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: SKELETAL VARIATION IN BIRD DOMESTICATION: LIMB PROPORTIONS AND STERNUM IN CHICKEN, WITH COMPARISONS TO MALLARD DUCKS AND MUSCOVY DUCKS
Review round: 1
Reviewer: 1

Output:
Basic reporting: overall the manuscript would benefit from proof readings to improve the general standard of English and sentence structure throughout, there are spelling errors that need correcting as well

I would like to see a broader introduction into the functional significance and importance of these areas of avian anatomy and morphology - there is no discussion of the role of the sternum in respiration that needs addressing for example - but a general broadening of the literature included in the introduction would better highlight the significance of this study - specific comments are detailed below.

tables could be improved as detailed - a numb hypothesis is stated but clearer aims and objectives would help
Experimental design: by incorporating the extra elements suggested in the introduction this will improve the definition of the research questions and better highlight how it fills a gap.

methods are ok but need to include more complete information as detailed 0 the sample sizes don't always seem to match up and swapping between common and species names is confusing at times
Validity of the findings: need clarification on comparisons made
Additional comments: overall the English could do with proof reading and corrections to sentence structure and spellings - too many sentence start with 'this' for example which is often non specific

line 35 correct mallard spelling

line 38 - this is unclear less intensive selection in which forms ?

line 52 - formatting error

line 53 use correct referencing format you don't need to give the title of the reference - its unclear if this is supposed to be a direct quote or not

introduction - this needs broadening to include a discussion of the role of the sternum in respiration

lines 99-100 - the language needs tightening in here, its unclear when you jump between common and breed strain names. what is the difference between fowl and chicken ? you need to specify what you are referring to here

line 116 - give model details and company name, country

line 119 - total length of the forelimb will depend on articulation of these bones not their overall length being summed it would db rare for the limb to be held completely straight - why was this parameter chosen for investigation ?

line 122 - be specific proportions of what?

line 123 - can you detail how the data from these groups were pooled or not pooled as the case may be

line 132 - why the variation in fore and hind limb numbers ?

line 132-141 - its hard to tally these numbers with the sample sizes that appear in the tables

line 194 - you state here that no differences were found in gallus but then on line 205 you choose to highlight 'the substantial size variation' surely they either are or are not different ?

line 208 -line 212 - jumping between common and species names is confusing do one or the other but not both

line 216 - again reference to the role in respiration would be appropriate here

line 219 - 222 - how can it be that you observe no differences but then when analysed individually you find differences - do you mean within and between groups ? needs clarifying what you analysed here

line 262 - this paragraph is confusing - the lack of a size difference is due to their being a lack of proportional differences - isn't this just trying to explain the lack of a difference by there being a lack of difference ? a functional explanation would improve this section

general comment - I think the difficulty in interpreting the data stem from the pooling of samples into wild and then everything else into domestic when clearly there are large variation in the domesticated breeds likely obscuring meaningful results

line 289 - 291- jumps between common and species names again

line 296 - are you referring to muscovy here ? 'this suggesting..' needs rewriting

line 300 how is this an analogous conclusion ?

line 300- linking or placing these changes in context of respiration needs to be done how would the sternum get damaged by resting when the muscles covers the bone ?

tables:

- adding sample size to table 1, table 2 should make clear if these samples are from the same Bird or different birds - it isn't clear what you mean by specimens and how these were pooled.

Table 2 why are some numbers underlined ? and what are there differences in fore and hind limbs measured

table 3 more information is needed in the legend

table 4 - the spacing of the table makes it hard to follow - are the r2 numbers spread over two lines ?

table 4 - the numbers and value don't need underlining if you aren't referring to anything specific

table 5 - nothing is bold in the table and if the names are in the table you don't need to specifically highlight RJF anyway - why do you only present these raw data ?

table 5 - why are numbers underlined ? why are the samples sizes different as you go across the table ?

figure 1 arrows not dots would be better and more clear, a fuller legends needs to explain what the figure is showing

figure 2 - p-value is written on the figure so it doesn't need explaining, im not sure the small images are helpful


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: SKELETAL VARIATION IN BIRD DOMESTICATION: LIMB PROPORTIONS AND STERNUM IN CHICKEN, WITH COMPARISONS TO MALLARD DUCKS AND MUSCOVY DUCKS
Review round: 1
Reviewer: 2

Output:
Basic reporting: Please find comments to the manuscript entitled “Skeletal variation in bird domestication: limb proportions and sternum in chicken, with comparisons to mallard ducks and Muscovy ducks” below.

The submitted manuscript is generally well written. The impact of domestication on the biometric measures is an interesting topic. In the introduction a brief mentioning of when the species subjected to this study was domesticated compared to other species (e.g. mammals) would provide evidence of what to expect in terms of skeletal evolution. Is the timeframe of domestication too short to change the skeletal properties in the included species?
In both the introduction and discussion, reference to flying ability of Muscovy is referenced by blog post and unrefereed web pages, that is not considered as appropriate referencing and may if extensive weaken the study.

However, the manuscript suffers from being a bit unstructured in particular in the results sections. Here the lack of structure is hampering the interpretation and the readability of the results, due faulty references to figures and tables, to little detail in the tables for getting an overview of the wild and domesticated birds just to give some examples. This section need a thorough and careful revision with a clear focus on structure and logic order in the presentation of the results. See also specific comments below (the list is not exhaustive). More words/description on where there are differences will be an improvement and help the reader.
By aligning the structure of Materials and methods section and the Results section the readers ability to interpret the results presents will be considerably increased. A suggestion could be 1) wild-domesticated all species, followed by 2) chicken related comparisons, including breed differences for both leg and keel bones.

There has been a mixup in updating figure and table numbers in many of the cross references. That should be avoided, as it is hampering the interpretation of the results and evaluation of the conclusions.
Experimental design: In general the design of the study is sound, however validation of the measurement accuracy and instrument uncertainty was not addressed. Details on this is described below in specific comments.

As the breeding of poultry is an ongoing event, it will be useful to include the year/date where the specimens have been collected and included in their respective collection. Additionally inclusion of a sufficient number of birds from modern commercial lines, both egg and meat type birds would have been very useful for displaying the potential impact of intensive breeding selection where selection criteria is meat and egg yield.
Validity of the findings: The analysis of the limb measures is found to be valid and sound. However, the analysis of the keel bone data is next to absent. This is a major weak point and is elaborated several places below in both general and specific comments
Additional comments: When specific attention was given to the keel bone of the chicken together with reference to keel bone damage (both fractures and deformities) it is hard to understand why specific measurements of various keel bone dimensions (e.g. length and height of the carina, total length, distances between distal/caudal ends of trabeculae etc.) was not performed and compared to limb properties and also between wild and domesticated birds. This information is next to necessary for the understanding of keel bone fractures in layer hens.
As described above the lack of detailed morphometric measures of keel bones, which could have been done at the same time as the initial registration, is weakening the results. It should be included, including comparative analysis to the limp data. If not possible, all sections regarding the keel should be taken out of the manuscript. At its present form it does contribute significantly to the rest of the study.
Furthermore inclusion of modern adult commercial layers and meat type broiler breeders would greatly increase the understanding of the selective pressure that these two types of chicken have been put through the last 3-5 decades.

