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: FUNGAL COMMUNITIES REPRESENT THE MAJORITY OF ROOT-SPECIFIC TRANSCRIPTS IN THE TRANSCRIPTOMES OF AGAVE PLANTS GROWN IN SEMIARID REGIONS
Review round: 1
Reviewer: 2

Output:
Basic reporting: Language
The manuscript is written in a way that one is able to follow the author’s intentions. However, the text should be revised for stringency, as there are numerous unnecessary words. Struggling with similar issues, I have started to look into options such as “Grammarly”, “Slick Write” or “Hemmingway Editor” for revising text. Potentially, these programs (all of them have a free version and there are plenty of other options) could help with avoiding lengthy sentences by assisting with punctuation and also with (limited) grammar suggestions.

Additionally, I would suggest to the authors to critically revise the structure of some paragraphs. In the Introduction and Discussion, it is sometimes difficult to follow the common thread within a paragraph and between sentences (e.g., line 60 ff.: This sentence describes what endophytes & epiphytes are and that they can play a crucial role in plant performance. The following sentence (line 64 ff.) describes the ambiguous relationship between mutualism and parasitism. Other examples: line 93 & line 95). Stating clearly defined research questions and following them could help structuring especially the Discussion. In the Methods section, the reader would profit by understanding at the beginning of the paragraph, why a specific analysis was performed. In several instances, this only becomes clear in the middle of the paragraph (e.g., line 155 ff. - Taxonomic classification, line 176 ff. – Orthologous gene analysis).

Intro & background
The introduction gives background on the diversity of plant-associated microbes, some factors structuring these microbial communities, and the study system agave (but see comments on stringency and common thread). It would be helpful to also find information on using metatranscriptomics to gain insight into microbe-plant interactions on a community level, as well as the difficulties faced by such approaches.

Figures
The photos in Fig. 1 should be at a higher resolution. Potentially they could also be bigger.
Figure 1 A: 	Only plant root-specific transcripts > Root-specific plant transcripts
		Only fungal root-specific transcripts > Fungal transcripts in roots
Figure 1 B & 2: There seems to be enough space in the figures in order to use unabbreviated cultivar names. It would be much easier for the reader to see which sample belongs to which cultivar
Figure 4: Provide only information for data shown in the figure. Also label the scale in the figure with “Number of proteins”

Legends and captions for the Supplemental Material are missing, making it in some cases impossible to use (e.g., Table S4)

Raw data
Is available in NCBI SRA.
Experimental design: Research question
The research questions should be stated in a more explicit way at the end of the Introduction. Potentially, they could be numbered. This would also help to structure the Discussion.

Methods
Methodology follows standard procedures and is described in a way that makes it possible to follow the approach (but see specific comments for methods where I think more information is needed).
Validity of the findings: As stated above, all underlying data have been provided.

The conclusions of the study are drawn on the taxonomic level of the divisions Ascomycota and Basidiomycota. At this level the differences in molecular function in my opinion are not overly meaningful, as both groups are very divergent when it comes to life styles. The molecular functions amongst members of both groups could be more divergent than between the two groups. It would be more helpful to discuss the findings on lower taxonomic ranks and it is surprising that the authors refrain from this. They clearly have performed the taxonomic assignments at lower levels (e.g., Figure 4; Kaiju and blastx analyses should also give better taxonomic resolution). Just looking at Fig. 4 shows that there are several fungal genera that are potentially mycorrhizal partners (Amanita, Pisolithus, Rhizopogon, Tulasnella), endophytes (Serendipita),pathogens (Fusarium, Verticillium) or yeasts (Zygosaccharomyces).

