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: DEVELOPMENT OF LIMB BONE LAMINARITY IN THE HOMING PIGEON (COLUMBA LIVIA)
Review round: 1
Reviewer: 2

Output:
Basic reporting: Basic reporting criteria are all met.
Experimental design: There are some details I missed from the description of the Methodology which are essential for the reader to understand the results and their interpretations. I indicated all these in the General Comments part.
Validity of the findings: Findings seem to be sound but I'd need to see and understand all the missing details related to the data analysis to be sure.
Additional comments: This MS is a very valuable, succinct piece of work for our field, as it lays down important new stones for the foundation of our understanding on the locomotion-related ontogenetic osteohistology. Furthermore, I have to emphasize the scientific value (also for future studies) of the online source of section images this study provided which reveals an unexpected microanatomical and histological diversity not only through ontogeny and across different elements, but also among homologous elements of different specimens representing similar ontogenetic stages. As such, I strongly recommend this work for publication, albeit with some revision and consideration of a few potentially important issues I listed below.

My major concerns are related to some missing details of the methodology and the need for a more elaborated discussion on the dynamic changes of osteonal development that may to some extent alter the original vascular architecture throughout ontogeny. Finally, I suggest that the authors devote a few more and deeper thoughts to interpret ontogenetic histological patterns in the context of precocial vs altricial development by comparing their results with those of other cited papers on birds showing precocial hind limb development (see more detailed comment on this below for Line 271-273).

These comments are also highlighted in the annotated pdf I prepared for easier context identification.

Best wishes,
Edina Prondvai

Specific comments by lines

Introduction

Line 55: “…generate large torsional loads occur…”
Delete either “generate” or “occur”

Line 57: ”If these loading patterns are stereotypical across birds,…”
Are you sure that the word "stereotypical" is the right one to use here?

Line 64-68: “Furthermore…development”
But this is not really surprising as in adult flying pigeons the outer cortex is mostly avascular OCL, so it is not only short of circular canals but also of any vascular canals. Hence, it is obvious that vascularity will be restricted to the deeper cortex which represents only a late juvenile stage (as most of the juvenile cortex is resorbed to keep the low relative cortex thickness characterizing avian bones).

Line 75: “Skeletal compensation occurs”
What type of compensation? Please, specify.

Methods

Line 106-108: Please, state where these sections are housed.

Line 117-118: “and vascular porosity (areal ratio of in-plane vascular canals to total cortical area)”
Only primary or also secondary canals?

Line 127: “Imin/Imax,”
Please, indicate here what I stands for.

Line 132-133: “between log10-transformed Zp and the log10-transformed product of body mass and bone length.”
Were these parameters log10 transformed to make them normally distributed or to linearize their relationship? Please, specify.

Line 146-149: “The image of ….for analysis.”
Did you do additional analysis to compare different octants within one section and homologous octants of homologous elements among different specimens? Or you averaged out the LI among the four octants? It may still be interesting to check patterns among homologous octants as they pertain to anatomical directions and hence potentially different loading regimes.

Line 191-192: “Robust PCA was performed separately for each element (Table 2).”
How do you mean that for each element you did a separate PCA? Or you mean each homologous element groups separately, that is for all humeri together, for all ulnae together, etc? Based on the content of Table 1 and 2 that seems to be the case, but please, clarify.

Results

Line 208: “the limb bones of the pigeon become increasingly compact with growth”
I would say limb bone cortex/cortices to avoid any misunderstandings (e.g. increasing compactness could be interpreted as happening all over the mid-diaphysis, including the medullary cavity).

Line 214: ”aged 2–4 weeks (Figs. 2 and 3). As individuals mature (4–9 weeks of age),”
What does this age interval mean in terms of body mass % of adults? So which section of the pigeon growth trajectory does this phase represent? Related to this, it would be useful to show a general pigeon growth trajectory (age vs mass) with indications of the different ontogenetic stages represented in your sample, or these described hallmark ontogenetic stages where histology shows these differences.

Line 215: “bony tissue with poor vascularization along the superficial half to third of the cortical wall”
Would you call this part an outer circumferential layer? Is it composed of largely circumferentially oriented collagen bundles or in which direction do the fiber bundles run? Please, specify, as this can be interesting information for the discussion of torsion-related histology.

Line 220-221: “The polar section modulus (Zp) of the humerus, radius, ulna, femur, and tibiotarsus increases with growth”
I looked up the general calculation of this parameter and I found this:

Zp= (π(D4 – d4))/16D

If this is the one you used, your result would mean that the relative cortex thickness increases with growth (because if d [in this case the diameter of the medullary cavity] gets larger, Zp gets smaller and vice versa). Is this right? If yes, it's interesting, as I would have expected the other way around, that is that cortex gets relatively thinner with maturation (at least that's what I found in duck bones through ontogeny). Be this the case in pigeons or not, it would be useful to give the calculation method of Zp here or in the SI, maybe along with a small sketch of a tube and the parameters in the equation for easy comprehension, so that the reader can think for him/herself about what this and your results actually mean in terms of mid-shaft microanatomy / bone geometry through ontogeny.

Line 233-234: “In the humerus…. PC1”.
If I look at your Table 2, I find dominance of Zp in the humerus, equal effect of Zp and length in the femur and dominance of bone length in all the rest of the bones.

Line 249-250: “We converted the PC1 coefficient into standardized coefficients of the original predictors (mass, length, and Zp).”
What kind of conversion does this mean? You mean the coefficients of the linear combination of the variables giving PCs, so the variable loadings? Please, specify.

Discussion

Line 266-267: “As such, juveniles benefit from relatively robust skeletal proportions (negative allometry),”
Which parts of the skeleton? And relatively robust compared to what?

Line 271-273: “Moreover…2012).”
And what about leg bones? In this paragraph you only discuss wing bones, whereas it would be crucial to include how these findings in the wing bones relate to your findings in the hind limb bones. The entire skeleton following altricial growth trajectory, I'd expect to find similar allometries and growth-related structural changes in both, forelimbs and hind limbs. Which would then contrast the other birds that have precocial hind limbs but altricial forelimbs, like the mallard, seagull, etc, so the birds you referred to as well. Please, provide details on this, as this information is not only very interesting but very important in the interpretation of your data in this locomotor ontogeny context.

Line 291-292: “even when richly vascularized as in the pigeon, torsionally-loaded bone is not necessarily more laminar.”
Maybe not in later ontogenetic stages, but don't the humerus, ulna and femur show high laminarity early in ontogeny, and they consistently have higher LI than radius and tibiotarsus at any ontogenetic stage?

Line 313-315: “torsion-induced shear strain might have a minor effect on laminarity, but ontogenetic effects clearly dominate in bones selected for locomotion.”
Which ontogenetic effects? I mean, locomotion-related shear strain is also an ontogenetically changing effect in terms of its relation with the growing body mass and improving locomotor capacities. So what do you mean here exactly?

Line 318: “oblique-to-transverse orientation”
In relation to the bone's long axis? So oblique would be like spiraling at high angles to the long axis, and transverse would be circumferentially oriented fibers? Also, are you referring here to what we usually call in birds the OCL? So the outermost cortex layer? Or this applies throughout the entire adult cortex the inner half of which is still vascularized? Please, specify.

Conclusion

Line 329: “This developmental pattern differs from a recent report in growing emus,”
It does differ in some respects but corroborates in others, like the conclusion that laminarity is only weakly related to shear strain and that other ontogenetic factors play a more important role.

Line 331: “adaptation to locomotor-induced torsion involves elevated bone laminarity is not supported.”
To me, the picture is not as clear as that. You say in individual bones you find a decreasing laminarity from about 2.5 weeks posthatching up to adulthood. However, the pattern still seems to hold true that bones loaded more in torsion among limb bones (i.e. humerus, ulna and femur) show consistently higher laminarity throughout ontogeny within the same skeleton than the radius and tibiotarsus.
Furthermore, it is not only laminarity that decreases with ontogeny, but also vascularity as a whole, and the structural properties are then dictated mostly by large-scale collagen fiber orientation which is, if I understood it correctly, still dominated by circumferential bundles in the outer half of the almost avascular cortex. And this circumferential orientation might just as well reflect torsional load resistance in a cortex that gets more and more compacted during ontogeny.
Finally, I’m wondering whether you would consider the following scenario: During cortical osteonal compaction, which gives the cortex more structural rigidity in the locomotor maturation process, vascular canals that may have been circumferential originally start to get more and more filled up. In this process, the transversely/circumferentially running mid-sections of the canals which interconnect the longitudinal canals into the network referred to as laminar architecture are preferentially filled up and thereby leaving mostly longitudinal canals open. So this may dynamically change the overall vascular canal architecture into less laminar in the older cortical regions, i.e. in the inner cortex. Closing to adult sizes, the outer cortex becomes less and less vascular, being organized into an OCL.
If this was the case, it would explain why Skedros & Doutre "overestimated" laminarity in those bones based on their CPL images which, by showing collagen fiber orientation in osteonal infillings, still reflect the original but filled up circumferential vascular canal orientations.
In addition, looking at all of your images from the online source (which also made me realize the baffling diversity among a lot of homologous bones at similar ontogenetic stages), a pattern occurred to me that may support this scenario of osteonal development of laminar canals turning them into separated longitudinal canals: That is laminarity is usually more expressed in the outer cortical half (in juveniles where no OCL is yet present) or middle cortex (where OCL is oresent), while the inner cortical half shows a more longitudinal architecture. If laminarity was a permanent condition once it’s there, I’d expect to find more laminar bone in inner cortical halves at >2.5 weeks age, which I couldn’t really see in any of these elements when I checked all bones in your online image library. I’ve cut out an image of MWU 275 Ulna (4.5 weeks of age) to show you what I mean: here I zoomed in on an area where inner cortex shows more longitudinal canals, while some circumferential canals forming laminar cortical areas in the mid cortex are being “transformed” into separated longitudinal canals by osteonal infilling of the circumferential sections (sorry, this image doesn't appear here, but check your section).

