge…sentence is a bit awkward…HCPs can still function adequately without an e-health environment…can you please explain further?

It appears authors have been using eHealth and e-health (see line 116) interchangeably. Should consider adopting one and use consistently throughout the manuscript

The motivation for the work could be made clearer. What is the goal of this study? Is it to understand the viewpoints of HCPs in SSA as regards the adoption of eHealth? Why is the Qmethodology important in this context?
Experimental design: Methods
Move the aim of the study (line 151) to the last paragraph of the introduction, if you can.
Use the methods section to focus on what you did to achieve this aim.

Describe the study setting or host institution/facility. What was the rationale for choosing this facility?
Line 155. What was the inclusion/exclusion criteria for your study cohort? What is the size of the general population from which the sample was drawn?

Can you provide further details to the Q-method? For instance, what is the meaning of an eigenvalue in the context of the Q-method? What is the value range and their meaning?
Explain the meaning of the statement numbering and corresponding ranking methodology—e.g., (statement number, ranking)—and what they mean to your factor analysis results, including its interpretation.
Validity of the findings: Results
Reconcile the labeling of Table 3 and “Table 1” (list of appended tables) to properly point to the reference on 206.
Line 223…you meant “accessing” instead of “access”?

The viewpoints of HCPs in one facility in Nigeria cannot be generalized for the the entire SSA, as portrayed in the title and conclusion. Please provide an explanation for this generalization.
Additional comments: In general, the paper is good. Please ensure that the study objective(s), methodology, findings, and conclusion are well-aligned.

Tables must be organized, and cited for the first time, in ascending numerical order, as recommended by PeerJ; the table numbering were confusing…too many of them were labeled as “Table 1”

What are the limitations and potential future work? A sample drawn from one institution in Nigeria cannot be blindly used to generalize for the entire SSA population.


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: EHEALTH ADOPTION AND USE AMONG HEALTHCARE PROFESSIONALS IN A TERTIARY HOSPITAL IN SUB-SAHARAN AFRICA: A QMETHODOLOGY STUDY
Review round: 1
Reviewer: 2

Output:
Basic reporting: Title
The title sounds like misleading. It should clearly reflect what the authors assessed i.e. factors influencing eHealth adoption and use among healthcare professionals. If left the way it is now, it would mean that the authors assessed also the actual use, which was not the case. Considering the small sample size of the HCPs and the institution involved in the study being one, creates a huge doubt whether that can represent SSA. Because of the existing diversities in ICT infrastructure in SSA countries I suggest the authors to edit their title (remove SSA). It is too much exaggeration of the representation of the study population.

Quality of writing
1.	The authors should also improve the quality of writing… there is a lot of long and hanging sentences. Hanging in the sense that, some sentences appear like paragraphs but are just single sentences. Example: Line 111 – 114; 115 – 118 etc.
2.	There are three table 1, no table 2 and the number of the tables are not in chronological order in the manuscript. The title of the tables should be well written.
3.	Chronology: data collection – ethics and – then analysis. You may need to consider rearrangement.

Discussion
Although the study was done in Africa (SSA), most of the references cited by the authors were those from outside Africa. Because of the differences in ICT infrastructure between SSA and western world, comparing assessments between these two different worlds may not be illogical. I recommend the authors to compare with studies done on the continent even if did not use the Q-methodology. You can also consider citing the following reference:	Nyamtema A, Mwakatundu N, Dominico S, Kasanga M, Jamadini F, Maokola K, Mawala D, Abel Z, Rumanyika R, Nzabuhakwa C, van Roosmalen J. Introducing eHealth strategies to enhance maternal and perinatal health care in rural Tanzania. Maternal Health, Neonatology, and Perinatology (2017) 3:3. DOI 10.1186/s40748-017-0042-4.
Experimental design: Methods
Study site and population: It was not clear which eHealth strategies were the HCPs experienced with. ?HMIS, teleconsultation, eLearning contents/ resources, soft copies of clinical job aids and management guidelines etc. It would have added value to the reader to know exactly the spectrum of the eHealth strategies which are referred in this study. Equally important, it was not clear how the eHealth strategies were introduced in this institution, whether there was any kind of training on it before and after introduction of the strategies. The failure to adopt and use of eHealth could be attributed to the methodology used to introduce it. Although, this may be beyond the scope of this study, the reader would have loved to know the actual use of the eHealth in this institution.
Validity of the findings: Limitations of the study
Limitations of the study based on the Q-methods and how the eHealth was introduced in this institution should be added in the discussion. The authors will agree with me that the purposive selection of experienced HCPs poses a threat to the trustfulness of factors influencing adoption and use of eHealth. The q-method depends on the cooperation and truthfulness/ honesty of the respondents. All these may create some degrees of bias.
Additional comments: No comment


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

Input:
Title: EHEALTH ADOPTION AND USE AMONG HEALTHCARE PROFESSIONALS IN A TERTIARY HOSPITAL IN SUB-SAHARAN AFRICA: A QMETHODOLOGY STUDY
Review round: 2
Reviewer: 1

Output:
Basic reporting: OK
Experimental design: OK
Validity of the findings: OK
Additional comments: I'm satisfied with the corrections by the authors.