Related to the previous comment might also be the fact that many conclusions and argumentations in the Discussion are not grounded on the results of the manuscript. Often the paragraphs become very speculative (e.g., line 435ff.: this paragraph describes the sugar metabolism, but in the whole paragraph the results of the study are only mentioned in one sentence (line 442), without going into details of the findings and what they could potentially mean).
Additional comments: Specific comments
Line 58: … and investigating them … -> … and investigating these …
Line 64: I would suggest using another reference than Faust & Raes 2012 to provide information on the diversity and function of plant-associated microorganisms. Faust & Raes 2012 is an article about microbial networks and models. There are plenty of reviews about plant-associated microorganisms (e.g., Vorholt 2012 Nature Reviews Microbiology; Fitzpatrick et al. 2020 Annual Review of Microbiology; Trivedi et al. 2020 Nature Reviews Microbiology)
Line 66: … plant biome … > … plant microbiome …
Line 71: … drought mechanisms, commercial uses, … bioenergy production … > These properties of agaves should be described in separate sentences, as drought mechanisms are physiological traits of the plant, whereas the other properties are the value that humans gain from the plant.
Line 90: In this context, for agave plants, we believe, tolerance to heat and drought stress is in part … > In agave plants tolerance to heat and drought partly might be due to …. This hypothesis needs more justification in the form of references. It should not be difficult to find these, as there are plenty of studies showing the influence of root-associated fungi on plant performance.
Line 105: … a cryptic species in A. niger (Duarte et al., 2018). > Please delete this part, as it sounds as if A. welwitschiae is not a species on its own.
Line 117: … these fungi communities … > … these fungal communities … This should be checked throughout the manuscript. It occurs several times.
Line 134: … at SRA (accession … > … at the NCBI SRA (accession …
Line 137: “The complete pipeline“ can unfortunately only be seen by readers that have a subscription to the journal “Industrial Crops and Products”. Please provide a sufficient description of the pipeline
Line 143: It is great to see that so much effort went into correctly identifying fungal and plant transcripts. However, I wonder why in subsequent taxonomic annotation the result of the Kaiju analysis was used and not the blastx/uniref90 approach. Additionally, no information is provided in the results how well the latter approach performed.
Line 144: Please provide more information (especially on parameter settings) on the Kaiju classification.
Line 155: Taxonomic classification. Please describe at the beginning of this paragraph in which way the two phyla were compared? This seems essential for the whole paragraph and is only described from line 169 onwards.
Line 174: The text in Methods S2 is nearly identical to the Methods text in the manuscript. I think differences could easily be incorporated into the main text. The results shown in Methods S2 could go into the Supplemental Material though (with a proper legend).
Line 209: Please explain why only the top 10 genomes of Ascomycota & Basidiomycota were used for HSP orthology analysis.
Line 210: If only the top 10 genomes from Ensembl plus three datasets from this study were selected, how does this add up to 26 datasets? Although the Methods section say 3 cultivars times three samples, but the results never show these 9 samples? Therefore, I was under the impression the individual samples for each cultivar were pooled.
Line 214 ff.: General sequence/assembly statistics at the beginning of the Results would be beneficial. Potentially, a table showing this would be good
Line 217: These plants were in … > I think the sentence either belongs into the Introduction or the Discussion, as it is not a result. Additionally, more justification (i.e., relevant literature) is needed for assuming that the fungi contribute to the phenotype of the plant.
Line 221: Are these transcripts now root-specific or transcripts found in all tissues of the different cultivars?
Line 224: Expression analyses need to be described in the Methods section.
Line 232: It is unclear what analysis is described. I think what is meant is a comparison of orthologous genes/transcripts based on nucleotide similarity.
Line 240: … probably indicating that > This should be moved to the Discussion, as it is already an interpretation of the results.
Line 348: … annotated as ACD. > What does the abbreviation ACD stand for. As it is highlighted in the text, the Discussion (line 465) should revolve around what ACD is and how it could be important.
Line 368: I would disagree that the study characterized the molecular functions of the transcripts of the fungal communities, as this entails detailed molecular studies in a laboratory. The presented work used bioinformatic tools to predict potential molecular functions.
Line 371: Why was the percentage of fungal transcripts a surprise? Already in the Introduction it is mentioned that in a previous companion study the authors found 10% of the transcripts to be fungal.
Line 376ff.: Although agaves contain a velamen as do many orchids, it might not have the same function. Especially, as the orchid in the citation (Deepthi & Ray 2018) is an epiphytic orchid while the roots of agaves are in the soil. It is also curious why the emphasis of this paragraph is on the velamen, as it is not functionally related to CAM. There are CAM plants without velamen and plants with velamen that are not CAM plants.
Line 385: Please explain how the intention to sequence the plant explains the low expression values of the fungus? I would argue that the low expression values of the fungi are related to the low fungal biomass in the plant compared to plant biomass.
Line 401: In which way where the communities very similar? Functionally, taxonomically, etc.)
Line 403: … previous findings that fungal communities depend vastly on the soil (Lee & Hawkes, 2020) … > This is exactly what the study by Lee & Hawks does not find. Their study indicates that there is little overlap between leaf and root fungal communities (their Fig. 1 A). Only 1.2% of ASVs overlap between the two tissues.
Line 456: Particularly … > Please revise the sentence as it is not logically coherent. The first part of the sentence deals with physiological properties of HSP in fungi, whereas the second part deals with how HSP can be used by humans to treat diseases.
Line 460: At what depths were the samples collected? This would be important to put the statement “soil surface can reach over 40°C” into context. Additionally, is the Nobel 2010 (Desert wisdom/Agaves and Cacti: CO2, water, climate change) the right reference for the surface temperatures at the sampling site?


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: FUNGAL COMMUNITIES REPRESENT THE MAJORITY OF ROOT-SPECIFIC TRANSCRIPTS IN THE TRANSCRIPTOMES OF AGAVE PLANTS GROWN IN SEMIARID REGIONS
Review round: 2
Reviewer: 1

Output:
Basic reporting: I think the authors have addressed most of the concerns raised in the previous round of reviews and the manuscript has improved significantly. Still, there are several aspects, which I think should be addressed before publication can be considered.

General comments & suggestions:
Please check the tenses throughout the manuscript, as these often switch between present and past tense even within individual paragraphs.

It would be helpful for the reader if the authors could be consistent with the naming of taxonomic groups. For instance, throughout the text Basidiomycota (e.g., line 343), Basidiomycetes (e.g., line 353) or basidiomycetes (e.g., line 352) can be found. These names do not necessarily represent the same taxonomic group and could be confusing to the reader.

As suggested by reviewer 1 the authors included now analyses of rRNAs for taxonomic assessments of the fungal communities. There was some interesting information obtained on potential fungi being present in these communities, for which ecological information is available. For instance, there are several yeast taxa (Cryptococcus, Saccharomyces, Malassezia), one lichen species (Verrucaria), but also a potential parasite (Erysiphe). However, this information is only sparsely used in the Discussion to obtain a taxonomic context for the transcriptomic results. Obviously, caution is needed to correlate the rRNA results with the transcriptomic results, nevertheless I think it would add the results into perspective.