Based on this pattern, I could hypothesize the following allocation system between growth and biomechanics:
1)	Fast growing juveniles need high vascular supply in their limb bones but they are still affected by the torsional loads due to their "training" in the increasing locomotor maturation period. Thus, they will develop some laminar vasculature in the bones most affected by torsional "training" loads.
2)	As the cortex grows radially, the innermost cortex gets more and more compacted, obscuring the once predominantly laminar vascular architecture, while the outer, i.e. newer cortex half keeps showing higher proportion of laminarity.
3)	Closing to adulthood, growth rate decreases, less and less vascular canals are needed to sustain fast growth and biomechanial constraints become dominating. When the almost avascular OCL starts to be deposited and make up half of the adult cortex, collagen fiber orientation may reflect torsional load distribution within the skeleton (taking over from vascular canal indicators).

So all in all, I think it would be worth to consider these osteonal maturation processes that may change initial vascular architecture through time as well as the larger scale collagen fiber orientation in the OCL in your biomechanical interpretations.

Figure 1
Caption title: You mean that the shape of the vascular canals is cylindrical? Please, clarify.
Caption: Please, indicate the ontogenetic stage of the scanned specimen. Furthermore, what does figure panel 'c' represent? It's missing from this caption and it's not really clear based on the figure itself, either.
Also, figure panel labels indicated in the caption should correspond with figure panel labels in the figure itself. In this case either both capitalized or both lower case letters.
Finally, the two small insets in panels D and E which show virtual section planes look to me the same. It seems that it's only the view of the section plane that is different, i.e. you rotated the angle of the view. Please, explain it here more accurately.

Figure 2
Are anatomical directions in wing bones defined in extended wing position?

Figure 3
Could you indicate ages in weeks to the listed body masses of specimens as well?

Figure 4:
“Positive allometric scaling of polar section modulus (Zp)”
Allometric scaling of Zp with body mass * bone length? Why multiply body mass with bone length? They are already correlated independent variables. So what will that multiplication do? Won't it increase their multicollinearity to even higher degrees and thereby give a false relationship between Zp and these size measures? So how can this X axis be interpreted exactly? Please, explain. Also indicate here in the caption the underlying method of this figure (i.e. linear regression).

Figure 5
Which method did you use for fitting these curves on the LI vs PC1 scatter plot? And how do these graphs reflect the beta regression with the logit link function that you said you used for characterizing this LI vs PC1 relationship? So how are these figures related to the output of your beta regression? Please, give explanation in here as well as in the methods section so that the reader can interpret the link between the beta regression with logit link function and these figures, as well as the results of this analysis.

Table 1
Add that it's based on linear regression.
Also, here my question is the same as in the graphical representation of the same scaling relationship in Fig. 4: why did you multiply body mass with bone length for the independent variable? Wouldn't this inflate the effects of these correlating predictor variables?

Online image source of sections:
MWU 275: Can the tibiotarsus of this specimen record some sort of pathological condition? I’m just asking because of those large resorption cavities present mostly in the inner cortical half.


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: DEVELOPMENT OF LIMB BONE LAMINARITY IN THE HOMING PIGEON (COLUMBA LIVIA)
Review round: 1
Reviewer: 3

Output:
Basic reporting: General comments:

This is a robust, and very well written, study dealing with an important topic in bone developmental biology and biomechanics.  The study is superbly crafted and conducted.  I did not identify anything substantial with respect to problems or shortcomings in the methods and results.  The authors have done an excellent job in describing their findings and have employed several different statistical approaches to evaluate and report the data.

I only have a few comments.  My main concern deals with some of the conclusions, which can be readily modified.

Specific comments:

Lines 131-135: I would like to have you add 1-2 sentences regarding the rationale for why the ‘product of body mass and bone length’ was chosen.  Provide a reference if available.  Also state exactly why isometry is a slope of 0.75.  Please make it clear to the less knowledgeable reader what the units are for both axes of Figure 4 so that the rise over run is 3 over 4 (correct?).

Lines 274-279:  Skedros and Doutre could have done a better job in stating that higher laminarity is suggested in their images mainly in the outer one-half to two-thirds of many of their images.  I was able to examine all of your sections that are available in the Paleohistology Repository.  Additionally, I was also able to examine all images from the Skedros study, which are not accessible to the public, unfortunately.  While their statement of “high laminarity” does appear to be overestimated, gross inspection of their unstained images generally resemble your MWU 273 image K.  Therefore, I would not change what you have written, but I do wonder if your pigeons, especially the older ones, had access to flight activities similar to those used by Skedros and Doutre.  In that study the pigeons were legally wild shot, and the first author shot several himself (this should have been reported in their Methods, but was not unfortunately).  It would be presumed that pigeons that experience natural flight would have “sufficient torsion” in their humerus to warrant tissue-level adaptation (though, as argued below, this might NOT be expressed as variations in laminarity).  Were your pigeons raised in cages, if so how large were the cases?  I have made a telephone call to Stromberg’s Chicks and Game Birds in Pine River, Minnesota, but I decided to not wait for the answer given that this review was late anyway.  Please make a comment on the flight activities (free ranging, caged, etc – size of cage or enclosure if known) of your pigeons, especially the older ones.  Below, for other reasons, I have encouraged you to examine and include as a reference a study of histology of ulnae from young and older turkeys by Skedros and Hunt (2004 J. Anatomy, volume 205: 121-134).  Note in that study the younger turkeys could actually fly for short distances even though they were a domesticated strain that were raised for consumption.  These flying domesticated turkeys (these were the subadult/younger ones) were found to clearly have histology that correlated with a different load history (bending) of their ulnae than the ulnae of the older birds (presumably more torsion), which could not fly because of their body weight.  How sure are you that your birds had sufficient NATURAL torsional loading to adequately test our main histological/laminarity vs. load history hypothesis?

Line 323: Examine Skedros and Hunt (2004, J. Anatomy) and note that they advanced the work of their 2003 paper.  This 2004 study focuses more on regional variations and ontogenetic/ changes in collagen fiber orientation (CFO) data as being, perhaps, more sensitive than laminarity for identifying prevalent/predomination torsion/shear loading from bending loading.  A similar conclusion regarding regional/load-history-related CFO variations is also stated in Keenan et al. 2017 (Am. J Physical Anthropology, vol. 162) – though this more recent study did not deal with birds or laminarity.  The emphasis on CFO as a ‘preeminent material characteristic’ for interpreting load history in these two studies (2004 and 2017) leads me to the main criticism I have with your conclusion, as stated in your last sentence (see below in Validity of Findings):
Experimental design: This research is within the Aims and Scope of the journal.

The research question is well defined and relevant and meaningful.

This is a rigorous investigation and is performed at a high technical standard.

Methods are robust and seem reproducible.
Validity of the findings: Lines 334-335: You conclude that “This result is consistent with previous findings and suggests …. more strongly at the gross anatomical level rather than at the histological level.”  This statement is premature because an adequate histological characterization of your bones has not yet been done/reported.  Until analyses of predominant CFO are completed across your growth/age range, you should consider adding a new final sentence that is something like: “However, additional studies that quantify ultrastructural characteristics of our bones that are thought, when compared to laminarity, to more strongly correlate with and reliably distinguish the relative prevalence/predominance of torsion vs. bending (i.e., predominant collagen fiber orientation) are needed to confirm this conclusion.”  This suggested sentence is perhaps too wordy, but please consider adding something about the fact that additional analysis of the bone MATERIAL is needed.