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: EHEALTH ADOPTION AND USE AMONG HEALTHCARE PROFESSIONALS IN A TERTIARY HOSPITAL IN SUB-SAHARAN AFRICA: A QMETHODOLOGY STUDY
Review round: 2
Reviewer: 2

Output:
Basic reporting: In my view the title is still misleading. What the authors did was to explore the viewpoints of healthcare professionals on the adoption and use of eHealth in clinical practice, but the title reads ‘eHealth adoption and use among healthcare professionals …’. By reading the current title the reader would expect to see results on the adoption and the actual use of eHealth. Unfortunately, this is not the case. The authors did not assess the actual use of eHealth and how the eHealth was adopted. The title should represent what the authors exactly did ‘viewpoints of HCP on … or factors affecting adoption and use of eHealth’ or something like that.
Experimental design: Acceptable
Validity of the findings: Limitation of study
The authors say that ‘The limitations for the study has been included (see line 500-503).’ On the contrary these lines do not talk anything related to the limitation of the study.
Additional comments: None


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: PHYSIOLOGICAL RESPONSES OF OXYRRHIS MARINA TO A DIET OF VIRALLY INFECTED EMILIANIA HUXLEYI
Review round: 1
Reviewer: 1

Output:
Basic reporting: Article well written and very interesting, but the results are ambiguous requiring additional experiments to validate the interpretations.
Literature, figures and tables are well presented.
Experimental design: Study requiring additional experiments to validate the interpretations as I elaborate below.
Validity of the findings: Please see my comment in the ' General comments for the author' section
Additional comments: In this study entitled ‘Physiological responses of Oxyrrhis marina to a diet of virally infected Emiliania huxleyi’ Goode and coauthors assessed the prey-predator interplay between the important, cosmopolitan bloom-forming phytoplankter, E. huxleyi, and O. marina a heterotrophic dinoflagellate frequently used as model microzooplankter in laboratorial assays. The results of the study performed in vitro using culturing and cell biology/biochemical approaches broadly showed, according to the authors, that O. marina displays higher grazing rates of non-infected E. huxleyi (Ehux) cells relative to infected cells, but contrastingly O. marina display higher growth rates when fed on infected cells. The mechanisms underlying these experimental observations are unknown, however the results are interesting and relevant leading the authors to suggest that virally infected Ehux can support larger growing populations of single celled heterotrophs and enhance carbon transfer to higher trophic levels.
From my view point this study highlights a very interesting phenomenon (although a previous study by Evans and Wilson have shown contrasting results - the authors acknowledge this fact) and clearly opens a new avenue of research. The study has various limitations when extrapolating to processes that might happen in natural bloom conditions. For example: 1) O. marina is not a common grazer during Ehux blooms (see Olson and Strom 2002) and therefore a more realistic response to natural predators remains unknown; 2) The Ehux strain (CCMP374) used is non-calcified. Yet, during bloom most cells appear to be calcified even during the bloom-demise phase and although noncalcified cells are produced probably as a result of viral infection these are minor in field populations (see Frada et al. 2012). A calcified strain should have clearly been used by the authors. Moreover, technically it does not seem to me that only 10 cells are sufficient to estimate significantly the average volume size of cells of a microbial population (see the large standard deviations obtained - How O. marina kept identical cell volumes while growing faster in the presence of infected cells? And it is not clear why infected Ehux cells were not included in the fatty acid (FA) analysis and how the authors distinguished between O. marina FA from the FA of prey FA in the food vacuoles during feeding conditions (where they starved prior to analysis?). Nonetheless, I consider that these represent some details that could either be mitigated by the authors here (some of them where discussed) or explored and developed in subsequent studies. However, what concerns me the most is the main result of the study, i.e. O. marina consumes less but faster in the presence of infected cells. As the authors suggested, this may indeed result from a higher nutritional value of infected cells or higher assimilation efficiencies, but it may have also resulted from additional processes that should have been considered by the authors. Specifically, O. marina can survive through the uptake of dissolved organic molecules in the laboratory and may be able to use this mechanism in saprobic environments (see Lowe et al. 2011) and can also feed on bacteria (Jeong et al. 2008). During a current lab or field Ehux infection, there are expected buildups of dissolved organic matter due to viral-mediated cell lysis, bacteria (cultures are not axenic) and off course virions in the medium (which can also be consumed by microzooplankton (see Gonzales and Suttle 1993), and possibly also by O marina as well). Thus, it is possible that the apparent reduced grazing on infected cells resulted in a shift in grazing preferences of O. marina to other highly abundant sources which could have supplemented enhanced growth rates of the grazer relative to cultures solely supplemented with non-infected cells. If this is the case the results and subsequent interpretation of the data will considerably change namely by emphasizing the importance of the viral shunt. Thus, I feel that an additional experiment testing this scenario assessing for example O. marina performances in conditioning medium from infections should be requested to the authors prior to publication.