Specific comments & suggestions

Line 49: Plants hosts many microorganisms and investigating their metabolism can … > Plants hosts many microorganisms and investigating the metabolisms of these can …
Line 68: … than other aspects still under investigation. > … than other aspects.
Line 86: … three papers … > … three studies
Line 107: … (2) identified their main phyla … > … (2) assessed their taxonomic affiliations …
Line 108: … including transporters …. > … focusing on transporters …
Line 109: … (4) investigated their heat shock proteins, as these appeared consistently as most expressed transcripts. > … (4) investigated potential heat shock proteins.
Line 110: Around 12% of the full transcriptomes … > I think these sentences should be removed, as they present Results and not Introduction.
Line 124: We have harvested … > We harvested … Please scrutinize the manuscript carefully as many instances occur where “have” can be removed.
Line 128: … 1ug … > … 1 µg …
Line 140: … we have done … > … we performed … (also in line 158)
Line 307: … it is found … > … is found …
Line 308/9 … also a domain present in HSPs. > … also an HSP domain.
Line 310: Regarding central metabolism … > Regarding primary metabolism
Line 350: Please explain in the text what is meant by “plant profile”.
Line 365: … Basidiomycota, considering the total for the three communities. > … … Basidiomycota in the three communities.
Line 389 ff.: Please explain to the reader how this part of the study (i.e., HSPs in fungal genomes) is relevant to the study. These are interesting results, but they seem to justify a separate manuscript.
Line 418: … experiments, we believe that … > … experiments, often …
Line 429: We understand that our dataset … metatranscriptome, but the number … transcripts is sufficient … > Although, our dataset … metatranscriptome, the number … transcripts seems sufficient …
Line 433: Nevertheless, … > Please revise this part, as it is not possible to see the connection to the previous sentence.
Line 437: … the plants are on the same field and climate conditions. > … the plants were grown on the same field.
Line 449: We too were not able … > This sentence seems to contradict itself, as 1.7% of the transcripts belonged to AMF lineages, but not AMF were identified.
Line 453: Please revise the sentence. Improving plant performance might be the main goal of this research route and the study certainly could give hints as to which genes/transcripts could be potential candidates for further research. However, due to the study being exploratory and not experimentally it is impossible within its frame to pinpoint actual function of certain transcripts.
Line 457: … nutrients delivery. > … nutrient delivery.
Line 470: On the other hand … > Please revise the sentence. It is not clear as to whether the presence of chitinases could be a defense mechanism of the fungi against mycoparasitic fungi (in which case it is not clear how this relates to the research topic) or whether it is a defense mechanisms that the authors assume to be a mechanism that these endophytic fungi use in order to defend their host plant.
Line 476: … symbiotic fungi can have fewer … > … symbiotic fungi on average have fewer …
Line 527: … study underlies the importance … > … study underlines the importance …
Line 529: In summary, … > Like for the sentence in line 453, I think this statement needs to be revised. In my opinion the present study can only hint to
Experimental design: I have no further additions to the first review
Validity of the findings: I have no further additions to the first review
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: STRESS COPING STRATEGIES USED BY NURSES DURING THE COVID-19 PANDEMIC
Review round: 1
Reviewer: 1

Output:
Basic reporting: 1.	The tables are professionally prepared, however the average age of respondents should be corrected (it is 3.3, and should be, as in the description 43.3).
Experimental design: 2.	The authors provide a well-formulated research goal in line with the conducted analyzes, nevertheless they should complete the research problem and the assumed hypothesis.
Validity of the findings: 3.	The first part of the conclusion is correct, but the part of the conclusion from line 423 to line 427 has no justification in the analyzed data.
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: STRESS COPING STRATEGIES USED BY NURSES DURING THE COVID-19 PANDEMIC
Review round: 1
Reviewer: 2

Output:
Basic reporting: The paper appears to be well-written but a modest improvement in the use of professional English language may be necessary. For example, the first sentence of  the Introduction Section states, “The risk of SARS-CoV-2 infection, as well as the sense of helplessness, loss of control and unpredictability, are among many elements that currently affect nurses' professional functioning.” This is a vague and not much informative opening statement and also lacks a proper reference.
  A better rendering could be “ The risk of getting SARS-CoV-2 infection,  worries about exposing loved ones, anxiety and frustration, emotional and physical exhaustion, burn out, a feeling of being overwhelmed, and struggles and challenges with parenting are a few among many factors that affect nurses’ personal lives and professional functioning.”
Experimental design: The research hypothesis/ research question(s) and objectives are not clearly articulated. The sampling type, sampling method, inclusion/exclusion criteria, and study population are not well defined.
Validity of the findings: The validity of the finding is questionable.  There is no baseline study conducted prior to the advent of the pandemic to compare with the results of this study conducted during the pandemic. The study appears to project mere speculation that all the stress faced by the nursing professionals is attributable to the pandemic. Nursing professionals are known to work under high stress even absent the added burden of the pandemic and this study does not quantify how much of the stress is caused by having to work during this pandemic. Moreover, the study does not provide the variance of the outcome variable expressed by each independent variable.   Despite the title of the study, “Stress coping strategies used by nurses in the time of the COVID-19 pandemic,”  the authors do not provide as part of the results of their study, what  established stress coping strategies are employed by the sample population (There is no mention of the methods of coping used by these nurses.)
Additional comments: The conclusions of the study appear to be causally unrelated to the stress of having to work during a pandemic.  Based on many years of lived professional experience,  this reviewer believes that the conclusions of this study would be valid in the absence of the COVID-19 epidemic, given the already high-stress levels among the nursing care providers.