A final comment is in order, which I offer as food for thought.  I was happy to see that you kept your focus on avian bone histology and did not try to generalize some of your findings/conclusions to other animals, especially to mammals that generally have appendicular bones with greater relative cortical areas in their mid diaphyses.  But there is something more fundamental regarding your last 3 sentences, especially line 332 and lines 334-335, that I would like you to think about --- and perhaps you have anyway, so forgive me if I am being pedantic.  In these final sentences you are conveying a very reasonable view that one should look closely at bone structure (“geometry”) when considering a bone’s load history. This seems very reasonable, and most readers would just think, in view of your data, ‘this seems correct and is very reasonable and consistent with conventional wisdom’.  But I just would point out that you are dealing also with animals that have, generally, ‘low volume limb bones’.  There is the idea that bones that might be constrained by being at the lower end of the range of the “stressed volume effect” – and typically do not remodel with secondary osteons perhaps, arguably, because of their relatively “low volume” --  would, ipso facto, seem to, or in fact do, adapt geometrically because material adaptability is not needed (this is discussed in more detail in a book chapter by Skedros 2012; Bone Histology, An Anthropological Perspective, ed. By Stout and Crowder).  In contrast, ‘higher volume bones’ would adapt more readily at the material level because of the need to accommodate microdamage, though this MATERIAL-level adaptability could still be missed if the histological analysis was not comprehensive.  Therefore, you are dealing with a paradigm of bone adaptability that seems to work well in avians of the size that you have studied, and you did not drift out of this paradigm.  Nevertheless, it is plausible that your bones are adapting at the sub-microstructural level (I said “ultrastructural” above, others would say “nanostructural” level) in ways that are elusive because a ‘geometric/structural bias’ that permeates studies of low volume bones makes one think that there ‘is no more to look for’ in terms of what a bone might employ to effect tissue-level developmental adaptation.  The likelihood that CFO and laminarity are dissociated in terms of downstream influences on bone tissue mechanics for local load history should be kept in mind, and I hope that you consider testing this hypothesis in the same bones of the present study.  Having said this I was very happy to see that in your pigeon bone images shown in the Paleohistology Repository that you have listed CFO on a check list for future analysis.
Additional comments: Please see my general comments in the above sections of this review.


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: DEVELOPMENT OF LIMB BONE LAMINARITY IN THE HOMING PIGEON (COLUMBA LIVIA)
Review round: 2
Reviewer: 1

Output:
Basic reporting: All four criteria/areas are perfectly met. Additional figures and videos in the revised version increase the standards of the manuscript even above what I expected.
Experimental design: Revised MS meets all four listed criteria.
Validity of the findings: Revised MS meets all four listed criteria.
Additional comments: I am very pleased with this revision, actually beyond my expectations. I did have though a few minor questions and comments that I indicated in the annotated pdf of the revised MS. These are, however, only for the authors to consider before publication (maybe could be modified even at the accepted stage) and not major critical points.
I congratulate to the authors for their nice, high-standard, meticulous work.

Best wishes,
Edina Prondvai


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: SPATIAL ECOLOGY OF LITTLE EGRET (EGRETTA GARZETTA) IN HONG KONG UNCOVERS PREFERENCE FOR COMMERCIAL FISHPONDS
Review round: 1
Reviewer: 1

Output:
Basic reporting: - The manuscript needs to be proof-read to ensure correct language and grammar are used throughout. Currently there are sentences that do not make sense leaving the meaning ambiguous (e.g. line 8, 25, 140). There are also a lot of very short sentences (e.g. line 31) disrupting the flow for the reader

- The introduction provides very little background to the broader relevance and importance of this study, very quickly narrowing in on the specifics of the study system. The first paragraph introduces the current pattern of natural wetland loss/conversion, using broad sweeping statements about impacts/benefits to wildlife. Then, in the second paragraph specific survey techniques are introduced.
The introduction needs to clearly outline the context for this study by discussing the importance of wetland habitats for birds and potential impacts if they are lost. Also, defining natural and human-influenced wetland habitats beyond just examples would be beneficial given the focus on these throughout the paper. Although these two habitat types are highlighted as being distinct the reader can not be sure if both offer equally suitable resources for waterbirds or if there is a cost involved in the conversion. Previous studies have investigated this for numerous waterbird species that rely predominately on human-influenced wetland habitat types (e.g. Richardson et al 2001, Tourenq et al 2001, Sundar 2006, Fidorra et al 2016), I would suggest incorporating this in the introduction as it would highlight the importance of this research

- The author provides very limited information on the ecology of the study species, specifically information on the population of little egrets in this area would provide greater context to the scope of this paper given a small sample size.

- While the discussion section outlines the key findings from the paper it is limited in the depth of the discussion around these points. The author should expand their focus beyond an explanation of the results, addressing broader ecological implications and outcomes.

- There is no discussion around the use of other habitat types beside wetlands despite this being mentioned in the introduction

- Line 16: note that GPS tracking is a type of satellite technology, ‘ARGOS or GPS’ might be a better example.

- Line 179: note it refers to PIC08, this individual is not listed in the study

- Raw GPS data was accessible and in a useable format

- Figure 1: only mentioned in the text in reference to the size of the home range, however individual maps are on different scales making it hard to compare size
Experimental design: - The objectives of this study are clearly defined but repetitive. Objective 2 and 3 largely reiterate Objective 1 just in more detail. Objectives 2 and 3 would benefit from having associated predictions helping the reader focus on the intended outcomes from this paper.

- The use of point fixes in the habitat analysis (Table 2) is potentially problematic with the majority of fixes recorded during winter (Dec- Feb), therefore biasing the result (line 140)

- A sample size of 9 is limiting but not uncommon in studies similar to this. However, I have concern over the analysis approach and specifically seek clarity on the GLMM. Seasonal effect is being tested however there is only n=2 for summer. This needs to be acknowledged in the methods and caveated when drawing conclusions from the results.
Validity of the findings: - There are no test statistics reported from the GLMM and subsequent ANOVA within the text of the results section, despite them being described. They are only referred to in Fig 3 and Table S2, making it hard for the reader to substantiate the results.

- The author has a tendency to make broad sweeping statements from the results that are later contradicted when the data is discussed at a finer scale.
e.g. Line 139 describes a preference for fish ponds across seasons then line 147 notes low occurrence at fish ponds in spring

- The penultimate paragraph in the discussion (lines 177-182) is the first time the author clearly acknowledges the seasonal gap in the data. Given that objective 2 aims to ‘examine seasonal patterns of movement and habitat use’ the author needs to address this earlier as it could have significant ecological implications. Instead the author uses language that alludes to cross seasonal data for all individuals (e.g. line 125/126).
As noted by the author the small sample size over summer also coincides with the breeding season, further highlighting the potential significance of this sampling gap. The paper might benefit from being reframed as a study on the non-breeding habitat selection of foraging little egrets accounting for this. It is problematic for the author to definitively conclude seasonal differences (line 153) with a limited summer sample size.
Additional comments: This study provides a novel contribution to our understanding of the importance of wetlands (natural and human-influenced) for waterbirds such as the little egret in Hong Kong. The conclusion is clear and concise.


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: SPATIAL ECOLOGY OF LITTLE EGRET (EGRETTA GARZETTA) IN HONG KONG UNCOVERS PREFERENCE FOR COMMERCIAL FISHPONDS
Review round: 1
Reviewer: 2

Output:
Basic reporting: In this paper, Pang et al. present novel data about spatial ecology and habitat use of little egrets inhabiting Mai Po Marshes and Inner Deep Bay in Hong-Kong (China). Authors characterize habitat selection and evaluate variation across seasons, discussing the results in the contexts of management of wetland systems.  The main novelty of this work comes from the use of GPS tracking with this species. Despite it is abundant and distributed worldwide, so far (to my knowledge) there is no research published providing tracking data of this species. Moreover, results may be relevant for management actions in a wetland system of priority conservation, so I congratulate the authors for that. The manuscript is succinct but the extension is sufficient to provide main results and draw conclusions. However, authors do not present any expectation or hypothesis.
Experimental design: Authors tracked 9 birds with GPS over a whole year recording high-resolution positioning data, used three different approaches to estimate home range, and perform resource selection analysis to evaluate the importance of each kind of habitat for the species. Methods are generally correct, though some clarifications should be provided or analysis corrected, specifically regarding linear mixed models.

Also, authors get into inter-individual differences in habitat use. Yet, to do so they merely discuss it from standard deviation of metrics calculated. As they use (G)LMM, it is possible to provide some more statistical support to such differences taking advantage of random effects outputs.

I provide more in-deep comments in this regards in the attached reviewed document.
Validity of the findings: Statistical methods used looks appropriate, though some clarifications should be done. Findings of the article are useful for conservation purposes, as they can provide guidance in management schedules in human-influenced wetlands. However, authors do not fully take advantage of results during the discussion to highlight such usefulness in the context of wetland management and conservation.
Additional comments: I have some general comments that may help to improve the manuscript and its value for the research community. First, since authors discuss the results within the framework of conservation and management of wetland habitats, I do think introduction could be improved in a more comprehensive way in that regard. Specifically, developing the text (and providing supporting references) indicating that some wetland systems (as it is the case of Mai Po), despite being all wetlands, contains a high diversity of micro- and meso-habitats and that in some cases human alteration/management of these systems may lead to increase such habitat diversity. It is once within this framework where measuring habitat-type selection spatially and over time, and ranking habitat-type use gains value, as you can use the knowledge gained to provide guidance in management. I feel this link is lacking in the text and clearly would improve the work.