Specific comments:
Line 52: At the start of the line, there is an empty gap. It is unclear whether some text/wording is missing or if it is merely a technical issue.
Line 71-73: References to the muscovy ducks’ flight capacity may benefit considerably by using scientific and/or textbook literature or other refereed information instead of using anecdotal information from internet blog posts. For example a comparison of allometrics of skeletal bones and potential ability to fly in muscovy and pekin ducks by Swatland (1980). In the paper by Converse and Kidd (2001), the muscovy duck is considered a migratory bird. Poultry breeding standard reference may also be able to provide information on ability to fly.
Line 82-88: Crookedness of keel was also described by Buckner et al. (1949).
Line 88-90: The impact of perches on keel bone deformities in laying hens was recently reviewed by the EFSA panel (EFSA, 2015). Please clarify that this relations is not purely historical.
Line 93-94: The proposed definition of a keel bone fracture seem somewhat imprecise. The definition of a fracture in medical terms is referring to a discontinuation (breaking) of the bone tissue resulting a variety of different fracture manifestations where the involved bone is completely or incompletely disrupted with or without fragments. Fracture definition and characteristics are well described in a text book chapter by Marchiori (2013).  A fracture will depending on the level of post fracture immobility and will heal more or less deformed with or without medical intervention. If a fracture is left unstable/insufficiently immobilized the bone repair tissue (callus) with “grow” excessively and new bone formation will appear close to the fracture line and “on top” of the bone surface as exostoses. This callus may in extreme cases be palpable for even the inexperienced after receiving a brief training. Callus and exostoses are always visible on x-ray. Please review the fracture definition accordingly. References to keel bone fracture characterization/visualization are to some extend available using different diagnostic modalities (e.g. imaging, histology, pathology) (Eusemann et al., 2018; Fleming et al., 2004; Richards et al., 2011; Rufener et al., 2018; Scholz et al., 2008; Thøfner et al., 2020; Tracy et al., 2019).
Line 115-116: To overcome the risk of applying unknown degrees of inaccuracy and uncertainty of the measurements and measuring equipment both replicate measurements and calibration of equipment is of great value. At present it is unclear whether a bone was measures several times (technical replicates). Please specify, and justify if not more than one measurement. What were the approaches/procedures for calibration of the calipers?
Line 119-121 : the total length of given limb “was taken”, “was defined” seems more appropriate as the tarsus/carpus and toes/phalanges were not included.
Line 122-123 (Table 1): The distribution of wild and domesticated birds is unclear for all three species. Please clarify. The pooling of all the wild and domesticated birds within each species is disturbing, especially in chickens, where many different breeds in all sizes are included. Please justify the reasoning. It would increase the immediate readability if table 1 was presented as an overview of the distribution and numbers of wild and domesticated birds within each species and note the breeds of mallard ducks and chickens, maybe including the mean length of the specific bones.
Line 133-136: It is acknowledged that juvenile modern meat type chickens were not investigated. Nevertheless, the study will benefit greatly by including adult broiler breeder birds, as these chicken line have been bred intensely for meat development. This has significantly altered their gait to a level where it is considered the new “normal” that broilers have increased gait scores compares to other breeds/line including commercial layer type hens, like the leghorn-derived lines.
Line 153 (table 3): Table 3 is referenced the manuscript, but the information provided in the manuscript in table 2.  Again, presentation of the result can be improved. The means/medians and the variation of each breed would help the reader to interpret where the differences between breeds may be present. Please consider illustrating by using a plot/figure instead.
Line 179-191: The absence of specific measurements of keel dimensions is weakening the results considerably with regards to the keel morphology and its potential importance to keel bone fracture development observed in in particular highly productive laying hens.
Line 197: reference to table 2 is wrong. The table 2 provided in the manuscript is on the different chicken breeds included in the study. Maybe it is the content of table 1 that is referred to. Including presentation of the mean properties for reference will greatly improve the presentation of the results. Maybe move up fig 8 for this part…
Line 202: Table 3 is referenced the manuscript, but the information provided in the manuscript in table 2 is presenting the results described. As mentioned above  (line 153).
Line 205-212: The lack of structure in the whole results section together with the faulty reference to figures and table numbering is impairing the review of this last paragraph too much.
Line 221-223: By including origin of breed formation, overall type of chicken (layer/meat, large/giant/dwarf size, height/weight span) in the table with the number of bird within the specific chicken breeds would substantiate the interpretation and discussion of the breed differences among the chicken breeds including RFJ. This can also be used for discussion the selective pressure of certain traits. In relation to that the absence of modern highly selected commercial lines, both layer and meat type chickens is weakening the conclusions. The selective pressure of the commercial lines may be considered the highest and fastest. Maybe visualized by creation of a small phylogentic tree including wild and domestic forms as well as breeds. Interactive Tree Of Life is an online tool for the display, annotation and management of phylogenetic and other trees (https://itol.embl.de/gallery.cgi) (Letunic and Bork, 2019).
Line 235-249 and fig 3 and 8: Some attention to the large variation in limp and bone lengths in both domesticated chicken and mallard breeds should be given. In particular as many of the different breeds have been bred for exterior traits including (body) size, therefore also known as “fancy breeds”. The observed differences may be partially explained by this. As mentioned above an overview of the standard size/dimensions, age of breed, intended produce (egg/meat) may help clarifying.
Line 266-288: Loss of ability to fly is discussed, however no comparison of the differences between the flying Muscovy duck and the non-flying mallard, in both domesticated Muscovy-mallard and the wild birds, has been presented. Maybe that may help supporting the conclusions.
Line 295: Take care in the use of refereed references. Same issue as in line 71-73 in the introduction. Again taking the length of the domestication age/time span into account may or may not support the conclusions made on the Muscovy ducks ability to fly. Maybe the timeframe for this evolutionary change is too short. A reference to known spatial relation to skeletal changes for comparison will substantiate the conclusions.
Line 310-324: The data handling and analysis of the keel bone observations included in the manuscript is almost absent apart from a “presence-absence” table on the 5 selected criteria, therefore it seems a bit strange that such a large part of the discussion is not based on solid grounds or sufficient data. This is a major weak point. As described above the lack of detailed morphometric measures, which could have been done at the same time as the initial registration, is weakening the results. It should be included, including comparative analysis to the limp data. If not possible, all sections regarding the keel should be taken out of the manuscript. At its present form it does contribute significantly to the rest of the study.

References
Buckner, G.D., Insko, W.M., Henry, A.H., Wachs, E.F., 1949. Rate of Growth and Calcification of the Sternum of Male and Female New Hampshire Chickens Having Crooked Keels. Poult. Sci. 28, 289–292. https://doi.org/10.3382/ps.0280289
Converse, K.A., Kidd, G.A., 2001. Duck plague epizootics in the United States, 1967-1995. J. Wildl. Dis. 37, 347–357. https://doi.org/10.7589/0090-3558-37.2.347
EFSA, 2015. Scientific Opinion on welfare aspects of the use of perches for laying hens. EFSA J. 13, 4131–4171. https://doi.org/doi:10.2903/j.efsa.2015.4131
Eusemann, B.K., Baulain, U., Schrader, L., Thöne-Reineke, C., Patt, A., Petow, S., 2018. Radiographic examination of keel bone damage in living laying hens of different strains kept in two housing systems. PLoS One 13, e0194974. https://doi.org/10.1371/journal.pone.0194974
Fleming, R.H., McCormack, H.A., McTeir, L., Whitehead, C.C., 2004. Incidence, pathology and prevention of keel bone deformities in the laying hen. Br. Poult. Sci. 45, 320–330. https://doi.org/10.1080/00071660410001730815
Letunic, I., Bork, P., 2019. Interactive Tree of Life (iTOL) v4: Recent updates and new developments. Nucleic Acids Res. 47, W256–W259. https://doi.org/10.1093/nar/gkz239
Marchiori, D.M., 2013. Trauma, in: Clinical Imaging: With Skeletal, Chest, & Abdominal Pattern Differentials: Third Edition. Elsevier Inc., pp. 625–765. https://doi.org/10.1016/B978-0-323-08495-6.00010-5
Richards, G.J., Nasr, M.A., Brown, S.N., Szamocki, E.M.G.G., Murrell, J., Barr, F., Wilkins, L.J., 2011. Use of radiography to identify keel bone fractures in laying hens and assess healing in live birds. Vet. Rec. 169, 279-+. https://doi.org/10.1136/vr.d4404
Rufener, C., Baur, S., Stratmann, A., Toscano, M.J., 2018. A Reliable Method to Assess Keel Bone Fractures in Laying Hens From Radiographs Using a Tagged Visual Analogue Scale. Front. Vet. Sci. 5, 124. https://doi.org/10.3389/fvets.2018.00124
Scholz, B., Roenchen, S., Hamann, H., Hewicker-Trautwein, M., Distl, O., Rönchen, S., Hamann, H., Hewicker-Trautwein, M., Distl, O., 2008. Keel bone condition in laying hens: a histological evaluation of macroscopically assessed keel bones. Berl. Munch. Tierarztl. Wochenschr. 121, 89–94. https://doi.org/10.2376/0005-9366-121-89
Swatland, H.J., 1980. Development of Carcass Shape in Pekin and Muscovy Ducks. Poult. Sci. 59, 1773–1776. https://doi.org/10.3382/ps.0591773
Thøfner, I., Hougen, H.P., Villa, C., Lynnerup, N., Christensen, J.P., 2020. Pathological characterization of keel bone fractures in laying hens does not support external trauma as the underlying cause. PLoS One 15, e0229735. https://doi.org/10.1371/journal.pone.0229735
Tracy, L.M., Temple, S.M., Bennett, D.C., Sprayberry, K.A., Makagon, M.M., Blatchford, R.A., 2019. The Reliability and Accuracy of Palpation, Radiography, and Sonography for the Detection of Keel Bone Damage. Animals 9, 894. https://doi.org/10.3390/ani9110894


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: SKELETAL VARIATION IN BIRD DOMESTICATION: LIMB PROPORTIONS AND STERNUM IN CHICKEN, WITH COMPARISONS TO MALLARD DUCKS AND MUSCOVY DUCKS
Review round: 2
Reviewer: 1

Output:
Basic reporting: the paper is much improved in terms of the writing and sente3nce structure and many of the small errors in logic have been corrected
Experimental design: clarifications to the methods and experimental design have been added
Validity of the findings: sensible conclusions drawn on the main findings
Additional comments: happy with the comments having been addressed appropriately and these have improved the submission


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: SKELETAL VARIATION IN BIRD DOMESTICATION: LIMB PROPORTIONS AND STERNUM IN CHICKEN, WITH COMPARISONS TO MALLARD DUCKS AND MUSCOVY DUCKS
Review round: 2
Reviewer: 2

Output:
Basic reporting: The authors have responded adequately to initial review comments and suggestions and thus really improved the manuscript. There are however still some typos and formatting issues, as well as minor comments and discrepancies in the presentation of the results. Please see specific comments below.
Experimental design: The authors have responded adequately to initial review comments and suggestions

It is acknowledged that the authors are not including broilers, as they are (as mentioned  by the authors) juvenile in their intended life span. However their parents, from which they inherit their traits, are fully matures when they reach the end of their normal production cycle (>60 weeks old). Therefore, it would still have been relevant to have had bones from these adult fully matured bird included, as was suggested in the first review of the manuscript. It is however recognised, that those birds, may have been hard to track down in museum collections.
Validity of the findings: The authors have responded adequately to initial review comments and suggestions.
Additional comments: Comments to the author:
Please ensure that you either italicize or not italicize all anatomical names (according to journal policy). At present anatomical names are in italic and not. Both are "legal" ways to write the names according to Smart P., Maisonneuve H. and Polderman A. (eds) Science Editors’ Handbook, European Association of Science Editors. www.ease.org.uk. Section 2.2: Anatomical nomenclature by Jenny Gretton. Here it is also stated that the writing should follow the specific journal policies. Another option could also be to use the common names for the bones/muscles/specific anatomical features.

Specific comments:
The comments below refer to the line numbers in the word version with highlighted changes. Comments to figures and tables refer to the full pdf version of the revised manuscript.