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: STRESS COPING STRATEGIES USED BY NURSES DURING THE COVID-19 PANDEMIC
Review round: 1
Reviewer: 3

Output:
Basic reporting: 1. In the present study, stress coping strategies used by nurses during the COVID-19 pandemic are described.
2. Throughout the paper, clear, unambiguous, and professional language is used, with only minor grammatical errors.
3. In this paper, there is sufficient background information to show how the work fits into the broader field of knowledge.
4. In general, the paper is self-contained and includes all results relevant to the hypothesis.
Experimental design: Experimental design:

Responses were collected from 220 nurses providing health care in both inpatient and outpatient settings in Poland's north-eastern region. The study was conducted online using the diagnostic poll method and a survey questionnaire containing the General Self-Efficacy Scale (GSES 10-40), the MINI-COPE questionnaire (0-3), and the Perceived Stress Scale (PSS-10).

The paper reports the following major findings:
- Major findings related to stress are:

1. There was a high level of stress among the nurses (PSS-10 - 20.9),

2. The stress was related to:
2.1. Work experience as a nurse (β -0.250, p=0.014),
2.2. Hours worked per month (β 0.156, p=0.015), and
2.3. Self-assessment of health (β -0.145, p=0.037))
3. There was an average sense of self-efficacy (GSES - 29.1), and it depended on the workplace (p=0.044).

- Strategies for coping with stress (Mini-COPE):
1. The younger nurses mentioned
1.1. Venting (p=0.010),
1.2. Instrumental support (p=0.011),
1.3. Sense of humor (p=0.013) and
1.4. Self-blame (0.031).

Strengths of the paper:

1. The paper includes clearly describes the introduction, methods, and major findings.
2. The paper performs sound statistical analysis and reports the details enough to replicate the study.
3. Conclusions are supported by the evidence presented in the paper.
4. There is a knowledge gap about the factors that induce stress and the coping mechanisms used by nursing staff during the COVID-19 pandemic. The article focuses specifically on this aspect.

For suggestions regarding improvement please see the comments below.
Validity of the findings: Suggestion for improvement:
1. The paper contains multiple typographical and grammatical errors. Proofreading can easily correct minor grammatical errors.
2. The study was conducted online, so a 55% response rate may have introduced some bias to the results. Stressed out respondents may have participated more in the survey, or vice versa.
3. As the paper compares multiple tests and results. However, these multiple statistical tests were not corrected for the multiple comparisons. I would suggest correcting the p-values for the multiple comparisons to prevent false-positive results. Although it won't affect negative findings, it will rule out false-positive findings. Benjamini and Hochberg's false discovery rate (FDR) correction method will be useful here for correcting the FDR. In order to establish the reliability of the results, it is vital to correct the p-values.
Additional comments: Overall comment: The paper is generally well written and has a logical flow. It provides enough details for reproducibility and many of its methods are sound.


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: STRESS COPING STRATEGIES USED BY NURSES DURING THE COVID-19 PANDEMIC
Review round: 1
Reviewer: 4

Output:
Basic reporting: Basic Reporting
•	Thank you for this relevant journal on an important topic. I would recommend updating the title to align with English language flow. The title, as it currently reads, is slightly awkward. For example, it would read better as “Stress coping strategies used by nurses during the COVID-19 pandemic”. I suggest you have a colleague who is proficient in English and familiar with the subject matter review your manuscript, or contact a professional editing service for consistency in the English language. Some other notable text needing revision are mentioned below:
o	In line 4, the running title, please incorporate the concept of stress or coping strategies as this is the main concept of the article.
o	In line 37, a stand alone paragraph of one sentence is not appropriate.
o	Line 185 is an incomplete sentence.
o	In the supplemental file of informed consent, the Polish to English translation is poorly completed.
•	In the abstract, please follow the journal guidelines (Methodology/Methods is not bolded). Also:
o	Subheadings must be bold, followed by a period, and start a new paragraph e.g.
Background. The background section text goes here...
•	The Raw data is a mixture of Polish and English and with 220 responses, it is difficult to remember which translation applies to the English translation. As the article is submitted in the English language, the raw data should also be provided 100% in English.
•	An extensive description of the PSS-10, Mini-COPE and GSES are not needed, in my opinion, as these are well-known and published questionnaires. A simple description and reference would be perfectly acceptable in this instance in the setting of such well-known tools.
•	In the Discussion, please avoid 1 or 2 sentence paragraphs. Appropriate paragraph length is 3-5 sentences.
•	The literature search is somewhat outdated. Few articles are included from 2021 and there are many new relevant articles that have been published in 2021 that can strengthen the literature review and discussion even further.
Experimental design: I agree that the research question is well defined, meaningful and relevant and fills a needed gap in knowledge. The methods and data analysis and appropriate ethics approvals are well-described.
Validity of the findings: In Table 1, if all 220 answered the question on M or F, the percentage should be 96.4, not 96.6. Kindly confirm all respondents answered this questions. 212/220 = 96.4%
In Table 2, please confirm this is correct
<!--[if !msEquation]--> <!--[endif]--> x¯ - mean, ± SD – standard deviation. Is this a typo?
Additional comments: I commend the authors for their work in this field in offering a comprehensive review of Polish nurses, a notable group not previously reported extensively in the literature. The statistical analysis is strong and well-reported and explained. For the main area of improvement, it is in the flow of English I have noted above) which should be improved upon before Acceptance as well as updating the literature review including more articles from 2021.
Finally, please provide all raw data fields in English.