Also, I have the feeling that the work would gain in robustness presenting expectations/hypothesis in the introduction. Indeed, the authors present hypotheses as two possible explanations of bird behaviour in the discussion. But these hypotheses could flow directly from habitat diversity of wetland system plus previous knowledge of the system. That is indeed a strength of this work in relation to habitat diversity, habitat use, and the lessons for management. If authors were familiar with the system studied in this work, and previously they knew that fishponds are drained in a specific time window annually -which promotes not only diversity but dynamism- they could make some expectations beforehand regarding spatial ecology of egrets. Doing so, the results would gain relevance as (1) enhance the importance of high-resolution remote-tracking to monitor individual use of habitat in complex systems at several temporal scales and (2) provide robust support to consider a possible impact (positive or negative) on bird community by habitat alteration or by modifying/shifting/quitting management tasks in human-influenced wetlands. This should be reflected in discussion obviously, enriching it. You may want to take these advices into consideration and make some extra-work in the text to improve it.


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: SPATIAL ECOLOGY OF LITTLE EGRET (EGRETTA GARZETTA) IN HONG KONG UNCOVERS PREFERENCE FOR COMMERCIAL FISHPONDS
Review round: 2
Reviewer: 1

Output:
Basic reporting: By restructuring and adding the suggested  detail to the introduction and discussion the author highlights the ecological relevance of their work, justifying the important role this research can play in the conservation and management of wetland habitats.

The author now provides clear, concise objectives, capitalising on the strength of this work, making the overall focus of the paper more coherent and the discussion of results more robust.

Figure 1: The edits made to this figure have significantly improved it, making the differences in home range size and available habitat types clear to the reader.

Figure 3: This figure is very confusing and missing a level of detail that would be useful for the reader. Firstly, the x axis would benefit from being labelled with the relevant units. Also, presenting the data as a difference from autumn (set as 0) is an unusual approach. Displaying all group means and standard error would make the figure clearer for the reader to interrupt. Lastly, from what I understand in 3C, the model estimates are being presented on the link scale instead of as the raw parameter estimates. I feel this should be back-transformed to avoid visually over representing the seasonal differences.

Figure 4: I am now wondering if the size (area^2) of drained fishponds should be considered for this figure rather than simply number drained. Although I am certain they are correlated, given spatial ecology is the overarching focus of this manuscript it might be more relevant to have it as a measurement of space rather than a count, as this is likely to effect food availability. However, this may be a matter of personal style

Line 104: grammatical issues with sentence

Line 263: grammatical issues with sentence
Experimental design: No comment
Validity of the findings: No comment
Additional comments: I very much enjoyed reading the resubmitted manuscript on the spatial ecology and role of wetlands habitat for little egrets in Inner Deep Bay, Hong Kong. The edits provided by the authors have significantly improved the manuscript. I feel the authors have dealt well with the feedback they received and therefore my suggested improvements are minor.


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: SPATIAL ECOLOGY OF LITTLE EGRET (EGRETTA GARZETTA) IN HONG KONG UNCOVERS PREFERENCE FOR COMMERCIAL FISHPONDS
Review round: 2
Reviewer: 2

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: Authors have carried out successfully the changes proposed in the first review. The manuscript has improved substantially, so I congratulate the authors. My unique important comment is that the hypothesis statement could still be improved to make it clearer. I attach the document with some comments and suggestions that will help to improve a little bit that weakness. Authors will also find more suggestions along the document that may help to make text clearer to the readers.


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: COMBATING SARS-COV-2: LEVERAGING MICROBICIDAL EXPERIENCES WITH OTHER EMERGING/RE-EMERGING VIRUSES
Review round: 1
Reviewer: 1

Output:
Basic reporting: .
Experimental design: .
Validity of the findings: .
Additional comments: Dear all
Greetings
I send in the attached file some suggestions and considerations.
Kind regards


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: COMBATING SARS-COV-2: LEVERAGING MICROBICIDAL EXPERIENCES WITH OTHER EMERGING/RE-EMERGING VIRUSES
Review round: 1
Reviewer: 2

Output:
Basic reporting: This manuscript could be improved significantly by removing the word “that” and “which”, both are used multiple times, some multiple times in a single sentence throughout the document.  In addition, the text could be more “active” which would increase reader interest.  The text lacks definitive recommendations and often leaves the reader wondering if the authors believe any of the content is efficacious.

Intro and background show context for Coronavirus Sars2 and an additional 9 viruses from a WHO list presented as table 1. The context is related to infection prevention and control and what physical characteristics these viruses share, and how these characteristics can be exploited in similar newly emerging viruses.  The literature review is adequate although some references do not confirm the authors presentation (influenza mortality of 60%) and other literature not cited (see below) provide survival results on environmental surfaces at high and low temperatures and high and low humidity. The authors list no data (wood et al) but there is plenty of data available. see references at end of text.

The article structure, and tables are fine although tables 1 and 2 could be condensed into one table.  As a review article there is no raw data to share. Figure 2 is a bit confusing, there are levels of antimicrobial activity scaled vertically but some of the levels are blank for an active antimicrobial making one wonder what the product was or why the indicated level was included starting with enveloped viruses.

The review is focused on infection prevention and control of enveloped viruses (and one nonenveloped virus) from a WHO list of emerging and re-emerging viruses. In our current climate of the SARS-COVID pandemic this article is relevant.

I found four reviews of the current COVID outbreak, two dealing with pharmaceuticals, one presented WHO guidelines and one was more general and included infection prevention and control as one section of the review.  This review focuses on infection prevention and control with examples of how the control measures have been used historically.

Introduction introduces the subject and identifies Infection prevention and control as the goal of the review. Motivation is to glean effective methods of disinfection and infection prevention using historical data from similar viruses especially when the virus is newly emerging.
Experimental design: The article content is within the Aims and Scope of the journal
Some of the statistics are misleading although derived from other literature, i.e. infection rate and morbidity rates for COVID19 are artificially high because testing was only done on symptomatic individuals and some deaths were incorrectly attributed to COVID-19.  When testing includes all individuals, symptomatic and asymptomatic  and all age groups the statistics change significantly from early reports. There was no discussion of this or how one should assess statistics early in an outbreak.
Since no actual data was collected the methods are not described.
All references are available through PubMed or Google Scholar and HTTP sites.
The review is organized in a logical and coherent subsections
Validity of the findings: This review does not address novelty or data since it is a review. No new data from this study.  It does address modes of transmission and historic use of disinfectants and their efficacy for enveloped viruses as identified from the WHO list presented in table 1
Conclusions supporting the “goals” of aiding the IPAC community in arriving at strategies for dealing with current or emerging viruses are weak. They do make a case for using, disinfectants for environmental surfaces but no new applications.  They do not discuss the importance of testing (individuals or surfaces) or isolation to reduce infection rates.  Likewise there is no discussion of testing contacts or contact tracing to reduce infection rates.  All their information is historical and used commonly to disinfect surfaces.  Strategies are tried and true, nothing new in this arena as well.  There is no definitive statements or strategies for how to deal with a new unknown emerging virus, what information should be sought early on and how to apply the information to developing a strategy for reduction of virus in the environment and how to reduce spread of the virus from person to person. Although animal to human transmission is mentioned there is no discussion or recommendations for prevention of transmission.  It is a review of current literature but is short on actual recommendations.
See above comments.
the conclusions do not identify unresolved questions/gaps/future directions.
Additional comments: In table 2 the mortality rate of influenza was listed as 60% and the following paper was cited.  The paper does not give a mortality rate for influencza.  Cieslak TJ, Herstein JJ, Kortepeter MG, Hewlett AL. 2019. A methodology for determining which diseases warrant care in a high-level containment care unit. Viruses 11(9): 404 773; doi:10.3390/v1109077

Influenza references for environmental surfaces.  The table indicated no data was available.
Survival of influenza viruses on environmental surfaces
B Bean, BM Moore, B Sterner… - Journal of Infectious …, 1982 - academic.oup.com
To investigate the transmission of influenza viruses via hands and environmental surfaces, the survival of laboratory-grown influenza A and influenza B viruses on various surfaces was studied. Both influenza A and B viruses survived for 24–48 hr on hard, nonporous surfaces  …
Cited by 547 Related articles
[PDF] asm.orgFull View
Survival of influenza virus on banknotes
Y Thomas, G Vogel, W Wunderli, P Suter… - Appl. Environ …, 2008 - Am Soc Microbiol
Skip to main content. ASM: Antimicrobial Agents and Chemotheraphy; Applied and Environmental Mircobiology; Clinical Microbiology Reviews; Clinical and Vaccine Immunology; EcoSal Plus; Eukaryotic Cell; Infection and Immunity; …
Cited by 136 Related articles