Line 76: comma is missing in reference
Line 79: breed names should be lower cased.
Line 80: semicolon and comma is missing in references
Line 87, 90 & 91: comma is missing in reference
Line 109: Should be Stratmann in reference.
Line 145: anatomical and/or osteological could be added after discrete to emphasize that it is mainly a descriptive analysis of the sternae.
Line 146: This is the first time red junglefowl is mentioned. Shouldn’t it be her the abbreviation is stated for the first time?
Line 163-165 (after …limb.): These two sentences fit better if put after the definition of the total limb length (at the end of the this paragraph).
Line 212-216: It is acknowledged that the authors are not including broilers, as they are (as mentioned  by the authors) juvenile in their intended life span. However their parents, from which they inherit their traits, are fully matures when they reach the end of their normal production cycle (>60 weeks old). Therefore, it would still have been relevant to have had bones from these adult fully matured bird included, as was suggested in the first review of the manuscript. It is however recognized, that those birds, may have been hard to track down in museum collections. Please add, information justifying for not using adult fully matured broiler breeder birds (parent stock birds).

Line 219: remove comma after 16
Line 259-262: In the text it is stated that breeds with 4 or more specimen are included to increase rigor. In table 1 and the number of specimens per breed are listed. When looking at this table, there are considerable discrepancies between the in-text listed breeds ≥4 specimens and the listed breeds in the table. Furthermore, kömpfer is not even included in table 1. Additionally, in table 6 and figure 8 it is stated that >5 (which should be replaced with ≥5, to fit data from table 1 accordingly) birds are included in the illustrations. However, neither cochin (n=6) nor seidhenhuhn (n=5) are included, and white leghorn have only 4 specimens.

Line 385: comma is missing in reference
Line 394: remove comma before et al.
Line 397, 398, 400, 402: comma is missing in reference
Line 440: insert space after cases

Figure 5: Is the lines in each box mean or median? What do the error bars present (SD, SEM, CI, interquartiles)? Please update accordingly.
Figure 8: >5 should be replaced with ≥5, to fit data from table 1 accordingly. Is the lines in each box mean or median? What do the error bars present? Please update accordingly. Note the comments for line 259-262 about the missing breeds in the figure.

Table 6: : >5 should be replaced with ≥5, to fit data from table 1 accordingly.


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

Input:
Title: LIPIDS ASSOCIATED WITH PLANT-BACTERIA INTERACTION IDENTIFIED USING A METABOLOMICS APPROACH IN AN ARABIDOPSIS THALIANA MODEL
Review round: 1
Reviewer: 1

Output:
Basic reporting: The manuscript needs language editing. The quality now is quite uneven. Some parts are good, whereas some others (methods in particular) are hard to follow. There are many abbriviations not commonly used in the pland pathogen interactions field. I would recommend using more commonly used terms to describe various treatments. Also check that all treatments are reffered to in a consistant manner: Control treatment is now reffered to as both "CK" and MgSO4.

References and litterature background is fair.

The raw data is shared. However, I do not understand how the raw data in the xls-table of lipid species is used to calculate the data presented in figure 3.

The manuscript contains a well defined and potentially intersting experiment. I do however have some concerns.
Experimental design: The manuscript contains original research that is well within the scope of the journal.

The research question is defined and relevant.

The methods are not well described. In particular it's hard to follow exactly what time points are used and how exactly the plants are inoculated with bacteria (needle less syringe?).

I have several major concerns that needs to be adressed:

1. The methods seems to indicate that a bacterial slurry is defrosted, suspended i Kings B medium and cultivated over night. Thereafter the slurry is diluted in MgSO4 to a low OD and used for inoculation. I worry that there is absolutely no way of knowing that this actually represent bacteria growing in exponential phase rather than mostly dead bacteria after freeze storage. It critically depends on how much of the culture was diluted. The methods seem to indicate that a "tube was just diluted in 5 ml Kings B. In my experience this experiment is usually done on bacteria growing freshly grown on solid medium suspended in MgCl2 och MgSO4.

2. The interpretation of the OPLS-DA plot (Figure 2. . The only comment in the main text is that there is a short distance between samples repesenting systemic leaves from plants inoculated with avirulent and virulent Pseudomonas. However, in fact there is also a short disatnce between these two and local leaf control. In fact the two clusters tha seems to space furthest apart are control systemic leaf local virulent treated. This needs to be better commented on to the very least. To me it seems to indicate that there might be fundamental problems with the experiment.

3. How is the data in the heat plot in figure 3 generated? It is presented as "fold differences", but what is it mormalized to. I cannot understand what it is normalized to as all possible treatments seems to be presented. Additionally, there seems to be quite large variations between the replicates for some of the treatments.

4. It's a bit strange that OPDA-containing galactolipids ("arabidopsides") does not show up in any treatment. Col-0 is well known to make large amounts of these when challenged with an avirulent strain of P. syringae.
Validity of the findings: Provided my aboive concenrns can be met, the findings are valid. However, the final conclusion is not actually supported anyway. Change in the level of a particular metabolite does not imply funtion in SAR. One can safely say that a particular change is associated with the state of SAR induction but nothing can be inferred about actual function.
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: LIPIDS ASSOCIATED WITH PLANT-BACTERIA INTERACTION IDENTIFIED USING A METABOLOMICS APPROACH IN AN ARABIDOPSIS THALIANA MODEL
Review round: 1
Reviewer: 2

Output:
Basic reporting: The manuscript is written clearly throughout, but requires thorough editing to erase minor errors (some are listed below). The introduction into the subject is sufficient, and literature is well referenced (but reference list should be checked again, I notice two missing references, see below. Literature is relevant, only sometimes more care may have been taken to cite the most appropriate (original) publications (see below).
The manuscript is well structured. Figures are of high quality and figure legends are informative. Relevant raw data is provided in the supplement, but could be improved. Specifically, the metabolite table should be supplemented with the respective retention times. QPCR raw data (Ct values) may also be provided as a supplement.
Experimental design: The research question is well defined and relevant. The experimental design (choice of time point, number of replicates, and choice of extraction method) has some limitations (see below) and description of methods is lacking some details (see below). Some phrases used are misleading and should be corrected, e.g. ‘Differentially expressed lipid metabolites (DELM)’ –metabolites are not expressed.
Please discuss your choice of extraction method: which previous publications used this method in plants/with Arabidopsis leaves, which lipid classes were targeted and why?
At which time point were the samples harvested for lipid analysis? This must be mentioned in the methods section, and the choice of this time point should be discussed with respect to previous publications, e.g. those on the timing of known SAR metabolites, such as NHP or SA in systemic leaves.
RT-qPCR: how were the relative expression levels calculated? It looks like different treatments were used as reference (or set to 1) for the different genes.
Line 169: why did you choose different cut-offs for up- or downregulated metabolites? 0.83 vs. 1.2?
Validity of the findings: Generally, a better understanding of the regulation of lipid metabolites during SAR is highly desireable and a welcome addition to existing knowledge of SAR.
The identification of metabolites (Suppl. Table 1) was probably performed in an automated way. Some predictions appear rather unlikely to me, given the previously reported abundance of these metabolites. Some PA levels appear rather high, in some cases these molecules may rather be designated as PE (?). Also PIP signals appear rather high in some cases (?) There are several unlikely predictions e.g. MGDG 29:10, TG 11:4. Hence, manual curation of this list seems to be necessary.
It should be stated (and discussed) that you used rather relaxed criteria for identifying regulated metabolites. With a cut-off of minimum up- or downregulation of 20%. When discussing the relevance of the detected metabolites, you should put the magnitude of regulation in context: How strong were differences in levels of these metabolites in previous publications? Are the magnitudes detected potentially relevant?

You used three biological replicates per treatment. Unfortunately, one of your control treatment samples, SL-CK3, appears to be quite different from the two other control samples (Figure 3). Please discuss. Did this influence the composition of your differentially regulated metabolite list?
To put your results in context, more detail on previous work, specifically: which lipid molecules have been found before in SAR metabolomics approaches, would improve the discussion.
In the discussion, please discuss if you found previously identified SAR-related lipids, and, if not, why? How about Glycerol-3-phosphate, or other molecules such as terpenoids (Attaran, Rostas, Zeier, 2008), sterols (Griebel & Zeier, 2010), tocopherols (Stahl et al., 2019), or the role of lipid peroxidation in SAR responses?
Conclusions are generally well stated, with minor amendments required. If one would be very critical, the title suggests that SAR-associated lipids are identified. Striktly speaking, locally and systemically induced metabolites were identified. If these metabolites play any role in SAR (enhanced resistance of the systemic tissue) is not tested in this study. Therefore, the statement in line 23-24 ‘during SAR’, or line 81 ‘important factor’, ‘essential roles’ are overstatements and should be removed.
Additional comments: 51-52 better cite most recent papers on this subject: . Ngou, B. P. M., Ahn, H.-K., Ding, P. & Jones, J. D. G. Nature https://doi.org/10.1038/s41586-021-03315-7 (2021).
54 bacteria-free parts?
58 Li et al. 2020a is not the most appropriate reference, as there are much earlier reports on PIP
80-82 ‘Various PAs, PCs, PEs, and TAGs were identified as important factors in plant immunity, playing essential roles in basal resistance and’ -whether the molecules are essential or not, has to be shown by functional studies
90-91 unclear meaning
104 please give rationale and references for the choice of extraction method
132-133 ‘types of software program’? Specify!
151 planthad
166 metabolites are not espressed. Please rephrase, also your abbreviation ‘DELM’
201 MgSO4-infected?
201-203 expression of GPAT1 is not indicating the importance of PA
206 There are earlier references on the general nature of SAR
231 Fernandez-Delmond et al. not in references
239 Gal?
245 Gao: missing reference
270-283 I do not understand the connection between elevated PE levels and the eut ‘story’ presented in this section...
285 Vir bacteria?
303 this should be expressed more careful and specific
310, 313 TGs were displayed enhanced abundance?
Cfu per mL per leaf disc –better to convert this to the actual area of the leaf disc.
Figure 1: format of column graphs is different in A and C
Figure legend 1: diameter 50 mm?
Figure 3 SL-CK3 appears very different from the two other control samples. D
Figure 5 legend: lipid-related genes?
Figure 5: number of replicates, how were relative levels calculated? Sometimes LL:CK, sometimes SL-CK is set to 1?
Legend to Figure 5: ‘lipid related genes’ ‘treatments with Avr’ These terms are not correct, please specify.