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: STRESS COPING STRATEGIES USED BY NURSES DURING THE COVID-19 PANDEMIC
Review round: 2
Reviewer: 1

Output:
Basic reporting: No comment
Experimental design: No comment
Validity of the findings: No comment
Additional comments: No comment


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

Input:
Title: STRESS COPING STRATEGIES USED BY NURSES DURING THE COVID-19 PANDEMIC
Review round: 2
Reviewer: 2

Output:
Basic reporting: NA
Experimental design: NA
Validity of the findings: NA
Additional comments: My points raised during the previous review have been addressed by the authors. I have no further suggestions.


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: STRESS COPING STRATEGIES USED BY NURSES DURING THE COVID-19 PANDEMIC
Review round: 2
Reviewer: 3

Output:
Basic reporting: The authors have made all suggested changes. The article reads very well. No additional changes requested.
Experimental design: The experimental design is appropriate and research question well defined. No changes requested.
Validity of the findings: All data has been provided and conclusions well-stated. No additional changes are requested.
Additional comments: Thank you to the authors for their very thoughtful and thorough revision to the manuscript. I fully support moving forward to publication of this important paper.


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

Input:
Title: COMPARISON BETWEEN HYDROXYAPATITE AND POLYCAPROLACTONE IN INDUCING OSTEOGENIC DIFFERENTIATION AND AUGMENTING MAXILLARY BONE REGENERATION IN RATS
Review round: 1
Reviewer: 1

Output:
Basic reporting: In this study, Luchman et al aim to investigate the capacity of two different synthetic scaffolds (HA and PCL scaffolds) in promoting in vitro and in vivo bone regeneration. Based on the data collected, they found HA scaffold works better on maintaining cell viability and promoting bone formation. And the authors concluded that HA and PCL scaffold geometry might have influenced on the bone regeneration capacity of MC3T3-E1 cells. However, HA scaffold shows better capacity in promoting bone generation in both in vitro and in vivo experiments, which indicates its great potential for application in bone regeneration.
This study is well-organized, experimental design is easy to follow, and the results are scientifically sound. The biggest highlight in this study is the application of surgical transplantation of MC3T3-E1-HA and MC3T3-E1-PCL, which gives directly evidence that HA scaffold showed better in vivo bone regeneration potential with enhanced new bone volume, increment of bone surface density, and new bone formation.
If it is applicable, this manuscript can be considered for publication in its current form.
Experimental design: Experimental design is good.
Validity of the findings: Findings are scientifically sound.
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: COMPARISON BETWEEN HYDROXYAPATITE AND POLYCAPROLACTONE IN INDUCING OSTEOGENIC DIFFERENTIATION AND AUGMENTING MAXILLARY BONE REGENERATION IN RATS
Review round: 1
Reviewer: 2

Output:
Basic reporting: The author first needs to decide the overall aims of the study and do the design according to that. The main issue in this manuscript is the lack of clarity of the overall aim. In biomaterials, scaffold geometry is referred to, i.e., scaffold pore size, scaffold curvature, and or design. Here, the authors compared different materials, not only differ geometrically.
Experimental design: The author used PCL and HA, then evaluated the effect of scaffolds geometry and surface roughness on cellular attachment; however, scaffold geometry is not the exact thing your study focused on. The study is missing an essential aspect of defining material in terms of chemical characterization, morphology for PCL pore size, and analysis of materials properties.

In line 295, the author stated, ‘The number of viable MC3T3-E1 cells on scaffolds was markedly higher than the control 2D culture plate, significantly during the initial day of culture (days 0) between MC3T3-E1-HA and MC3T3-E1-2D (p = 0.034).’ the authors need to realize that MTT assay reflects the metabolic activity of cells, not a number.
Validity of the findings: The authors referred to a question that was not fully supported by the study design. The title and aim of the study need to be more specific toward the materials used.
The results need to be supported with more material characterization analysis, including SEM images for scaffolds before cells seeding, AFM data to indicate surface roughness, EDS data for elemental analysis.
Additional comments: The author stated that "All of the surviving animals were sacrificed six weeks"-not clear what was the total surviving number and whether it has affected the n=6 indicated in their in vivo study.
The author stated that "It has been proved that the surface topography of rough-textured capable to enhance the cellular adhesion and production of more mineralized matrix during osteoblast differentiation of cells." -Missing reference.
The author stated that "The obvious reduction in cell viability on HA and PCL scaffolds in comparison with control 2D culture plate may be due to the transition of the cells from a proliferative phase into the differentiation phase induced by direct interactions with the scaffold" -Missing reference.
Masson trichrome staining is critical to identify and define new bone formation


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: COMPARISON BETWEEN HYDROXYAPATITE AND POLYCAPROLACTONE IN INDUCING OSTEOGENIC DIFFERENTIATION AND AUGMENTING MAXILLARY BONE REGENERATION IN RATS
Review round: 1
Reviewer: 3

Output:
Basic reporting: 1- A proof reading for English language is suggested.
2- The title should be changed. Please see my first comment on "Validity of the findings" section.
3- Some minor revisions are suggested. Please see the last page of the annotated manuscript which I upload (After line 664).
4- Authors are suggested to use marks like arrows and arrow heads in figure 7. For example in the high magnification PCL image, authors placed NB near to an osteoid structure, and the writing is on connective tissue. Inexperienced readers may misunderstand this image therefore, putting pointing marks could be beneficial.
Experimental design: 1- Although some additional techniques might be used in the study, the used methodology is acceptable for answering the research question.