Survival of influenza virus on hands and fomites in community and laboratory settings
DV Mukherjee, B Cohen, ME Bovino, S Desai… - American journal of …, 2012 - Elsevier
… Four environmental surfaces were then sampled, including 2 hard, smooth surfaces (indoor doorknob and telephone [cellular or land line]) and 2 absorbent surfaces (pillowcase … To evaluate how viral concentration and surface properties affect survival of influenza virus, we …
Cited by 32 Related articles
[PDF] asm.orgFull View
Effects of air temperature and relative humidity on coronavirus survival on surfaces
LM Casanova, S Jeon, WA Rutala… - Appl. Environ …, 2010 - Am Soc Microbiol
Skip to main content. ASM: Antimicrobial Agents and Chemotheraphy; Applied and Environmental Mircobiology; Clinical Microbiology Reviews; Clinical and Vaccine Immunology; EcoSal Plus; Eukaryotic Cell; Infection and Immunity; …
Cited by 165 Related articles
Find it @ UF
Virus survival on inanimate surfaces
MC Mahl, C Sadler - Canadian Journal of Microbiology, 1975 - NRC Research Press
… Rosalind Stanwell-Smith. 2008. Advice for the influenza season: infection control in the care home … 2006. Survival of Two Avian Respiratory Viruses on Porous and Nonporous Surfaces … International Journal of Environmental Health Research 15:3, 225-234. [Crossref] 24 …
Cited by 87 Related articles


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: COMBATING SARS-COV-2: LEVERAGING MICROBICIDAL EXPERIENCES WITH OTHER EMERGING/RE-EMERGING VIRUSES
Review round: 1
Reviewer: 3

Output:
Basic reporting: Line 62: No comma needed
Line 63: Comma needed between “SARS-CoV-2” and “as”
Line 122: Should read “…dependant on a number of factors…” (“on” is missing)
Line 123: No comma needed after “immune”
Line 154: Comma needed between “excretions” and “including”
Line 179: Remove “etc.” from between “infectivity half-life” and “infectious titer”
Line 183: Add an apostrophe at the end of coronaviruses (coronaviruses’) to indicate the reviews are referring to the survival of different coronaviruses.
Line 208: Should read “…susceptibility of human…” (“of” is missing)
Line 219: Add “they” between “although” and “have”
Lines 231-232: Should read “…and, therefore, are comprised of host cell…” (commas needed around “therefore” and wording changed to correct the meaning of the sentence)
Line 236: Should read “…envelope, therefore, is…” (commas needed around “therefore”)
Line 240: Should read “…detergents, such as soap,…” (commas needed around “such as soap”)
Line 261: Should read “…coronaviruses that, therefore, could…” (drop the first comma and “and”)
Line 263: No commas needed around “PMCX”
Line 273: Insert a period at the end of the sentence.
Lines 279-280: Should read “Factors, such as the addition of an alkaline agent, were found…” (commas needed around this example)
Line 282: Comma needed between “viruses” and “such as”
Line 284: Need commas around “most recently”
Line 285: Comma needed between “viruses” and “such as”
Line 288: Comma needed between “sufficient” and “as”
Line 296: Replace the misused word “amendable” with the intended word “amenable”
Lines 298-299: Remove the parentheses from around the listed virus families, as these names are essential to the sentence, and add a comma between “Phenuiviridae” and “indicate”
Line 311: Comma needed between “outbreaks” and “such as”
Lines 313-314: Recommend: “The WHO has posted a webpage “Coronavirus Disease (COVID-19)
 advice for the public (WHO, 2020b).””
Line 316: No is comma needed between “rub” and “and.” A comma is needed before “especially”
Line 318: Comma needed between “Table 2” and “for which”
Line 327: A comma is needed after “(Fauci, 2005)” to begin his quote.
Line 329: Comma needed between “diseases” and “as well as”
Line 337: Remove the first “nonenveloped virus” from the list.
Line 361: Comma needed before and after “therefore”
Line 364: Close the parentheses after “2020”
Line 696: Change “genotypically” to “conformationally” to convey the proper meaning.
On page 25 of the PDF, remove the period from in front of the title for Table 1.
Pages 25-28: The titles for Tables 1 and 2 need a comma between “viruses” and “including”
Pages 29-30: The title for Table 3 needs a comma between “viruses” and “including” and after “SARS-CoV-2.” There should be a semicolon between “available” and “the result”
Page 32: The title for Figure 1 needs a comma between “infactions” and “such as”
Page 33: “Clostridioides” is missing the first “d” and “Hepatitis C” is missing the first “ti”
Page 34: Change “genotypically” to “conformationally” to convey the proper meaning. “Hepatitis C” is missing the first “ti”
Experimental design: What keywords were used in the literature search?
What source(s) was searched?
What date(s) was the literature search performed?
Validity of the findings: The references I looked up appeared reputable.

In line 343, the authors are perplexed about why many emerging viruses are enveloped. Enveloped viruses use host cell membrane components to evade the immune system and to facilitate host cell entry. I recommend reading and commenting on the information from the following article:
Watanabe Y, Bowden TA, Wilson IA, Crispin M. Exploitation of glycosylation in enveloped virus pathobiology. Biochim Biophys Acta Gen Subj. 2019;1863(10):1480-1497. doi:10.1016/j.bbagen.2019.05.012
Additional comments: I enjoyed reading this review and thought it was well done.


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: OVEREXPRESSION OF KIAA1199 IS AN INDEPENDENT PROGNOSTIC MARKER IN LARYNGEAL SQUAMOUS CELL CARCINOMA
Review round: 1
Reviewer: 1

Output:
Basic reporting: 1. The writing of this manuscript is far from satisfactory. The authors need to find someone good at English to correct all the grammatical errors and misuse of terms including but not limited to sentences with missing verbs (e.g. Line 102), excessive use of clause (e.g. Line 246), and sentences making no sense at all (e.g. Line 245).
2. The authors need to define some of the explanatory variables used in their statistical analysis such as “Pathological Differentiation” and “Clinical Stage”. They need to briefly explain what these factors are, why they are included in their analysis, what are the levels of these factors, etc. They also need to describe by what standard do they classify someone as being drinking, i.e. how frequent does one need to drink to be classified as so.
3. Writings between Line 76 and Line 84 are contradictory. On one hand, the authors claim that “over-expression of KIAA1199 contributes to resistance to cell immortalization and cancerization” (Line 76-78). In the next sentence, they say “KIAA1199 is over-expressed in different cancers”. Does it means that over-expression of KIAA1199 is a part of our body’s defense mechanism to prevent cancer cells from becoming more malignant and invasive? If so, higher expression level of KIAA1199 should be beneficial instead of being correlated with low survival rate as suggested by the results of this paper.
4. There are some missing citations such as in Line 109, it needs a reference to the Helsinki Declaration. Line 141, a reference to the original immunohistochemistry protocol adopted by the authors. Line 173-174, reference to the original (or the version adopted by the statistical software used in this report) Cox regression and Kaplan-Meier survival analysis.
5. In Line 149, spell out the abbreviation DAB.
6. Wrong number in Line 202, “… in LSCC tissues were 52.4%(54/105, Table 1) …”. According to Table 1, this number should be 55/105.
7. In all tables, the levels of the factor “Clinical Stages” do not show up properly.
8. The authors use the term “independent predictor” several times (such as in Line 234). This should be changed to “statistically significant predictor”.
9. The figure legends need to be moved from figures to the end of the main text.
Experimental design: 1.  What is the sample size for the RT-PCR and western blot data shown in Figure 1? 10 or 105? If it were ten, how did the authors choose which samples to use?
2. Why did the authors exclude patients with recurrent and multiple cancers?
3. How did the authors select the 105 patients other than excluding cases with recurrent and multiple cancers?
4. The number of female patients is too small (only two) to make any valid statistical inference. Should just remove gender as a potential predictor in all statistical analysis reported here.
5. In Line 101 “No anticancer therapy was given before surgery”. Is it ethical to deny patients with anticancer therapy before surgery? Is it out of consideration of achieving the best treatment results or just for the sake of this study?
6. The authors conducted a series of Chi square association tests to evaluate the relationship between expression level of KIAA1199 and other factors. For the results to be valid, the authors need to check chi-square test assumptions regarding the underlying distribution of the data. Because the chi-square test is based on asymptotic approximation with normal distribution, it is generally required that for any entry of the n*m way table, the expected count should be at least five. If this assumption were not valid, the authors need to consider alternative non-parametric methods to test for association such as the Fisher’s exact test.
7. The authors need to check the assumptions of using Cox proportional hazards analysis: one such assumption is that the hazardous ratio is assumed to remain constant over time. This can be visualized by plotting Kaplan-Meier survival curves for each of the categorical variable. Paralleling survival curves are a good indicator of constant hazardous ratio.
8. Overall survival rate (OS) is usually complicated by competing events such as death not due to the disease of interest. I wonder how did the authors deal with such kind of complication?
Validity of the findings: 1. The hazardous ratios reported in the paper lack information of the base level of the corresponding factor. Without it, it is hard to evaluate how those significant factors affect the survival time.
2. The error bars for NC are missing in Figure 1 B&C. Moreover, it is hard to believe comparison shown in Panel C is significant given the huge error bar associated with CA.
3. There are a few counter-intuitive points drawn from the results. One is that as shown in Table 3&4, smoke is a non-significant predictor for the survival time of these 105 patients. This is in contrast to previously published results such as this one: ORL J Otorhinolaryngol Relat Spec. 2012;74(5):250-4.
4. Another one is that one learns from Table 2 that the expression level of KIAA1199 appears to change from being largely low (48 patients out of 70) in T1/T2 stage to largely high in T3/T4 stage (33 out of 35). And then, it drops to largely low (50 out of 85) in N0 stage and rises back up to predominantly high (18 out of 20) in N1-N3. The fluctuation of the expression level appears to be very unusual. Could the authors comment on it?
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: OVEREXPRESSION OF KIAA1199 IS AN INDEPENDENT PROGNOSTIC MARKER IN LARYNGEAL SQUAMOUS CELL CARCINOMA
Review round: 1
Reviewer: 2