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: LIPIDS ASSOCIATED WITH PLANT-BACTERIA INTERACTION IDENTIFIED USING A METABOLOMICS APPROACH IN AN ARABIDOPSIS THALIANA MODEL
Review round: 1
Reviewer: 3

Output:
Basic reporting: The manuscript is clearly structured and easy to follow. Some sentences need english editing. The introduction with references is suitable. Raw data are supplied.
Description of figures can be improved:

Fig. 2: Would you have not expected that the mock treated local and systemic leaves are in close proximity? Please comment/explain. Did this affect the list of metabolites?

Fig. 3: What do we learn from Fig. 3? There is no further comment on this figure except that this heatmap is there.

Fig. 4: add the observation that the virulent and avirulent strain have more overlap in lipids than local and systemic of the same strain (not surprising but noteworthy)
General: the occurence of several compounds, especially proposed esterified fatty acids, are rather surprising (Suppl. Table 1, Fig, 3, text), for example TG 14:1_10:4_11_4), TG 8:0_11:2_20:5, TG 4:0_18:2_22:6 or TG 6:0_10:3_18:3. Please re-evaluate the data to remove errors generated by automated identification.
Experimental design: The research question is well defined and the data are new. The methods are sufficiently described.
Validity of the findings: Investigations aiming to identify compounds and mechanisms involved in SAR are certainly valuable. However, this manuscript is descriptive resulting in some (new) compounds which show alteration in levels upon pathogen treatment in local and distal leaves. The identification of these metabolites should be reliably correct (see "basic reporting"). Also, the conclusions are rather limited, an overinterpretation should be avoided (see below).
The discussion can be improved:
Especially conclusions that lipids are involved in immune responses (line 291) or even resistance (line 311, 316) cannot be drawn.
paragraph line 250: the conclusion is not clear. Since PCs are present in bacteria, is it possible that the locally detected differencial PCs derive from the bacteria?
For considering a potential relevance of the accumulating compounts please comment the amount and amplitude of change of the metabolites.
Please comment on the timing of responses to Pseudomonas. Which responses are described at which time points and which time point was chosen here? This might have a big impact on the results.
The comparison with published metabolites should be improved. It should be included whether you found an accumulation of lipophilic compounds described by other groups e.g. steroids, tocopherols or oxidized lipids.
Additional comments: specific points:
line 97: bacteria are sedimented are pelleted but not precipitated.
line 164 and legend Fig. 5: MgSO4-inoculated, it is not an infection
Line 166: change the term „differentially expressed lipid metabolites (DELMs)“. Genes are differentially expressed, but not lipids.
line 176: „related to local resistance“: the relation to resistance is not clear.
line 203: PA is an important lipid factor in plant immune processes – unvalid conclusion.


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: LIPIDS ASSOCIATED WITH PLANT-BACTERIA INTERACTION IDENTIFIED USING A METABOLOMICS APPROACH IN AN ARABIDOPSIS THALIANA MODEL
Review round: 2
Reviewer: 1

Output:
Basic reporting: The specific points mentioned in the first review have been addressed to a large extent. The manuscript text, especially those passages added in the resubmission, is not ready for publication as language need to be improved and a number of minor errors/typos are present.

The Gao et al. 2020 reference should be mentioned in the introduction, as the data in this study is from the same experiments already described in Gao et al 2020.
Experimental design: not applicable
Validity of the findings: 1) Heat map (Figure 3):
Why is one sample missing in the heat map? I believe that the same set of samples should be used for all anlyses.

Compared with the previous version of the heat map, 5 candidates are missing. Why?

There is no explanation how the heat map was generated, what the values represent, and how the shown metabolites were selected.

I have difficulties understanding your answer in the rebuttal letter:
‘We found out from the raw data that some metabolites were not identified in SL-CK-3 group. The expression pattern of SL-CK-3 was different from the other two groups. The result did not influence the composition of our differentially regulated metabolite list because the list of metabolites is made up of 18 samples. "

-	I don’t see a causal relationship here? Actually, 17 different metabolites were designated as differentiall abundant in your manuscript.
-
‘If some metabolite is not identified, the value of this metabolite is “0”. ‘

-	I don’t understand the argument.

‘Moreover, it may be caused by taking different systemic leaves of locally MgSO4-treated plant (leaves of developmental stages 7-9 as sample).’

Didn’t you use a pool of several leaves for your samples? Please specify this in the methods section.

2) Unlikely metabolite predictions have been successfully removed.

3) The issue with one of the control treatment samples, SL-CK3, has not finally been resolved (see above). Removal from the heat map calculation is not a good option in my opinion. Either this sample is discarded from all analysis or it is included in all. If the sample remains, please mention the impact for the results obtained in the discussion (e.g. differentially regulated metabolite list).
Additional comments: Line 132 Reference missing for the software ‘software program Lipidsearch 4.2.21.’
Line 134-135 using types of software: SIMCA-P14.1.

Line 164-166 All samples in OPLS-DA were within confidence intervals; and this OPLS-DA plot can show the differences in groups. (Figure 2).

Line 173 P19001-Lipidmedian corr).??
Line 174 We found from new heat map ??
Line 107 can refer to
Figure legend to Fig. 3 is difficult to understand: ‘The phylogeny lines on the left represent the clustering analysis results of different samples.’?
Language: 	 -MgSO4 -> MgSO4
-there is positively correlated between PE
-‘normal metabolites’?


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: LIPIDS ASSOCIATED WITH PLANT-BACTERIA INTERACTION IDENTIFIED USING A METABOLOMICS APPROACH IN AN ARABIDOPSIS THALIANA MODEL
Review round: 2
Reviewer: 2

Output:
Basic reporting: The manuscript has overall improved. Some sentences still need english editing.
Description of figures can be improved:
Fig. 2: I still think that it needs to be mentioned that the mock controls (LL-CK versus SL-CK) are so different. Is this due to a wounding response? Or to the use of younger leaves for the systemic response?
Fig. 3 and its description have strongly improved. However, it is still not clear what is shown. The legend says content of lipids. How have the content values between 20 and 35 been generated? Please explain in the legend. Why is the sample SL_CK.3 missing in the figure?
Fig. 4A and B: The number of DLMs is the same as in the last version even though the number of metabolites was reduced from 173 to 127. For example TG (5:0_18:2_18:3) was removed but the number of DLMs mentioned in the text is still the same. Please re-check this figure and the corresponding paragraph.
Experimental design: no comment
Validity of the findings: The discussion has improved.
Conclusions: line 342: this was not changed compared to the last version even though the 20 and 13 DLMs refer to Table 2 which only shows a total of 18 metabolites. Please re-check.
Additional comments: line 174: remove „new“
lines 181, 342 and 346: change DELM to DLM


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: LIPIDS ASSOCIATED WITH PLANT-BACTERIA INTERACTION IDENTIFIED USING A METABOLOMICS APPROACH IN AN ARABIDOPSIS THALIANA MODEL
Review round: 3
Reviewer: 1

Output:
Basic reporting: Thank you for your revisions. The revised manuscript has been greatly improved, and carefully addressed my concerns from the previous review. I thank the authors for their detailed responses to my comments. The updated manuscript with the new Figure 2 (OPLS-DA), Figure3 (heat map), Figure4 (venn) and differentially regulated metabolite list (table 2) is much clearer. I think this is an interesting publication and the paper will be a useful reference.
I still spotted some occasional typographic errors (e.g. MgSO4 in figure legend to Fig.5.), and the term ‘Differentially lipid metabolites (DLM)’ maybe should be changed into ‘differentially regulated metabolites’, but I am not a native speaker, and I believe this will be taken care of when checking the proofs. The somewhat low resolution of Figure 2 and Figure 4 in the pdf file seems to be not present in the original file. I assume this will be taken care of by the production department.
Experimental design: OK
Validity of the findings: OK
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: LIPIDS ASSOCIATED WITH PLANT-BACTERIA INTERACTION IDENTIFIED USING A METABOLOMICS APPROACH IN AN ARABIDOPSIS THALIANA MODEL
Review round: 3
Reviewer: 2

Output:
Basic reporting: I do not have any further comments
Experimental design: I do not have any further comments
Validity of the findings: I do not have any further comments
Additional comments: I do not have any further 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: WINTER DIET OF BURROWING OWLS IN THE LLANO LA SOLEDAD, GALEANA, NUEVO LEÓN, MÉXICO
Review round: 1
Reviewer: 1

Output:
Basic reporting: 'no comment'
Experimental design: 'no comment'
Validity of the findings: 'no comment'
Additional comments: The study deals with the dietary niche breadth of burrowing owls Athene cunicularia in northern Mexico. The authors consider prey type, frequency of occurrence, and biomass during three consecutive winter periods. The sample size was adequate and authors identified more than 800 prey items. Vertebrates accounted for the majority of the consumed biomass with Cricetid rodents being the most important prey species. With regard to invertebrates, beetles were the most commonly found in pellets. Based on the figures of niche breadth the burrowing owl is regarded as a generalist species though a significant association between relative biomass of rodent species and invertebrate families was discovered. The authors justify this pattern as a result of rainfall variations. Overall I find the study interesting and meriting publication. The manuscript is well written, the sample sizes are adequate for statistical analysis and the analytical procedure is indicative for dietary studies of raptors. I have a number of general comments that could improve the manuscript and assist the reader to extract the most of its value:
1)	In all tables apart from a grand total per season, relevant subtotals per taxa would be interesting to be depicted.
2)	Generalist vs. specialist division with regard to winter diet in nocturnal birds of prey is very common in all food ecology studies. However I believe that many studies misjudge dietary shifts as food specialization. I wonder if the authors have data on the abundance of the potential or actual prey species on the study area. Moreover data on their availability would be mostly valuable. I would suggest to make a short paragraph on the ecology of the prey species such as their breeding cycle, or when population peaks are expected and connect them to the findings of the study.
3)	In the discussion section, it would be interesting to make some comments on the owl’s feeding behaviour and hunting strategy. I wonder if rainfall have an indirect impact on the species due to the vegetation height or structure that could impede foraging.
4)	Last perhaps all the data on the biomass, prey items, niche breadth, the study area and the reference from different studies could be included in a table. This would make comparisons much easier.