2- Is it possible for authors to use more appropriate staining technics for paraffin sections if you still have unstained sections or blocks available? Staining techniques such as Masson Goldner trichrome staining, Movat’s pentachrome staining, etc. are commonly used staining technics to see tissue transitions and identify all tissue types relevant with the osteogenesis which would allow you to address different tissue types much better. For example, in the last image (high magnification MC3T3-PCL) of figure 7, some of the NB areas are probably consist of cartilage and there’s a fibro-cartilage transition area as well but H&E staining is not providing enough details to fully identify these structures.

3- For my detailed comments and further questions, please see the last page of the annotated manuscript (After line 664).
Validity of the findings: 1- The title of the manuscript is indicating the effect of “scaffold geometry”, but I believe the used methodology and obtained results are not sufficient to say anything about the effect of geometry. HA and PCL are entirely different materials and as it is mentioned in the manuscript, they have some different qualities effecting the osteogenesis. If the HA and PCL had the same geometry, could we expect to get similar results between the experimental groups? The geometry of scaffolds indeed proved to have good amount of influence but scientists usually use different geometrical patterns with same type of material like using HA scaffolds in hexagonal geometry vs HA scaffolds in square geometry. Therefore, the title must be changed and all the statements related with scaffold geometry should be discarded.

2- Line 420: Line 420: To evaluate cellular adhesion, other technics should be taken into consideration. If you’re making this statement depending upon your electron microscopy imaging results, please refer this analysis in the sentence. Otherwise, it feels like your MTT results provided this knowledge of enhanced adhesion.

3- Line 444: "and once they form multilayers they start mineralization (Yu et al., 2017)."
In this article Yu et al. mentions double-layered cell transfer method (https://www.nature.com/articles/srep33286 ). There’s no mentioning of that the cells start mineralization when they become multilayered. Please revise this statement.

4- Line 466: Changing “successful osteoblast formation” to “successful induction of osteogenic differentiation.” could be a better choice. There are several cellular steps throughout the osteogenic differentiation starting with mesenchymal stem cells to osteocytes and beginning from the early osteoprogenitors, those cells express ALP gene and produce ALP enzyme. Therefore, saying successful osteoblast formation is a bit assertive as these cells could be of any cell type from an early progenitor to a mature osteoblast.

5-Line 522-523:”The infiltration of connective tissue resulted from the interaction between MC3T3-E1 cells and the osteoconductive property of HA scaffold”
Which results are refering to this statement? Connective tissue normally does not infiltrate into an area. In normal physiological healing, cells do migrate to defect area and they differentiate form a fibrous tissue. Afterwards cells do condensate and form fibro-cartilage tissue parts which would enlarge and form a cartilage later on. Cells keep on differentiating and due to vascularization and ALP activity, osteoid structures occur in the defect site. Please change the statement for this part.

6- For my other comments please see the annotated manuscript that I upload (After line 664).
Additional comments: Authıors had investigated the qualities of HA and PCL scaffolds seeded with MC3T3 cells both in vitro and in vivo. Although the study is well planned, I believe the manuscript could use some improvements and some changes also had to be made starting with the title of the study.


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

Input:
Title: COMPARISON BETWEEN HYDROXYAPATITE AND POLYCAPROLACTONE IN INDUCING OSTEOGENIC DIFFERENTIATION AND AUGMENTING MAXILLARY BONE REGENERATION IN RATS
Review round: 2
Reviewer: 1

Output:
Basic reporting: The authors have adequately answered all requests.
Experimental design: The authors have adequately answered all requests.
Validity of the findings: The authors have adequately answered all requests.
Additional comments: The quality and clarity of the paper are much improved and I have no further comments at this time. Very nice job!


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: COMPARISON BETWEEN HYDROXYAPATITE AND POLYCAPROLACTONE IN INDUCING OSTEOGENIC DIFFERENTIATION AND AUGMENTING MAXILLARY BONE REGENERATION IN RATS
Review round: 2
Reviewer: 2