Output:
Basic reporting: The experiments are solid, and the observations are very clear.
Experimental design: No comments
Validity of the findings: No comments
Additional comments: There are several grammatical and minor spelling errors in the manuscript, which need to be improved


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: OVEREXPRESSION OF KIAA1199 IS AN INDEPENDENT PROGNOSTIC MARKER IN LARYNGEAL SQUAMOUS CELL CARCINOMA
Review round: 1
Reviewer: 3

Output:
Basic reporting: In this study, the author explored the relationship between KIAA1199 expression and laryngeal squamous cell carcinoma (LSCC) progression using Western blotting, RT-PCR and IHC. However, the roles of KIAA1199 in other cancer types have been extensively discussed, like oral squamous cell carcinoma (Pitak Chanthammachat, et al., 2013), breast cancer (Nikki A. Evensen, et al., 2013), gastric cancer(Shinji Matsuzaki, et al., 2009), colorectal tumors (K Birkenkamp-Demtroder, et al., 2011; Lawrence C. LaPointe, et al., 2012), prostate cancer (Eriko Michishita, et al., 2006), ovarian cancer(Fan Shena, et al., 2019) and hepatocellular carcinoma (Zhengchen Jiang, et al., 2018), which renders the shortage of novelty of the present study. The author should conduct some mechanistic studies to explore how KIAA1199 participates in LCSS, affecting cell proliferation, invasion or migration? Other than novelty, the language issues were spread in the whole manuscript, which should be corrected before submitted to scientific journals, the author should seek help from academic editors or scientific researches with profound writing experience. The detailed language issues were listed below.

Why does this study recruit only 2 females, but 103 males, it may cause gender bias of the study.
Real time RT-PCR is wrong written, should be real time quantitative PCR (RT-PCR).
“Western blotting” or “Western blot”, please be consistent throughout the manuscript.
In Abstract, the author described “we collected tissues from 105 cases of
laryngeal squamous cell carcinoma (LSCC) to investigate the relationships between
KIAA1199 protein expression and clinical factors.” Did the author compare LSCC tissues with noncancerous tissues (control), what’s the number of control tissues?
Citation in the context is not consistent, blank should be placed between context with citation. Such as Line 66, 68, 69. All of these should be revised throughout the manuscript.
The writing from Line 70 to line 71 should be rephrased.
Line 75, “Nowaday” should be changed to “Nowadays”.
Line 95 “arrange” and “follow-up” should be separated.
In line 99, A total of 105 patients were recruited, how about the control tissues and where are they from, please specifically identify in the section of Materials.
In line 97, “We collected 10 pairs of fresh specimens and their matched adjacent
98 noncancerous specimens”, what’s the specific specimens were collected, please list the details.
In line 140, for IHC, what’s the control tissue?
In line 155, the section of “Immunohistochemical staining results” is redundant, please combine with subtitle “Immunohistochemistry”.
In line 223, what’s the meaning of “OS”? When it appears at the first time, please give the full name and designate the abbreviation.
Comma is not corrected used in many cases, like line 253.
Line 259, “draw” should be replaced with “drawn”.
Line 316, “We” should be replaced with “we”.
Experimental design: Why does this study recruit only 2 females, but 103 males, it may cause gender bias of the study.
Validity of the findings: In this study, the author explored the relationship between KIAA1199 expression and laryngeal squamous cell carcinoma (LSCC) progression using Western blotting, RT-PCR and IHC. However, the roles of KIAA1199 in other cancer types have been extensively discussed, like oral squamous cell carcinoma (Pitak Chanthammachat, et al., 2013), breast cancer (Nikki A. Evensen, et al., 2013), gastric cancer(Shinji Matsuzaki, et al., 2009), colorectal tumors (K Birkenkamp-Demtroder, et al., 2011; Lawrence C. LaPointe, et al., 2012), prostate cancer (Eriko Michishita, et al., 2006), ovarian cancer(Fan Shena, et al., 2019) and hepatocellular carcinoma (Zhengchen Jiang, et al., 2018), which renders the shortage of novelty of the present study. The author should conduct some mechanistic studies to explore how KIAA1199 participates in LCSS, affecting cell proliferation, invasion or migration?
Additional comments: In this study, the author explored the relationship between KIAA1199 expression and laryngeal squamous cell carcinoma (LSCC) progression using Western blotting, RT-PCR and IHC. However, the roles of KIAA1199 in other cancer types have been extensively discussed, like oral squamous cell carcinoma (Pitak Chanthammachat, et al., 2013), breast cancer (Nikki A. Evensen, et al., 2013), gastric cancer(Shinji Matsuzaki, et al., 2009), colorectal tumors (K Birkenkamp-Demtroder, et al., 2011; Lawrence C. LaPointe, et al., 2012), prostate cancer (Eriko Michishita, et al., 2006), ovarian cancer(Fan Shena, et al., 2019) and hepatocellular carcinoma (Zhengchen Jiang, et al., 2018), which renders the shortage of novelty of the present study. The author should conduct some mechanistic studies to explore how KIAA1199 participates in LCSS, affecting cell proliferation, invasion or migration? Other than novelty, the language issues were spread in the whole manuscript, which should be corrected before submitted to scientific journals, the author should seek help from academic editors or scientific researches with profound writing experience. The detailed language issues were listed below.

Why does this study recruit only 2 females, but 103 males, it may cause gender bias of the study.
Real time RT-PCR is wrong written, should be real time quantitative PCR (RT-PCR).
“Western blotting” or “Western blot”, please be consistent throughout the manuscript.
In Abstract, the author described “we collected tissues from 105 cases of
laryngeal squamous cell carcinoma (LSCC) to investigate the relationships between
KIAA1199 protein expression and clinical factors.” Did the author compare LSCC tissues with noncancerous tissues (control), what’s the number of control tissues?
Citation in the context is not consistent, blank should be placed between context with citation. Such as Line 66, 68, 69. All of these should be revised throughout the manuscript.
The writing from Line 70 to line 71 should be rephrased.
Line 75, “Nowaday” should be changed to “Nowadays”.
Line 95 “arrange” and “follow-up” should be separated.
In line 99, A total of 105 patients were recruited, how about the control tissues and where are they from, please specifically identify in the section of Materials.
In line 97, “We collected 10 pairs of fresh specimens and their matched adjacent
98 noncancerous specimens”, what’s the specific specimens were collected, please list the details.
In line 140, for IHC, what’s the control tissue?
In line 155, the section of “Immunohistochemical staining results” is redundant, please combine with subtitle “Immunohistochemistry”.
In line 223, what’s the meaning of “OS”? When it appears at the first time, please give the full name and designate the abbreviation.
Comma is not corrected used in many cases, like line 253.
Line 259, “draw” should be replaced with “drawn”.
Line 316, “We” should be replaced with “we”.


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: OVEREXPRESSION OF KIAA1199 IS AN INDEPENDENT PROGNOSTIC MARKER IN LARYNGEAL SQUAMOUS CELL CARCINOMA
Review round: 2
Reviewer: 1

Output:
Basic reporting: NA
Experimental design: NA
Validity of the findings: NA
Additional comments: The authors have made some effort in addressing some of the concerns and questions raised by the three reviewers, and yet some other questions are left either uncommented or insufficiently addressed.

I would like to see the authors to address all the questions and concerns from Reviewer 1 regarding the validities of the findings which are left uncommented. In particular, I would like to have your comments on the first two questions which are directly related to the credibility of the reported data and conclusions drawn from them in this paper.

Response to the third reviewer’s questions seems to be quite haphazard making me wonder if the authors proofread the rebuttal letter before submitting it.

In addition to these obvious shortfalls, I also notice that for some questions, although changes and corrections were promised by the authors, they have not been incorporated into the current version of the manuscript. These include Q3 (Basic reporting, Reviewer #1) and Q6 (Experimental design, Reviewer #1).

For the sake of clarity and reproducibility, I urge the authors to incorporate the information they provided in addressing the reviewers’ questions, in particular, in Q2 (Basic reporting, Reviewer #1), Q2 (Experimental design, Reviewer #1), and Q3 (Experimental design, Reviewer #1).