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: WINTER DIET OF BURROWING OWLS IN THE LLANO LA SOLEDAD, GALEANA, NUEVO LEÓN, MÉXICO
Review round: 1
Reviewer: 2

Output:
Basic reporting: The manuscript is well written, with no need for major corrections, but some sentences are a little confused.
There is a vast literature on the subject (burrowing owl diet) and, although the authors have cited many relevant papers, I think it was necessary to add some studies carried out in the Neotropical region (I suggested some to the authors).
Figures 1 and 3 do not present relevant information and can be deleted. The species is well known and images can be found easily in an internet search. Images of some remains of the prey, just as an example, do not add anything relevant to the manuscript.
Tables 1 and 2 can be merged. The information on Biomass and the frequency of ccurrence can be presented in a single table.
Table 5 can be deleted and information added to the body of the manuscript.
Experimental design: The study is basically descriptive of the species' diet, and although the authors have proposed to analyze the temporal variation and the relationship of the diet to the year rainfall at the study site, the analyzed period was too short to assess temporal patterns. Only 3 years were evaluated, with one year differing from the others. Result that can be merely casual, not reflecting a pattern in the diet, especially in the case of a generalist and opportunistic species, in which changes in the diet would be expected according to changes in prey availability.
The analisys of biomass surely undenderestimates data for invertebrates, sice the authors considered only one individual per pellet of each prey identified (Methods, ln.102-103). Also, the authors do not explain how they estimated the biomass for birds, most of which have not yet been identified at the Family level.
According to the manuscript, 358 samples (pellets) were collected, however, the authors did not reported how many nests and/or individuals the sample is representative. If the representativeness is low (1, 2, 3 couples of birds/nests) the result does not have much power to represent a pattern of the species' diet, even for the studied region.
Validity of the findings: At several parts on the discussion, the authors apparently use FO as an index of relative abundance (or relative density). For example: 1) Line 153 - “Invertebrates represented 90% of prey items consumed…”; 2) Line 157 - “Insects represented 84% of the items in the diet…”; 3) line 197 - “Vertebrates represented 10% of the remaining prey items consumed by the Burrowing Owls…”. The study presents Frequency of Occurrence data, therefore, the percentage is an indication of how commonly a certain prey is consumed by the owl, but it does not mean that it is greater or less representative in terms of the quantity of items consumed, as suggested by the sentences above.
Additional comments: Dear authors, I would like to congratulate you for the study, which brings new information about Bowl's diet during the non-reproductive season, a period in which we find less information in the literature about the species' diet. The manuscript is well written, with some confusing sentences that could be rewritten. However, there are some issues that I would like to discuss with you, which I think weaken the study, but which can be easily resolved.
The first issue is the temporal assessment of the diet. Although the results showed a relationship between diet and rainfall, the study covered only 3 years, a short time to establish a pattern of diet variation. Since the pellets where collected every 3 days, perhaps refining the analysis for a monthly assessment would reinforce the results found for the interannual analysis. I think the result presented is relevant, but as it is one of the main objectives of the work (Introduction, ln. 66-68), it weakens the study as a whole. I suggest maintaining the description of the Burrowing owl diet as the main objective of the study, presenting the temporal analysis and relationship with rainfall, but maintaining caution when analyzing these data.
Regarding the analysis of the diet, my main criticism is in the calculation of biomass assuming that there was only one individual per sample (Methods, p. Ln.102-103). Would not invertebrates be totally underestimated in this analysis? Although invertebrates tend to have less representativeness in terms of biomass, they are generally numerically higher (relative density), and the absence of relative density data (e.g., minimum number of items consumed) skews the result. It is relatively easy to estimate the minimum number of individuals consumed based on single structures (skulls, heads, pronotes), paired structures (elytra, jaws) or multiple (limbs, wings) and would add more information to the diet description giving a result more robust for estimating biomass and relative importance of each type of prey in the Burrowing owls diet. The use of Frequency of Occurrence (FO) alone to analyze the diet is also complex, especially when comparing the results with to other studies. On the one hand, FO reduces the effect of opportunistic consumption of aggregate prey (e.g., ants and termites), on the other, it gives equal weight to prey that is more abundant and to those that are rarer. If both type of prey (rare and abundant) are consumed regularly (eg, FO> 70%), but according to their availability, a prey that is little consumed because it is scarce (eg, relative density = 0.5 ind./pellet), but regularly consumed ( eg, FO = 85%), will appear to be as relevant as abundant prey (eg, relative density = 20 ind./pellet) also consumed regularly (eg, FO = 90%). My suggestion would be to include information on the relative density (RD) of individuals and calculate a composite index such as the IRI (Pinkas et al. 1971. Calif. Fish Game 152: 1-105), which aim to equalize FO, RD and Biomass. Compound indices can be problematic, but when presented together with FO, RD and biomass, they can be of great value (e.g., Cortés 1998. Canadian Journal of Fisheries and Aquatic Sciences 55 (12): 2708).
Still in relation to biomass, it is not explained in the manuscript how the mass was estimated for the birds, most of which have not yet been identified at the Family level. This information is highlighted at the end of the results (lines 145 and 146), so the way in which the birds' biomass was estimated should be reported.
Finally, I would like to know how many individuals or nests were included in the analysis. You collected 358 samples (pellets), however, more important than informing the total samples collected is informing how many nests and/or individuals the sample is representative, as this affects the amplitude of the results. If the representativeness is low (1, 2, 3 couples of birds/nests) the result does not have much power to represent a pattern of the species' diet, even for the studied region. Especially being one of the regions with potentially high density of Burrowing owls (according to the authors themselves, the region "... the most extensive and continuous habitat in terms of burrow and food availability for the Burrowing Owl in northeastern Mexico".).
Below I present some specific questions that need clarification or that can be improved in the manuscript.

1)	Introduction
There is a vast literature on Burrowing owl, especially on diet, many of which were cited in the manuscript, however, I would like to suggest some inclusions.
Lines 34 -37 - Several causes related to the decline of the owl populations in the Northern Hemisphere are listed, however based on data from Canada (Environment Canada, 2012). I’d like to suggest consulting Enríquez and Peréz (2017 - Los Búhos de México - In Enríquez (ed). Neotropical Owls: Diversity and Conservation) who on page 487-490 comments on the effect of pesticides on eggs (Garcia-Hernández et al. 2006); on illegal trade (Sosa-Escalante 2011); and on loss of habitat and other threats (Chávez-Ramírez 1990. Rodríguez-Estrella y Granados 2006, GarcíaHernández et al. 2006). I also suggest citing Rodriguez-Estrella and Granados (2006 - page 191, Table 3; In Rodriguez-Estrella (ed) Current Raptor Studies in Mexico).
Lines 38-39 - Although “Food availability is one of the most important natural limiting factors in populations during winter (Newton, 1998; McDonald et al., 2004).”, I don’t thing that is the case to Burrowing owl. It’s an opportunist and generalist species that will basically changes from one type of prey to another according to their availability. Also, you didn’t evaluated the food availability, so I don’t think that this sentence is related to the study or justifies the study as it seems the authors intended to including it in the Introduction.
Lines 40-42 - I suggest including more references on the species' diet in addition to Plumpton and Lutz (1993) and Poulin (2003). When writing “The winter diet of the Burrowing owl has been studied in the United States in Texas, Nevada, and California, as well as in other countries in North, and South America and consists of invertebrates, small mammals, and reptiles (Plumpton & Lutz, 1993). ” you suggests that information about the diet of Burrowing owls for these regions is scarce, when in fact it is vast. While “Invertebrates are consumed most frequently (Poulin, 2003) but mammals compose most of the biomass (Andrade et al., 2004; Littles et al., 2007; Nabte et al., 2008; De Tomasso et al., 2009; Andrade et al., 2010; Carevic et al., 2013). ”, suggests that only Poulin (2003) identified higher consumption of invertebrates, while many more studies found higher consumption of vertebrates, which is not completely true. Vertebrates may be more relevant in terms of biomass, but invertebrates are more representative numerically (in the discussion you highlight that most of prey eaten by the Burrowing owl is invertebrates). I strongly suggest that you rewrite these sentences so as not to induce the reader to a misunderstanding about the knowledge of the Burrowing owls diet.
Lines 66-68 - I wonder what your hypothesis is about the relationship between diet and rainfall. “We predicted that in a year with high rainfall, diet composition will be different than in drier year…” is a very broad hypothesis. How would you expect the diet to change in relation to local railfall? For example, what kind of prey would be most consumed in a drier year? And in the rainiest? What is the theoretical support to expect this variation? The hypothesis should be well defined and supported by the literature, which was mentioned briefly, in your Introduction, in the previous paragraph.

2)	Study Area and Methods
Lines 80-83 - “The Llano La Soledad… represents the most extensive and continuous habitat in terms of burrow and food availability for the Burrowing Owl in northeastern Mexico.” - is this conclusion based on any study or is it an inference of the authors?
Lines 90-92 - “Annual rainfall for 2002-2004 years, was obtained from the closest (~ 6 km) meteorological station in La Carbonera (19032; 92 CONAGUA, 2019).” - this information is loose in this paragraph. I suggest putting in the topic “Statistical Analyzes”, explaining that an analysis of the diet was carried out with the average precipitation of the years studied.