Output:
Basic reporting: 1.	Authors are suggested to avoid self-citations unless it is absolutely necessary (Ariffin et al., 2017; Abdul Wahab et al., 2020; Wahab et al., 2020; Yazid et al., 2019; Yazid et al., 2018).
Experimental design: 1.	Which solution is used for primary antibody dilutions in IHC? If you used commercial antibody diluents, please refer the product and company names and if you prepared your own conservative solution please mention it.
2.	Addition of IHC to the paper definitely added value to the manuscript. Although addition of anti-OCN and anti-ALP stainings are enough to bring good results into the table, there’s still room for improvements. Is it possible for authors to perform a quantitative study for IHC results by using softwares such as ImageJ or at least making a semi-quantitative evaluation by measuring the power of immunoreactivity in the defect zone (like +=weak, ++=moderate, +++=strong reaction)? I believe after gathering quantitative results, investigating if there’s any significant difference between ALP and OCN reactions between the groups would definitely strengthen the manuscript.
Validity of the findings: Authors had made a statement about cellular adhesion regarding to their MTT results. MTT assay is being utilized for measuring the toxicity of used reagents in vitro, yet the logic of the assay also helps researchers to make measurements about cell viability and proliferation in some cases. When I checked the articles, I see that researchers are using MTT assay to evaluate the effect of used material in cellular proliferation rates and using SEM imaging to prove cellular adhesion.
I do understand that having positive results in MTT assay also would mean that the cells are indeed adhered to the material but you are already utilizing  electron microscopy imaging for this purpose and we clearly see that the cells are adhered to the surface of materials. So, I believe you should discuss cellular adhesion depending on your electron microscopy results rather than MTT since it’s not a very appropriate technic to show cellular adhesion.
Additional comments: Dear authors;

Thank you for your detailed response to all reviewer comments. I would like to say that I believe adding IHC after making necessary changes in the manuscript really improved the value of your paper. Although it's not vital, I still belive if you can find some time make quantitative or semi-quantitative measurements for IHC staining would add much more value to your discussion. You can see my other minor concerns in my report.


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: COMPARISON BETWEEN HYDROXYAPATITE AND POLYCAPROLACTONE IN INDUCING OSTEOGENIC DIFFERENTIATION AND AUGMENTING MAXILLARY BONE REGENERATION IN RATS
Review round: 3
Reviewer: 1

Output:
Basic reporting: 1-Lines 308-311: Please place a citation for the used scoring system for IRS, if applicable.
2- "Multiple comparisons for in vitro cell viability and osteoblast differentiation potential of scaffolds as well as in vivo bone morphometric and histology grading analysis were evaluated using Bonferroni-corrected by one-way analysis of variance (ANOVA). "

Please mention the used statistical test for IRS. If the same test had been used, please mention that IRS is also subjected to that test.
Experimental design: The authors have answered all requests.
Validity of the findings: The authors have answered all requests.
Additional comments: Thank you for your efforts and answering all the requests in a positive manner. After these 2 little editings I believe there's no further concerns available for your manuscript.
Best Regards.


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

Input:
Title: PREDICTING SURFACE ABUNDANCE OF FEDERALLY THREATENED JOLLYVILLE PLATEAU SALAMANDERS (EURYCEA TONKAWAE) TO INFORM MANAGEMENT ACTIVITIES AT A HIGHLY MODIFIED URBAN SPRING
Review round: 1
Reviewer: 1

Output:
Basic reporting: My suggestions for improving basic reporting:
1. More details are needed for the CJS mark-recapture models. For example, did the authors use goodness-of-fit tests?
2. Is there evidence that using pigmentation for Eurycea is a reliable way to identify individuals? I’m surprised that the Discussion framed sampling the species as “rare” rather than “rarely on the surface”. There may be a large population below the surface and only rarely do individuals surface because of the conditions (e.g. being flushed from flow or because of changes in dissolved oxygen, conductivity, or temperature).
3. The code and data the authors supplied do not include the capture histories or analyses for the mark-recapture section so I could only evaluate the details in the main manuscript.
Experimental design: My suggestions for improving the experimental design:
1. The research question is defined in the Introduction, but I was confused why the authors chose to model how rain, dissolved oxygen, and conductivity affect salamander counts. Right after stating this in the Introduction, the authors describe why these three variables are likely related to each other. The authors should state clearly why they included variables if they predicted they were related (or being driven by the same underlying mechanisms). Further to the research question, please clearly state how research fills an identified knowledge gap.
2.	More justification and explanation is needed for how the generalized linear models (GLMs) were fitted. Several variables were multicollinear or expected to be (e.g. dissolved oxygen and specific conductivity appear negatively correlated, and the Introduction establishes why some predictor variables are likely related).
3. Further, fixing missing values or "outliers" to a mean using imputation risks masking real patterns. The authors changed the minimum value of conductivity to the mean, but if the hypothesis is that conductivity affects surface activity of salamanders then this will mask a real pattern. With only 39 points and about half having a 0 value for the response, changing the values of predictors seems riskier than simply removing those observations from the model. I recommend the authors further explain why they imputed values. Testing whether those changes affect conclusions would further convince me this analytic choice is not leading to spurious results.
4.	The model selection for the GLM considers various measures of rainfall accumulation (varying the days prior to the survey that are included). However, the authors did not compare models without some of the other variables. Because there are so few observations and that some variables are likely correlated, this might affect conclusions.  For example, I tested a model that just included rainfall accumulation 1 to 90 days prior to the survey (the most supported rainfall variable), conductivity, and the offset predictor. That model had more support than the next best-supported model in the paper (AICc 4.53 units lower) and both conductivity and rainfall accumulation significantly and positively affected the number of salamanders detected. Why did the authors not consider model subsets with fewer variables given the low sample size (39 maybe less if missing values are removed) and high number of predictors (5)?
Validity of the findings: My main concerns over the validity of the findings:
1. Not all raw data are provided (mentioned above in Basic Reporting).
2. Some of the conclusions are not supported by the analyses presented by authors. For example, in the Conclusion section the authors say: “The findings of this study indicate that by carefully considering recent rainfall patterns in the area, maintenance could be scheduled to avoid instances when the presence of E. tonkawae within surface habitat is predicted.” The data and model predictions are in Figure 5. The model predicts 1 salamander will be detected when rainfall exceeds 32 cm in previous 90 days (where the red horizontal line crosses the model prediction). However, the raw data show that 12 of 17 surveys when salamanders were detected had less precipitation than this value. Thus, suggesting maintenance when precipitation values are less than 32 cm does not seem supported by the raw count data the authors present.
Additional comments: This short paper examined a small population of Eurycea living in an urban environment to 1) estimate detection and survival, and 2) test what environmental conditions predict the number of detected salamanders in a culvert. I think the manuscript is interesting and contains unique data. I applaud the authors on their efforts working with such a challenging system where animals are hard to detect. I think this paper can make a valuable contribution, but I have several major concerns (outlined in previous sections) about the analytic approaches and the conclusions drawn from them.