A few more details:
Line 102: why O. marina was not maintained with Ehux 374 pior to the experiments? Please clarify.

line 43: Incorrect sentence. Modelling work – see Weitz et al. 2014 ISMEJ - show that viral infections led to an increase in productivity. Or are you referring productivity of the specific (target) host cells?

line 75: Full stop missing after “Martínez Martínez 2006)”.

Line 216: Please remove “and” between images were … measured.


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: PHYSIOLOGICAL RESPONSES OF OXYRRHIS MARINA TO A DIET OF VIRALLY INFECTED EMILIANIA HUXLEYI
Review round: 1
Reviewer: 2

Output:
Basic reporting: Introduction
The introduction provides a good background to the study context, however there are a few references missing. For instance, on line 78 – 80, where microzooplankton contribution to grazing is mentioned, there is no reference to the Meta-analyses conducted by Calbet and Landry, 2004 or Schomker et al., 2013 that demonstrated that microzooplankton consume 62 – 67% of daily primary production.
Line 43: There has been some recent papers which have shown that viral lysis can lead to enhancement of primary production by releasing limiting nutrients. See (Shelford & Suttle, 2018, Biogeosci, 15, 809-819, Poorvin et al., 2004, Limno. Oceanogr, 49, 1734-1741, and Shelford et al., 2012, Aquat. Microb. Ecol. 66, 41-66).

Figures
Figure 1; Fig 1 should have a legend for each of the shapes and virus: host ratios. The data from this figure contrasts with line 244 – 246 where it is said after 24 hours close to 100% of E. huxleyi cells were infected, whereas this figure shows <60% were at 20h p.i.
Figure 2; On Fig 2b the letter c is above the third bar, it is not explained what this means, or if it should just be a b.
Figure 3; Fig 3d and e show the predicted model of growth and ingestion on E. huxleyi. The concentrations after 5 days for E. huxleyi ingestion (~3 x 106 (again assumed cells mL-1)) suggests an almost endless supply of E. huxleyi. It is also not clear whether the results are cumulative or per time point as O. marina abundance increases. This should be made clear in the text or figure legend.

Results
Line 273; it is not immediately clear from the Figure that the different experiments represent different end points of the experiment (if this is correct). I would suggest the authors add a comment to this in the figure text.
Line 291 – 292: In table 2 there is no data shown on recently infected E. huxleyi populations to back up this statement as the results have been combined.
Experimental design: Methods
Line 94 – it is said that the cultures are non-axenic, however there is no mention throughout the manuscript on bacterial abundances throughout experiments. This has a number of important implications, particularly as O. marina is able to feed on heterotrophic bacteria (see Jeong et al., 2008., Euk. Microb. 55,4, 271-288). This could affect the growth rates of O. marina if they are ingesting both prey types. Particularly as the release of DOM from lysed E. huxleyi cells could stimulate bacterial production.
On line 109-110 the method for cell and virus concentrations is reported, however the method for counting viral particles is not specifically reported (typically through SYBR-green or another stain, followed by heating and enumeration on a flow cytometer).
I think it would be useful, if O. marina was counted by flow cytometry to see an example plot in the supplementary information. From literature, and personal experience, it can be difficult to gain accurate numbers of dinoflagellates without using probes (e.g. acidotrophic stains).
It is not mentioned throughout the method section the volume of the experimental manipulations, rather just cell concentrations are reported. It would be useful to know this information, particularly to see if the volume removed from sampling is significantly reducing the volume in the flasks used.
Line 122 – 123; the authors did not specify where they used a 50:1 virus: host ratio for experiment 4, whereas all others used the 100: 1. This would complicate comparisons between experiment 4 and 1, 2 and 3. In a similar manner, E. huxleyi : O. marina ratios were different in experiment 1 which would affect the grazing rates measured. If there was a particular reason for this, it needs to be made clearer to the reader.
Line 125 – 126; the sentence is confusing with the use of the word “immediately”, this implies that there was a T0 sample taken, however this does not seem to be the case, and samples were only take 5h and 24h p.i. If T0 samples were not taken, it should be specifically mentioned in the text that the concentrations were assumed based on volume.
Line 177; the population size of O. marina is assumed 6,000 cells mL-1, but it is not defined here.
Line 177 – 178; the initial conditions used for predictive modelling of population changes uses a number of assumptions, which are not described. Firstly, the use of 6,000 O. marina cells (assumed as mL-1) as a starting density is very high, given that within field populations during E. huxleyi blooms (See Fileman et al., 2002) the maximum observed dinoflagellate abundance is 57 cells mL-1, and maximum within Olson & Strom, 2002 was ~200 cells mL-1. Secondly, the authors assume prey saturation, but provide no value for this. These conditions would need to be better defined if this is to be used within the manuscript.
On line 212 the authors report they decided to use the fixative 70% ethanol, and the results were similar to nickel sulfate in Menden-Deuer et al. (2001). Unfortunately, no comparison of methodologies was carried out, but it could be useful for the authors to compare with published values of O. marina sizes, to help prove their method.
Line 216, a word is missing between “Images were ( ) and ….”
It is not specifically mentioned if the authors included a monoculture of O. marina as a control population for any of the experiments. In addition, a control of O. marina with EhV lysate, just to be sure there is no impact of the heterotrophic dinoflagellate on the viral populations.
Validity of the findings: Results
Line 244 – 246: The authors mention that close to 100% of E. huxleyi cells were infected, even if not evident from FCM. It is not clear how this is assumed? An erroneous assumption here would be that all viruses produced are infectious, as there has been no evidence to suggest this (to my knowledge).