3)	Results

Tables 1 and 2 can be joined. For each item you can inform in the table cell: FO (B) (e.g., Peromyscus maniculatus 0.7 (10.7), or the reverse B (FO)).
Table 5 can be deleted and the information mentioned in the text.
Line 128 - “Niche breadth measures were wide, indicating, as expected, a generalist species,…” - I suggest removing “as expected” because it is a term that “discusses” the result.
Lines 129-130 - “… with consistent overall estimates for both frequency of occurrence (FT = 0.77; 95% CI = 0.74-0.80) and biomass (FT = 0.74; 95% CI = 0.70-0.77).” - although FT refers to Smith's measure of niche breath, it is possible that some readers are unfamiliar with this measure, so I suggest including this information in the first mention, eg ”... both frequency of occurrence (Smith's measure [FT] = 0.77; 95% CI = 0.74-0.80) and biomass (FT = 0.74; 95% CI = 0.70-0.77). ”
Lines 145-146 - as I mentioned earlier, how did you calculate the biomass of birds?
4)	Discussion
At several parts on the discussion, the authors apparently use FO as an index of relative abundance (or relative density). For example: 1) Line 153 - “Invertebrates represented 90% of prey items consumed…”; 2) Line 157 - “Insects represented 84% of the items in the diet…”; 3) line 197 - “Vertebrates represented 10% of the remaining prey items consumed by the Burrowing Owls…”. The study presents Frequency of Occurrence data, therefore, the percentage is an indication of how commonly a certain prey is consumed by the owl, but it does not mean that it is greater or less representative in terms of the quantity of items consumed, as suggested by the sentences above.
Line 177 - 179 - “Conversely, in South America, although coleopterans have been found to be highly consumed and preferred by the burrowing owl, their relative abundance was higher in agricultural areas than in vegetated sand-dunes (Cavalli et al., 2013) … ”- Coleopterans are an important item in the Burrowing owl diet in South America and it is worth mentioning more studies that support this in addition to Cavalli et al (2013). As a suggestion, you could mention some of these studies:
ANDRADE, A., M.J. NABTE, AND M.E. KUN. 2010. Diet of the Burrowing Owl (Athene cunicularia) and its seasonal variation in Patagonian steppes: implications for biodiversity assessments in the Somuncurá Plateau Protected Area, Argentina. Studies on Neotropical Fauna and Environment 45: 101–110.
CADENA-ORTIZ, H.F., C. GARZÓN, S. VILLAMARÍN-CORTÉZ, G.M. POZO-ZAMORA, G. ECHEVERRÍA-VACA, J. YÁNEZ, AND  J. BRITO M. 2016. Diet of the Burrowing Owl Athene cunicularia, in two locations of the inter-Andean valley Ecuador. Revista Brasileira de Ornitologia 24(2): 122-128.
CAREVIC, F.S, E.F CARMONA, AND A. MUÑOZ-PEDREROS. 2013. Seasonal diet of the burrowing owl Athene cunicularia Molina, 1782 (Strigidae) in a hyperarid ecosystem of the Atacama desert in northern Chile. Journal of Arid Environment 97: 237-241
DELGADO-V., C.A. 2007. La dieta del Currucutú Megascops choliba (Strigidae) en la ciudad de Medellín, Colombia. Boletín SAO XVII: 111-114.
MOTTA-JUNIOR, J.C.. 2006. Relações tróficas entre cinco Strigiformes simpátricas na região central do Estado de São Paulo, Brasil. Revista Brasileira de Ornitologia 14: 359-377.
NABTE, M.J., U.J.F. PARDIÑAS, AND S.L. SABA. 2008. The diet of the Burrowing Owl, Athene cunicularia, in the arid lands of northeastern Patagonia, Argentina. Journal of Arid Environments 72: 1526–1530
TOMMASO, D.C., R.G. CALLICÓ FORTUNATO, P. TETA, AND J.A. PEREIRA. 2009. Dieta de la Lechucita Vizcachera (Athene cunicularia) en dos áreas con diferente uso de la tierra en el centro-sur de la provincia de La Pampa, Argentina. Hornero 24: 87-93


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: WINTER DIET OF BURROWING OWLS IN THE LLANO LA SOLEDAD, GALEANA, NUEVO LEÓN, MÉXICO
Review round: 1
Reviewer: 3

Output:
Basic reporting: 1.)	Although not bad in general, a careful review of the entire manuscript is needed in order to correct the numerous English errors (i.e., sentence structure, spelling, etc.).

2.)	I’m comfortable with the Introduction and background as it relates to the burrowing owl (BUOW) diet literature. However, as this manuscript is intended as a full scientific paper, I’d suggest a brief description of the owl, and perhaps a description of its distribution and habitat preferences.

3.)	Conformation of the manuscript structure with peerj standards seems good.  However, placing a comma after the author’s name and before the parentheses, in a citation (e.g., Smith, (2015) is not correct format.  I’d suggest removing these commas.

4.)	Figures are acceptable in general, although captions, which belong below the figure, should be edited for clarity, and then shortened.  Specifically, for Fig. 1, I’d suggest cutting out the term “study species”, Fig. 2 seems fine, Fig. 3 is good, and Fig. 4 is ok, but the axes must be labeled.

5.)	The Literature Cited section is extensive, which is usually a very good thing.  However, perhaps a reduction in the number of references might be warranted here.

6.)	More reference to tables and figures in the Discussion would help reduce the amount of text.

7.)	Some of the terminology used for diet parameters is confusing, and therefore, because of its relative importance, I will mention this issue again.  As an example, on line 44 of the PDF version of the manuscript, as well as in Table 1, the authors use the term “frequency of occurrence”.  Frequency data (not presented) refer to the number of individual prey items in a sample from that prey taxon.  Frequency values would be whole numbers.

Table 1 data are presented as percentages.  I believe these values to actually be “Percent of Total Prey Items”.  The authors should review these data.  Then, a “Frequency” column and perhaps even a “Percent Occurrence” column could be added to Table 1.  Percent Occurrence shows the percentage of all pellets containing that prey item.  All tables should be carefully reviewed by the authors.
Experimental design: 1.)	Primary research seems to be within the scope of this journal.

2.)	The specific research questions and hypotheses should be clarified.

3.)	The results from this study seem to fill a regional knowledge gap.

4.)	Description of study area is good.

5.)	Following pellet collection, all information relative to diet analysis, such as pellet dissection, prey identification, and methods of quantification are left to the literature. At least a brief summary of techniques used during this process is critical.

6.)	Seems that prey identifications were primarily made using taxonomic keys, or plates in field guides and other references.  Making Species and sometimes even Genus-level IDs using the literature can be challenging at best.  The best way to make such IDs requires the use of comparative Osteological and Invertebrate collections.  I see no evidence that the authors did this.

7.)	Quantification methods for the diet analysis must be clarified.  The authors should present data as Frequency, Percent Occurrence, Percent of Total Prey Items, etc.  Check the literature for published examples of how other authors have presented their diet data.

8.)	For any tabular listing of prey taxa, those taxa need to be presented in proper phylogenetic order.  For example in Table 1, Peromyscus, Reithrodontomys, and Neotoma are Genera within the same Family Cricetidae.  Two Heteromyids and a Sciurid separate these taxa at this time.  Please make these changes.  Also, tables should also include common names of all prey taxa that have them.
Validity of the findings: 1.)  The primary diet data have been presented, or they are at least available to the authors.  For example, I’d suggest that Frequency data for each prey taxon should be included. Also, remember that without the use of comparative collections, species-level identifications of osteological (skeletal) remains will always be in question.

2.)	Conclusions seem sound.

3.)	Once again, I believe that the results of this study, once the manuscript is edited for errors, and once additional prey parameters are included, represent a significant addition to the knowledge of the BUOW winter diet.
Additional comments: No Comments


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

Input:
Title: WINTER DIET OF BURROWING OWLS IN THE LLANO LA SOLEDAD, GALEANA, NUEVO LEÓN, MÉXICO
Review round: 2
Reviewer: 1

Output:
Basic reporting: Reporting is clear and unambiguous.
Experimental design: The research question is adequately defined and its analysis well designed.
Validity of the findings: OK the results are meaningful.
Additional comments: No comments fro 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: WINTER DIET OF BURROWING OWLS IN THE LLANO LA SOLEDAD, GALEANA, NUEVO LEÓN, MÉXICO
Review round: 2
Reviewer: 2

Output:
Basic reporting: No comment
Experimental design: No comment
Validity of the findings: No comment
Additional comments: Dear authors, I believe that in this new version of the manuscript most of the questions that I pointed out in the first review were resolved and the changes made resulted in an improvement in the manuscript. However, I still have some suggestions for the manuscript and some issues that I think are still unclear in relation to biomass assessment, which I have pointed out below.
Sincerely.

Felipe Zilio

INTRODUCTION
In the first paragraph, I’d like suggest a few changes about distribuiton and movements of the Burrowing Owl (BOWL). – “The North America races of Burrowing Owl (Athene cunicularia Molina 1782) are distributed from southwest Canada, through the west and central USA (but also in Florida) and Mexico, although most of the northern populations migrate to southern USA and Mexico (Marks et al. 1999).”
Lines 29-30 – “... and a great number of individuals spend part of their yearly life cycle in the three countries some portion of the year...” – This might be deleted
Line 33 – “The Burrowing Owl has diurnal activity, although hunting mainly at dawn and dusk (Coulombe 1971). - I understand your point, but BOWL is not a fossorial species. It uses burrows to make nests and protect itself from predators, as you mentioned later.

STUDY AREA AND METHODOS
Pellet Collection and Analyses
Line 107 – “We collected pellets at active burrows located along 20 random transects of 1 km x 200 meters where selected at random and each one measured 1 km long by 200 m wide...” – you repeated the information about transects.