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: OVEREXPRESSION OF KIAA1199 IS AN INDEPENDENT PROGNOSTIC MARKER IN LARYNGEAL SQUAMOUS CELL CARCINOMA
Review round: 2
Reviewer: 2

Output:
Basic reporting: The current manuscript fails to answer all of the questions raised by reviewers, and in the context of the response to reviewers, the author includes some characters not in English, which makes it totally unreadable. Thus, highly recommend to reject the paper for publishing on scientific journal.
Experimental design: The logic and questions raised in the first round of review regarding "Experimental design " haven't been corrected.
Validity of the findings: The validity of the findings is problematic, which has been explained in the first round of review. However, the author ignores the comments and fails to correct it.
Additional comments: The revised version of manuscript fails to answer all of the questions and concerns raised in the first round of peer-review. Thus, the current manuscript should be rejected for publication.


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

Input:
Title: CHARACTERIZATION OF THE TRANSCRIPTIONAL RESPONSE OF CANDIDA PARAPSILOSIS TO THE ANTIFUNGAL PEPTIDE MAF-1A
Review round: 1
Reviewer: 1

Output:
Basic reporting: It
Experimental design: i don't think it could be explain the mechanism by RNA-seq
Validity of the findings: some problems about novelty.MAF-1A  was previously reported. Antimicrobial peptides was used as  antifungal is an idea that has already been explored by other investigators
Additional comments: Cheng et al. report MAF-1A report MAF-1A peptide regulates the transcriptional response ofCandida parapsilosis. While the results reported are interesting, they do not clear the novelty threshold for publication . The  MAF-1A  was previously reported. Antimicrobial peptides was used as  antifungal is an idea that has already been explored by other investigators. i don't think the authors could demonstrate the mechanism by RNA-seq  .


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

Input:
Title: CHARACTERIZATION OF THE TRANSCRIPTIONAL RESPONSE OF CANDIDA PARAPSILOSIS TO THE ANTIFUNGAL PEPTIDE MAF-1A
Review round: 1
Reviewer: 2

Output:
Basic reporting: The Authors investigated the effect of the MAF-1A antifungal peptide against Candida parapsilosis. Cheng and colleagues determined the MIC value of the peptide against one of the reference strains of C. parapsilosis and determined the kinetics of growth at MIC level and compared that to the one of the peptide-free control. RNA-seq analysis was performed at two time points (6 h and 18 h) to reveal the transcriptional changes the treatment induces. They determined the differentially expressed genes and predicted protein-protein interactions. According to these changes and using KEGG pathway analysis, the Authors concluded the potential effect of MAF-1A against C. parapsilosis. The basic structure of the manuscript meets the standards of the journal.
Experimental design: Due to the emerging resistance of Candida species against the already applied antifungal agents makes this topic relevant and current, and the fundamental experimental design is correct. I found that the experiments had been carefully designed (especially the qRT-PCR analysis). Most of the methods are clear and described well.
Validity of the findings: I would recommend the Authors to be more careful when phrasing the discussion and conclusion parts of the manuscript. In summary, I think the data in this manuscript is valuable, supported with statistical analysis, but the Authors need to correct grammar mistakes, use a more elaborate English, restructure specific sections and discuss the results more carefully.
Additional comments: Due to the numerous errors in terms of grammar and style, and because of the inaccurate and incomplete presentation of the data, I do not recommend this paper to be published at this stage (see my comments later). First, I would strongly suggest the authors to perform a spellcheck by using one of the openly available online tools or to ask a native English speaker or maybe to use a dedicated service to correct these issues. There is some missing information and inconsistency in the way of presenting the literature and the results. For instance, the third paragraph of the „Introduction” mentions first AMPs, then MAF-1A and then returns to the general characterization of AMPs once again in the next paragraph. For an „Introduction” it is always advised to follow a „from the large to the specific” kind of point of view. It is also advised to mention some more AMPs from other sources. Investigation of the AMPs produced by molds really is a hot topic. To provide broader sight to the reader, it would be useful to describe how their structure and mode of action is related to the ones of AMPs from insects and specifically MAF-1A itself. It might be a convergent evolution, and they do not share much in common, but still, this would be an additional reason why to focus on AMPs produced by insects. I would have mentioned the currently applied antifungal agents, their mode of action, and how resistance have emerged against them to support better the necessity of alternative antifungal agents, such as AMPs.  In the „Materials and Methods” section 2.4. subsection (Line 117) the title is „Total RNA extraction” however this paragraph also contains the description of sequencing, the title should be changed. I did not find the order of the subsections appropriate in the „Results” part either. 3.2 subsection is only 5 sentences, it could have been presented together with 3.5 subsection. The Authors might consider changing the order of the subsections as follows: 3.1. MIC assay and time-kill curves; 3.2. Transcriptional stress response and enrichment analysis of MAF-1A treated C. parapsilosis; 3.3. Verification of differentially expressed genes; 3.4. Protein-protein interaction (PPI) network analysis. I think the results part describing DEGs should be more detailed. For instance DEGs at 18 h are completely missing, and the Authors did not mention those 32 and 24 genes that were down- and upregulated at both time points respectively.
After reading the manuscript, I could not find the title appropriate. „MAF-1A peptide regulates the transcriptional repsonse of Candida parapsilosis”. The peptide regulates the response of C. parapsilosis to what? Being aware of the results I think this is an overstatement. No matter what happens to a cell, its transcriptional profile will be changed. According to the findings of Cheng et al., MAF-1A induces stress that will evidently have an effect on the global transcriptome of the fungus. Unfortunately this was not evaluated in detail in comparison with stress responses triggered in even C. albicans by other types of antifungals, that data, however, exists in the literature (See references below). Similar comparison with C. parapsilosis would be more evident, but it is obviously encumbered by the lack of such data. However the Authors mentioned in the „Introduction” part that they would also investigate the difference in the response of C. parapsilosis and C. albicans to MAF-1A, the actual comparison is also missing from the later sections of the manuscript, in turn it would strongly strengthen the quality of the paper. If the Authors have that data, I do not understand why they did not perform the analysis. These all would have provided more solid data on the MAF-1A specific fungal response and would have greatly increased the quality of the paper. Considering all these, my opinion is that this effect should not be called „regulation”. Regulation is a process by which something is specifically and tightly controlled. The described alteration in the transcriptome here seems to be a general stress response due to the interference of MAF-1A with basic biological processes (membrane integrity, protein synthesis, mitochondrial function) as the Authors concluded according to their in silico analysis. However the fungal transcriptome is likely to undergo this or similar alteration to respond these indirect effects rather than getting directly regulated due to the presence of MAF-1A. It can not be excluded however that there are genes that are MAF-1A dependently regulated (either up or down), but this can not be concluded due to the lack of the aforementioned comparisons. Regarding the title, I suggest something similar as the followings: „MAF-1A peptide alters the transcriptional profile of C. parapsilosis” or „MAF-1A peptide triggers global changes in the transcriptome of C. parapsilosis” or „Characterization of transcriptional response of C. parapsilosis to the antifungal peptide MAF-1A”. The discussion is a bit misleading, because there are AMPs mentioned from various sources that possess specific effects that were actually experimentally validated. The fact that MAF-1A treatment leads to changes in the expression of genes related to these, does not necessary mean that a 26 amino acid long peptide possesses all these properties that these peptides from different sources have.

References for transcriptome analysis:
Liu et al. (2005) Genome-Wide Expression Profiling of the Response to Azole, Polyene, Echinocandin, and Pyrimidine Antifungal Agents in Candida albicans. Antimicrobial agents and chemotherapy
Dhamgaye et al. (2012) RNA sequencing revealed novel actors of the acquisition of drug resistance in Candida albicans. BMC Genomics
And a review
Pais et al. (2019) Microevolution of the pathogenic yeasts Candida albicans and Candida glabrata during antifungal therapy and host infection. Microbial cell

Here are my remarks and comments.
It would be better to use higher resolution pictures in Figure 2; 4; 5; 6 and 7, if it is possible.
There are sentences that are difficult to understand, like:
Line 23-25 „When treated with MAF-1A from six to 18 hours, 42 genes were no longer elevated and 25 genes showed a reversed expression pattern.”
Line 52-53 „These interspecies specificities may affect host identification and clearance, in addition to antifungal drug efficacy.” Does „host identification” mean „recognition by the host”?
Line 168-169 „A total of 2439 DEGs were detected at 6 h and 18 h time points for MAF-1A treated C. parapsilosis compared to controls (Figure 2).”
Line 179-180 „The DEGs in C. parapsilosis were treated for 6 h with MAF-1A and used to construct a PPI network based on STRING.”
Line 211-212 „In recent years, C. parapsilosis infection has risen to the second or third commonly detected species of the Candida genus.”

Inconsistent spelling
Line 76, 78 RNA-seq, then Line 82 RNA seq then Line 290 and Figure 3 RNAseq
Line 102 MAF1A, the rest is MAF-1A
In the main body of the manuscript it is „qRT-PCR” but the description of Figure 3 says qPCR
Figure 3 description says Material and Methods, while in the body it is Materials and Methods
Figure 3 description still P value „=” or „<”
The title of the subsections are sometimes written as a regular sentence (only the first letter is capital) sometimes all words start with a capital.
Sometimes word with capital letters appear in the middle of the sentence e.g. Line 251 Cysteine, Line 252 Ribosomes Line 237 Ergosterol
Table S1 „Primers sequences” -> „Primer sequences”
etc.