Discussion
Line 307 – 309: The fact there were no measures of bacterial abundance within the experiments means the authors cannot discredit the impact this may have had on O. marina growth rates. It should be specifically mentioned here.
Line 315 – 317: Did the authors measure a viral lysis rate from control E: huxleyi and EhV controls? This could be removed from the measured results to estimate the grazing impact of O. marina. This loss of E. huxleyi could also lead to enhanced DOM and bacterial production, as mentioned above.
Line 323 – 324: Although E. huxleyi do lose their coccoliths during infection (as seen in Frada et al., 2008) this was due to the shift from a diploid to haploid life style (“Cheshire Cat hypothesis”).
Line 337 – 338: Reference missing here of Hunter et al., 2015. Front. Mar. Sci. 2, 81 regarding lipidome changes during E. huxleyi infection.
Line 341 – 342: Same comment as above regarding sphingolipids.
Line 345 – 346: This conclusion is not properly shown in the results, especially given that they appear to be combined in Table 2.
Line 353 – The paper above by Hunter et al., 2015 displays changes in lipids during infection of E. huxleyi, therefore these could contribute towards the growth rate differences, however this paper isn’t referenced.
Line 378 – 379: Population and bloom dynamics of E. huxleyi by microzooplankton has been shown in a paper by Mayers et al., 2018. Prog. Oceanogr. doi: 10.1016/j.pocean.2018.02.024, in bloom and non-bloom conditions within the field.
Line 400 – 401: Trophic upgrading was not shown for all microzooplankton (currently only dinoflagellates). No trophic upgrading was displayed for a ciliate feeding on Dunaliella (Klein Breteler et al., Mar. Eco. Prog. Ser. 2004. 274, 199-208.
Additional comments: This paper by Goode et al, provides evidence for the impact of grazing by the dinoflagellate Oxyrrhis marina on virally infected and non-infected populations of the coccolithophore Emiliania huxleyi. This is an important study, as it focuses on the dual nature of different mortality pathways, which are often considered in isolation during laboratory experiments. Their results that O. marina growth rates are enhanced when feeding on infected cells is interesting, giving that this will affect E. huxleyi population dynamics within bloom scenarios.
The English is in general, well written throughout the manuscript, and ideas are conveyed well to the reader.
There are however a few methodological questions which are raised, primarily because bacterial abundance was not quantified throughout the experiment. The lysis of infected cells could lead to enhanced DOM release and enhanced bacterial growth, which could be leading to the higher growth rates observed by O. marina. However, no mention to this is present throughout the paper, in the methods, results or discussion.
In general, the methods section was a little difficult to understand on a first read, and they could perhaps be better explained to guide the reader through the procedures used.
In the methods section it is not described what piece of equipment the experiments are conducted in. The authors also do not describe if the incubation apparatus is maintained on a shaker, or is occasionally mixed to reduce settling of the material within the experiment.
With the fatty acid analysis conducted, the samples collected after 3 days would be a mixture of E. huxleyi and O. marina, as no size fractionation was conducted of the culture. This makes it difficult to compare with the initial analysis and to draw conclusions on how the fatty acid profiles differ. It should be specifically mentioned in the results and discussions section that we could not draw a conclusion from which portion of the community the results are.
Greater controls are needed within this experiment, for instance, having O. marina cultured with EhV filtrate. This would help eliminate any questions regarding whether O. marina is affected by viruses, or vice versa. This could be due to chemical mechanisms, or whether the feeding currents generated by O. marina could lead to virus ingestion or movement, which could influence predator-prey dynamics.