Lines 116-117 – About FRO.
Now I understand what you meant when you referred to the Frequency of Occurrence. I usually refer to the Frequency of occurrence as the percentage of pellets that contain a certain type of prey. The total of prey items (or individuals) found in the sample I know as a Relative Density (or Relative Frequency), so I was confused when you presented your results using phrases like “Invertebrates represented 90% of prey items consumed…” or “Insects represented 84% of the items in the diet…”. The raw data also helped me to understand what you meant by FRO. So, many of my comments related to this subject (biomass measurement, etc.) in the previous review no longer make sense. However, although it is clear to me now, I still think that using Frequency of Occurrence or FRO instead of Relative Density might confuse and induce readers to misinterpret their results.

Satatistical analyses
Lines 147 – “Average anual precipitation is 427mm (INEGI, 2005) - If this is the average rainfall for Llanos La soledad, it should be in the Study Area section. Otherwise, I don't see why to bring this information here, since on lines 163-165 you showed the average rainfall over the years sampled.

RESULTS
Lines 157-159 – “Insects, primarily from the Orders Coleoptera (IRI=41%; 54%) and Orthoptera (IRI=10%; 26%), represented 84% of consumed items but contributed only to 12% of the biomass (Table 1).” -  the second value refers to FRO? Table 1 does not show Biomass data.
Lines 166 – “There was a highly significant and small correlation between winters and prey items...” – I suggest: “There was a highly significant, but small correlation between winters...”
Lines 177 – “...were very stable among years with a relative biomass between 11 and 13% (Table 1).” -   Did you delete the biomass information from Table 1 or use biomass as Volume? If you excluded, I think you should include biomass in Table 1. In the first review, I asked how you estimated the biomass of unidentified birds and I still don't understand. On lines 123 to 128, you explained how you estimated biomass for idenfied species, but not for unidentified species. Unless you measured bones or feathers and inferred the biomass of each bird, then calculated the total biomass of unidentified birds for each year, how did you estimate the biomass of a specimen you don't know what it is using a reference collection of a museum?
Table 1 – You should include the Biomass data and I suggest changing the P (presence) for N (Number of itens or individuals).

DISCUSSION
Lines 236-241 – “According to IRI (Pinkas et al. 1971), the main food component was invertebrates (IRI = 98%; 90%) Insects (IRI = 97%: 96%) with the highest Coleoptera consumption (IRI = 41%; 54%) and Orthoptera (IRI = 10%; 26%). Now, there are either large prey that are eaten infrequently or predominate in the samples because they are slowly digested such as vertebrates (IRI = 1.7%; 10%) and Arachnida (IRI = 0.44%; 2%), as mentioned by Hart. et al. (2002) (Table 1).” - I think that the IRI data are results of your study, so they should be in the Results, not in the Discussion. Here, just present the name of the taxon you are referring to. The reader, if necessary, will go to the Table or the results to check the values.


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: POLYPHENOLIC EXTRACT FROM PUNICA GRANATUM PEEL CAUSES CYTOSKELETON-RELATED DAMAGE ON GIARDIA LAMBLIA TROPHOZOITES IN VITRO
Review round: 1
Reviewer: 1

Output:
Basic reporting: 1) The summary is unclear:
a) It remains to be argued why pomegranate peel extract is a good option against Giardia trophozoites. Why should it have activity against Giardia, if previously it has only been shown to have antibacterial activity?
b) The results do not describe the compounds identified by HPLC.
c) It is necessary to improve the discussion of the results.

2) The quality of figure 4 is low, the resolution needs to be improved.
3) What is the most relevant contribution, if in the introduction section they mention that the activity of pomegranate peel extract against Giardia was previously demonstrated in an in vivo model?
4) With the above, I believe that there should be a rethinking of the hypothesis.
Experimental design: In relation to the experimental design, I consider that it is well planned for the proposed objectives, I would only observe the following:

1) in section 2.6 Adherence inhibition assay: what concentrations of the extract were used?
2) in section 2.7 The Effect of pomegranate peel extract on morphology by Scanning Electron Microscopy (SEM): The SEM methodology needs to be further detailed.
Validity of the findings: The proposed hypothesis lacks solidity based on the referred antecedents.
The discussion is weak, mainly in the integration of the results, for example, why does the increase in the expression of tubulin destabilize the cytoskeleton? Why are the polyphenolic compounds identified decisive in the decrease in adherence or growth of Giardia?
A cell viability assay after treatment and the evaluation of other molecular markers of cell viability would help to reach more decisive conclusions.
Additional comments: No additional comments


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

Input:
Title: POLYPHENOLIC EXTRACT FROM PUNICA GRANATUM PEEL CAUSES CYTOSKELETON-RELATED DAMAGE ON GIARDIA LAMBLIA TROPHOZOITES IN VITRO
Review round: 1
Reviewer: 2

Output:
Basic reporting: NC
Experimental design: Dear authors,

The overall methodology is logic and acceptable. However, there are comments (all are in yellow highlighted colors) in the attached file for further improvement of this part.
Validity of the findings: Regarding Results, Discussion and References, comments are in yellow highlighted colors for further improvement.
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: POLYPHENOLIC EXTRACT FROM PUNICA GRANATUM PEEL CAUSES CYTOSKELETON-RELATED DAMAGE ON GIARDIA LAMBLIA TROPHOZOITES IN VITRO
Review round: 1
Reviewer: 3

Output:
Basic reporting: It is an interesting and well written and structured study; the introduction presents relevant information related to the need for new giardicidal agents and the importance of phenolic compounds as antiparasitic agents. Previous studies (Al-Megrin, 2017; El-Kady et al., 2021), using a methanolic and ethanolic  extract from P. granatum, demonstrated the reduction of G. lamblia cysts and trophozoites using a giardiasis model as wells as the induction of nitric oxide (NO). However, the characterization of the possible therapeutic targets of the promegranate extract has not been determined. In the present study, the in vitro giardicidal activity of an ethanolic extract obtained from pomegranate peel was evaluated. Authors identified the polyphenolic compounds contained in the ethanolic extract ( ellagic acid and punicallagins, among other components)  and their inhibitory effect on parasite growth and adhesion capacity, as well as its effect on the morphology and tubulin expression of parasite are presented. Figures are relevant, and figure legends are well described.
Experimental design: There are some considerations to the present study.
Different concentrations of the PP extract were tested, it is shown that parasite growth was affected by all the concentrations, as well as the adhesion capacity with the exception of 125ug/mL. Further assays were performed from 150 ug/mL to 200 ug/mL, demonstrating that the extract induced several morphological changes such as holes on the membrane and shorter flagella, among others. In addition, they showed that at 150 and 200 ug/mL, the ethanolic extract has different effect on the alpha tubulin expression demonstrated by Western blot analysis.
- It is convenient that authors state the reason they had to exclude the lowest concentration for ultrastructural and tubulin expression assays. Although the lowest concentration did not affect parasite adhesion, it inhibited the parasite growth, then, it could be of interest to evaluate if the lowest concentration induced any effect on alpha tubulin expression (decreased or overexpressed) or at the ultrastructural level. Besides, authors mentioned that “the structural and functional affectations caused by pomegranate may be due to the change in the expression of tubulin”.  In this regard, it is confused to correlate SEM and Western blot analysis; since the three concentrations evaluated (150, 175 and 200 ug/mL) affected the parasite morphology in a similar way, however, their effect on alpha tubulin expression is completely different.. One important feature that requires to be mentioned is how the alpha tubulin expression at different concentrations was normalized?  Besides, figure 4 quality needs to be improved.
 It could be interesting to include parasites treated with commercial ellagic acid and analyze the effects on parasite at the ultrastructural level and alpha tubulin expression, supporting the suggestion that the ellagic acid of the ethanolic extract induces alterations on the parasite cytoskeleton.
Validity of the findings: Authors mentioned that “These reports lead us to suppose that polyphenols, possible ellagic acid, in PP extract alters Giardia cytoskeleton”.  However, data presented herein do not give enough evidence that the extract or the ellagic acid alter the parasite cytoskeleton. No strong evidence supports the effect on parasite microtubule organization (disk alteration, lack of funnis, among others).
In addition, authors discussed the fact that 150 µg/mL and 200 µg/mL exposure caused opposite effects in α-tubulin expression. They mentioned that “paclitaxel-resistant human lung cancer cell lines overexpress α- and β-tubulin” and on the other hand “reduction of α- tubulin expression level in Giardia has been documented with curcumin, another polyphenolic compound, causing changes on its distribution”.
-In this regard, how can authors explain that both concentrations, together with 175ug/mL (that in Western blot analysis do not affect alpha tubulin expression), induce similar morphological alterations?
Additional comments: In general terms, the present study shows data about the effect of ethanolic extract on the growth and adhesion of Giardia trophozoites; in addition, the mechanism of action is analyzed by SEM and Western blot assays however, data presented are preliminary and do not clearly demonstrate the effect on the parasite cytoskeleton, a fact that authors suggest.
 To give more experimental support to their suggestion about the effect of the extract on the tubulin network, it is important to demonstrate the effect on the ventral disc as well as other cytoskeleton components.  SEM and immunofluorescence studies could give this evidence; SEM assays showing the ventral side of parasite could give relevant information


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: ALTERATIONS IN SARS-COV-2 OMICRON AND DELTA PEPTIDES PRESENTATION BY HLA MOLECULES
Review round: 1
Reviewer: 1