Incorrect format or type and typos
Line 19, 36, 37, 41, 54 etc. Candida -> Candida
Line 40 C. albicans -> C. albicans
Line 18/61 Musca domestica -> Musca domestica
Line 76, 78, 81, 82, 84, 112, etc. C.albicans/parapsilosis -> C.albicans/parapsilosis
Line 77-78 An accidental „enter”
Line 88-89 „C. parapsilosis was streaked on” -> „C. parapsilosis was streaked on”
Line 258 Thermus thermophilus -> Thermus thermophilus
Line 268-269 Saccharomyces cerevisiae -> Saccharomyces cerevisiae
Line 226 C. albicans through its -> C. albicans through its
Line 135 There is a dot missing from the end of the sentence
Line 137 P valueS -> P values
Line 157 p = <0.05 Is it „equal” of „less than”?
Line 176 the Authors mention „6 h and 8 h”, it was „6 h and 18 h”
Line 194-195 „Cell cycle-yellow”, Why yellow is there?
Line 224 E. coli -> Escherichia coli
Line 254 After „coworkers” no need to write a dot.
Line 257 Bac71-35 -> Bac71-35
The right part of „Figure 4” is not visible (CPAR2_4048..)
„Conclusion” section has a different format from that of the other sections.
etc.

Space is missing
Line 113, „100µl”
Line 116 „for24h”
Line 153 „95°Cfor”
Line 231 andthat
Line 282 C.parapsilosis
Figure 2 description „….(CPBC).The expression…”
Table S1. „Table S1.Primer”
Table S2. „Table S2.The”
etc.

The first paragraph consist of five sentences (8 lines) but the word „infection” pops up eight times. I would kindly ask the Authors to rephrase the sentence, and use a more elaborate style.
References should be put at the end of the sentences. It would not break the phrase.
Version number of softwares should be put right after the name of the software without brackets, and then the reference.
Line 184-185 Supplemetary figure 1 does not refer to the enrichment analysis, is it not Supplementary Figure 6 instead?
Line 265 What is NHD?

Inappropriate phrasing
Line 213 „Some C. parapsilosis are …” -> „Certain C. parapsilosis isolates are…”
Line 215-216 „Moreover, drugs against C. parapsilosis infection have been reported in the literature.” – I do not think this sentence fits here.
Line 216 „Antimicrobial peptides have excellent antimicrobial properties” This is a self-explanatory phrasing, it should be avoided.
Figure 1/Table R1 „Time-kill curves of MAF-1A under MIC concentrations for C. parapsilosis.” It is concentration and not concentrations. There is just one MIC of a drug against one isolate.
Table 1/2 „Significantly enriched KEGG pathways for genes with increased/decreased expression after 6 h.” -> „Significantly enriched KEGG pathways for genes with increased/decreased expression after 6 h of MAF-1A treatment.”

Grammatically inaccurate phrasing (only examples)
Line 82-84 „RNA seq was used to elucidate the mechanism(s) MAF-1A and its
associated changes in gene expression during early (6 h) and late (18 h) treatment stages, investigated according to time-kill curves of C. parapsilosis growth” -> „…mechanism(s) of MAF-1A…”, „…at early (6 h) and late (18 h) time points…”
Line 164 „downwards trend for the first 8 h” -> downward trend during the first 8 h
Carefully check plural and singular form of verbs and subjects.
Figure 2. description „Volcano plots depict the log2 FC (fold change).” The dot should be a comma.
Figure 2 description still, the term ”compared to” in sentences like „The expression of 101 genes significantly increased compared to 151 genes that decreased” is misleading. Expression was compared to the control, here „in contrast to” would be more scientifically precise.
Figure 2 description still in the text it is „CPAS vs. CPAC-up”, but on the diagram it is „CPAS vs. CPAC_up”
I suggest a different colour for the venn diagram because the colour for „RG2 genes” is almost identical with the one of „CPBSvsCPBC_up” group.
Table R1 „compared with control” -> „compared to the control”
Table R1 „fungus growth e percentage” What does „e” mean?

Writing the gene names correctly
The 3.4 subsection of the „Results” summarizes the predicted protein-protein interactions, however the Authors mention „genes” in the phrases. So, are those proteins or genes? If these are proteins, then the nomenclature says, that only the first letter of the protein should be capital, the rest is minuscule like Ubi1, Cdc28 etc., and not UBI1, CDC28. On the other hand it is not suggested to use C. albicans gene/protein names for ones of C. parapsilosis. Instead, the most precise writing is as follows for genes CPAR2_809000, or CpUBI1 (and this latter need to be italicized) and for proteins „protein encoded by CPAR2_809000 (or CpUBI1)”, Candida parapsilosis Ubi1, CpUbi1 or CpUbi1p. This is also true for the „Discussion” part (e.g. Line 235 ERG1 should be CpERG1). (See nomenclature: http://www.candidagenome.org/Nomenclature.shtml#systematic)
Line 245 Improper writing of a C. parapsilosis gene. „CPAR2-807700” -> „CPAR2_807700”

Scientific questions
Why did the Authors apply 35 °C for cultivation and performing the experiments. This temperature is neither the optimum for C. parapsilosis nor it is the physiological temperature of humans.

In the „Materials and Methods” section the Authors say (Line 100) that they have adjusted the cell number to „approximately 0.5x103-2.5x103”. Why is this range? How was the cell number determined?

Line 114 „10 µl of aliquots were streaked” Was it really 10 µl and not 100? I suppose it is the latter. What does „streak” mean exactly? Was this suspension plated on an agar plate or streaked using an inoculating loop?

Line 164-165 „the curve steepened to levels comparable to the control group with 3x108 fewer colonies recorded.” Is there a calculation or comparison for the growth rates of the fungi cultivated with or without MAF-1A? What time point does „3x108 fewer colonies” refer to? These all should be presented in the results.

In the subsection 3.5 the DEGs at 18 h are not even mentioned. So were not those 24 and 32 genes that are up and down regulated at both time points respectively. How do they fit in the rest of the results?

Why did the Authors measure the absorbance at 492 nm. Absorbance measurements for Candida are always performed around 600 nm. (See references: PMID: 27627759, 29941885, 29995344)

The authors themselves have that transcriptome data of C. albicans treated with MAF-1A, why did they not include the comparison? It would strongly increase the value of the „Discussion” and „Conclusion” sections and could identify C. parapsilosis or C. albicans specific MAF-1A response.

Taking all the DEGs into account at the two time points, do these results reveal or suggest any global adaptation process to the MAF-1A treatment in C. parapsilosis?


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

Input:
Title: CHARACTERIZATION OF THE TRANSCRIPTIONAL RESPONSE OF CANDIDA PARAPSILOSIS TO THE ANTIFUNGAL PEPTIDE MAF-1A
Review round: 1
Reviewer: 3

Output:
Basic reporting: This paper is easy to read and follow. English is correct.
Some revision of the figure captions are needed (Consider changing the caption of Figure 3 to explain what the figure presents. The explanation about the statistics can stay in the material and methods section). Also, does Supplementary Figure 1 relate to the protein-protein interaction or to q-PCR? I would suggest fixing the figure or the text accordingly.
Experimental design: no comment
Validity of the findings: no comment
Additional comments: In the manuscript “MAF-1A peptide regulates the transcriptional response of Candida parapsilosis”, Cheng and colleagues employ RNA-seq data to explore the mode of action of the antifungal peptide MAF-1A against C. parapsilosis. They report a number of genes to be differentially expressed after 6 and 18h of treatment. They find that genes that are differentially expressed are involved in amino acid synthesis and metabolism, oxidative phosphorylation, sterol synthesis and apoptosis. They conclude that MAF-1A’s mode of action involves disruption of membranes and normal organelle function.

This manuscript is well written and is easy to follow. Moreover, in a time when antibiotic and antifungal resistance is a widespread problem, this manuscript addresses a pressing and important issue. However, I have some comments. For instance, the authors have performed an enrichment analysis of the C.parapsilosis DEGs upon treatment with MAF-1A at the 6h timepoint and 18h timepoint. While the authors start describing the DEGs in both timepoints (section 3.2), they only describe enrichment analysis at 6h. It may be worth also performing this analysis at 18h.
It is quite interesting to note that there are some genes that are upregulated at 6h and downregulated at 18h, and all the way around. The authors focus much of their manuscript on this issue, but I feel it is equally important to discuss which genes are differentially expressed by the MAF-1A treatment itself, independently of time points (24 upregulated genes, and 32 downregulated genes, per the venn diagram).

Minor comments:

Authors chose 20 DEGs to validate RNA-Seq data by qRT-PCR. What was the rationale to choose those 20 genes?
Species names are often not italicized, both in the text and in the references