Output:
Basic reporting: Stepan Nersisyan and collaborators offer an interesting evaluation of alterations in SARS-CoV-2 Omicron and Delta peptides presentation by HLA molecules. The manuscript looks professionally written, excepting some fine-minor mistakes in grammar and typos.
In the introduction section, the authors just mention HLA class II viral-antigenic presentation; I recommend reinforcing this background since viral antigens are presented in the HLA class I context.
The last paragraph in the introduction regarding two updates to the T-CoV portal is out of the section; please relocate to the results section and avoid duplicating this information.
Figures look well and are highly informative. Please consider being more descriptive for titles for supplementary materials or include a legend describing their contents.
Although several pieces of research have described association studies related to genetic susceptibility with the HLA system, results vary between populations. Also, some alleles have been associated with protection in other viral respiratory diseases, particularly interesting those associated with class I alleles (example: doi: 10.1155/2018/3174868); please comment.
Experimental design: In my opinion, the submission defines properly the research question, which is relevant for the scientific forum.
Regarding the methods employed, the authors should expand the description for HLA-DRB1*03:01/peptide binding experiments section, beyond the ELISA standard protocols. Including trademarks/distributors and country.
Line 240 says: we then manually adjusted the results for HLA-DPA1/DPB1 and HLA-DQA1/DQB1 links. Please explain briefly in the text this adjustment.
Validity of the findings: Definitively, the higher strength is the validation employing peptide binding experiments. The authors compared peptide presentation profiles for the Omicron, Delta, and Wuhan SARS-CoV-2 variants in this manuscript. Only HLA-B*35:03 had one more Omicron/Delta tight binding peptide in NSP12 protein. Also, they predicted the complete absence of T-cell immunity evasion at the level of the whole virus.
When the analysis was limited to the Spike protein, eight alleles showed the enhanced ability of Omicron/Delta peptide presentation. In comparison, the four remaining alleles showed a significant decrease in the number of tight binders.
Data analysis apparently includes all information, no repository is mentioned, also, two updates to the T-CoV portal are described.
In a very recent paper, Zhang Y (doi.org/10.1038/s41431-022-01070-5) provides new insight into allelic influence on the HLA expression in association with SARS-CoV-2 and other common viral infectious diseases. Please discuss these findings in the context of your results.
Also, Xiao C (doi: 10.1016/j.isci.2022.103934) shows that most of the predicted CD8+ T cell epitopes showed proper binding with HLA-A2, whereas epitopes from B.1.1.7 had lower binding capability than those from the ancestral strain. In addition, these peptides could effectively induce the activation and cytotoxicity of CD8+ T cells. Please analyze and discuss these findings regarding your work.
The same group (doi: 10.3389/fimmu.2021.764949) previously described that the variation of a dominant epitope will cause the deficiency of HLA-A*02:01 binding and T-cell activation, which subsequently requires the formation of a new CD8+ T-cell immune response in COVID-19 patients. Please analyze and discuss these findings regarding your work.
Additional comments: Minor comments:
Please include the official description for variants the first time are described: Delta (B.1.617.2) and Omicron (B.1.1.529), etc.
Line 195, correct Oсmicron by Omicron.
Line 226: and a whole HLA haplotype is inherited from each "patient"...  did you mean "parent"? Please correct.


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: ALTERATIONS IN SARS-COV-2 OMICRON AND DELTA PEPTIDES PRESENTATION BY HLA MOLECULES
Review round: 1
Reviewer: 2

Output:
Basic reporting: The authors compare viral peptide presentation for the three variants: Wuhan, Delta and Omicron, and the comparisons were separately conducted for the whole virus and Spike protein peptidomes. The authors employed T-CoV database to obtain the lists of viral peptides which were predicted to be tight binders (affinity 50 nM) for the worldwide prevalent HLA class I and class II alleles.
Experimental design: The experimental design by the authors is sound. The presented work is interesting and it provides useful information for researchers investigating the dynamical aspect of peptide presentation combining computational with experimental results.
Validity of the findings: The key finding of the authors are:
•	absence of significant differences in the presentation of SARS-CoV-2-derived peptides by the most frequent HLA class I/II alleles and the corresponding HLA haplotypes
•	destructive effect of the Omicron mutations on PINLVRDLPQGFSAL for HLA-DRB1*03:01 allele
•	N211 deletion, L212I substitution and EPE 212-214 insertion dramatically declines the binding affinity of HLA-DRB1*03:01 and this peptide
Additional comments: The limitation of the study is that only set of peptides originating from the Spike proteins of the SARS-CoV-2 variants have been considered.

There are some points that should be addressed before the paper to be accepted:

1)	The authors should describe briefly how the binding experiments were conducted, and not just referring to their previous work.
2)	The author mention that not a single HLA class II allele passed 25% difference threshold when the analysis was performed at the level of the whole virus peptidome. It is not clear how did the authors choose hla-drb1*03:01 for further investigation?
3)	Line 194: predictions obtained by netMHCIIpan, all corresponding “Oсmicron”. Correct this typo, and check thoroughly the paper.
4)	Line 214:  There was another HLA class II allele with differential peptide presentation. Which is the HLA class II allele? This is not clear.
5)	In Figure 1 : -100% or -33% mean?


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: ALTERATIONS IN SARS-COV-2 OMICRON AND DELTA PEPTIDES PRESENTATION BY HLA MOLECULES
Review round: 1
Reviewer: 3

Output:
Basic reporting: Literature references were sufficient, the authors provided context and the article has structure. The conclusion section needs a rewriter and deleting the hypothesis.
Authors should be writing the aim in the introduction section.
Experimental design: The research show originality, bioinformatic analysis was rigorous, and peptide binding experiments had strong results.

Figure 2. Can you include the p-value in comparisons between Omicron and Delta Variants?

Figure 3. Can you include the p-value between HLA-DRB1*03:01+ and HLA-DRB1*03:01-

Line 171. “HLA-B*07:02, HLA-B*27:05, and HLA-A*32:01 had more than 50% difference in the number of presented Omicron/Delta peptides”; however, in figure 2, HLA-B*07:02 is the same value in both variants.

Figure 2. HLA-B*44:03 or HLA-B*08:01 show differences similar to HLA- A*32:01; do these alleles have biological importance? please discuss.
Validity of the findings: Line 315-317, authors add hypothesis; however, this is a conclusion section, please, delete the sentence and change to section discussion.
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: ALTERATIONS IN SARS-COV-2 OMICRON AND DELTA PEPTIDES PRESENTATION BY HLA MOLECULES
Review round: 2
Reviewer: 1

Output:
Basic reporting: The authors have improved the overall presentation of the paper, as suggested by the Reviewers.
Experimental design: The Material and Method section has been improved.
Validity of the findings: The results obtained from this work are sound and important particularly in the context of Covid-19 pandemic that we all are facing.
Additional comments: Accept in its current form


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: ALTERATIONS IN SARS-COV-2 OMICRON AND DELTA PEPTIDES PRESENTATION BY HLA MOLECULES
Review round: 2
Reviewer: 2

Output:
Basic reporting: The introduction shows context and structure.
References are sufficient.
The figures are well-described.
Experimental design: The results are novelty and robust.
Validity of the findings: Methods are described with sufficient detail.
Additional comments: The authors heeded the observations made previously.


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: EXOSOMES AND FERROPTOSIS: ROLES IN TUMOUR REGULATION AND NEW CANCER THERAPIES
Review round: 1
Reviewer: 1

Output:
Basic reporting: In this study, the authors aim to review the exosome-mediated regulation of ferroptosis and its related pathways. Generally speaking, this review is good. However, some improvement will be recommended before it can be accepted for publication. Following are the comments:
1. In this review, language is acceptable.
2. Since this review focuses on the regulation of exosome-mediated regulation on ferroptosis, I would suggest the authors to shorten section one which mainly focuses on the role of exosome in tumors. They don’t need to separate this session into different parts. One comprehensive summary of the current research progresses on how exosome promotes cancer aggressive phenotype both in vitro and in vivo will be fine enough. One summarized chart of how exosomal cargos regulate cancer will be needed. This chart should include type of cargo (lncRNA, miRNA, lipid, protein…), regulated cancer types, regulated biological phenotype (proliferation, migration, invasion, angiogenesis…), related signaling pathway (growth factor pathways…). It would be recommended that the authors provide evidence showing that one specific cargo can regulate aggressive phenotype or tumor formation of at least three types of cancers.
3. The last three section are better-organized. However, some improvements are needed. Some subsections are extremely succinct. For instance, subsection 3.1.2, 3.2.2, 3.3 and 5.3 are too succinct. For the mechanism of inhibiting ferritinophagy, the authors only mentioned the role of NCOA4. I believe, far more regulators are involved in ferritinophagy, besides NCOA4. Subsection 3.1.2, 3.3 and 5.3 have the same issue. More comprehensive reviewing is needed.
4. A chart summarizing the current approaches using exosomal inhibitor plus ferroptosis inducer will be recommended.
Experimental design: Please see details in Basic reporting.
Validity of the findings: Please see details in Basic reporting.
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: EXOSOMES AND FERROPTOSIS: ROLES IN TUMOUR REGULATION AND NEW CANCER THERAPIES
Review round: 1
Reviewer: 2

Output:
Basic reporting: The manuscript submitted by Shi et al. summarizes the role of exosomes during ferroptosis. This is a quite new topic, however, I was only able to find one single publication from one of the two corresponding authors regarding the topic of this review article. I am always a bit sceptic regarding reviews written by non-experts.
The review is interesting and provides novel insights of potential future therapies. There is no problem with language and grammar. Figures are also informative and nice.
Experimental design: The first part of this article summarizes the effects and therapeutic potentials of exosomes on tumors. The second part is dedicted to ferroptosis, especially in tumors. Afterwards, the authors describe how exosomes affect ferroptosis and potential resulting anti cancer therapies.
This is a logic way to organize the review.
References are cited.
Validity of the findings: The aim of the review is cleary reported in the introduction, covered by the text and summarized in the conclusions. In addition, the conclusion names future research areas related to the topic.
Additional comments: Unfortunately, for readers it is not entirely enjoying to read the manuscript. It is quite obvious that different people wrote the text and sometimes it is just a row of sentences that are not clearly connected. It seems that the respective authors summarized one publication within one sentence and the next one within another sentence without connecting these sentences. Therefore, many proteins are not introduced, e.g. ZEB1.
I recommend to improve the text in this regard.

Minor points:
Line 215/216: Loss of xc- leads to ferroptosis because it reduces intracellular levels of cystine and thereby GSH synthesis and GPx4 activity.
Sometimes I got the impression that the authors mixed expression and activity, e.g. why should erastin downregulate GPx4 expression?
The strange acknowledgement should be removed.