Below, I outline a few additional specific suggestions for improvement.

L43: I’d recommend clarifying that it is the United States of America’s Endangered Species Act and definition of “take”.

L123: Change “Teas” to “Texas”.

L176: The R package RMark sends a call to program MARK and I recommend the authors cite MARK in addition to the R package.

L182: What was the number of survey periods in your study? Did you use data from both the spring run and the box culvert? It seems by definition the capture probability would decrease after 2018 when the amount of habitat you surveyed decreased. I know you do not have much data, but why did you not test variables that would affect detection probability that you collected? For example, DO, SC, or a seasonal effect? You later state that Eurycea in Texas are known to change surface activity seasonally, so I expected that it would have been included in the CJS model.

L186: Did you conduct goodness-of-fit tests for the CJS model? Please report the results here.

Table 1. According to the Methods and Results, there were 39 surveys (17 with, 22 without salamanders). Why are these numbers different? Are these the number of surveys only considering those with the water measurements?

Table 3. Please update to limit significant digits (i.e. use number of digits that correspond to precision).


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: PREDICTING SURFACE ABUNDANCE OF FEDERALLY THREATENED JOLLYVILLE PLATEAU SALAMANDERS (EURYCEA TONKAWAE) TO INFORM MANAGEMENT ACTIVITIES AT A HIGHLY MODIFIED URBAN SPRING
Review round: 1
Reviewer: 2

Output:
Basic reporting: Overall the manuscript is well written, very interesting, and as someone that works with endangered species I applaud the tremendous effort that must have been exerted to collect this data!

I think the main thing that can be improved is the discussion. Currently there is a major lack of flow. The first two paragraphs of the discussion read like they belong in the introduction. The following paragraph has a lot of repetition from the study-site description and not much of a link to your findings. The next paragraph is mainly focussed on the limitations of your study, but this usually comes later, after you have discussed all your findings. Then, when discussing rainfall models there is no mention of the unimportant predictors (and why they are unimportant). Lastly, the ‘conclusion’ section is actually ‘management implications’ and feels a bit disjointed from the rest of the text at present – I think it would be better to blend management implications into the previous paragraphs as appropriate, and have a more general conclusions paragraph that summarizes your findings and perhaps why your study is interesting to researchers who study other endangered amphibians.

My second main concern would be the lack of citations to relevant literature. There is a huge body of work that deals with the issue of low detection probabilities in wildlife populations, including amphibians. Here are some that you might consider to strengthen the paper:

Ficetola, G. F. (2015). Habitat conservation research for amphibians: Methodological improvements and thematic shifts. Biodiversity and Conservation, 24, 1293–1310.
Gu W.D. and Swihart R.K. (2004) Absent or undetected? Effects of non-detection of species occurrence on wildlife habitat models. Biol Conserv 116:195–203.
Kellner, K.F. and Swihart, R.K. (2014). Accounting for imperfect detection in ecology: a quantitative review. PloS one, 9(10), p.e111436.
MacKenzie, D.I., Nichols, J.D., Sutton, N., Kawanishi, K. and Bailey, L.L. 2005. Improving inferences in population studies of rare species that are detected imperfectly. Ecology, 86(5), pp.1101-1113.
Mazerolle, M.J., Bailey, L.L., Kendall, W.L., Royle, J.A., Converse, S.J. and Nichols, J.D. 2007. Making great leaps forward: accounting for detectability in herpetological field studies. Journal of Herpetology, 41(4), pp.672-689.

Similarly, there is a wealth of studies that investigate the environmental correlates of amphibian surface activity that would be good to reference in the intro and/or discussion. For example:

Canavero, A., M. Arim, D. E. Naya, A. Camargo, I. D. Rosa, and R. Maneyro. 2008. Calling activity patterns in an anuran assemblage: the role of seasonal trends and weather determinants. North-West Journal of Zoology 4: 29–41.
Jaeger, R.G., 1980. Microhabitats of a terrestrial forest salamander. Copeia, pp.265-268.
Sanchez, K., Grayson, K.L., Sutherland, C., Thompson, L.M. and Hernandez-Pacheco, R., 2020. Environmental drivers of surface activity in a population of the Eastern red-backed salamander (Plethodon cinereus). Herpetological Conservation and Biology, 15(3), pp.642-651.
Semlitsch, R. D. 1985. Analysis of climatic factors influencing migrations of the salamander Ambystoma talpoideum. Copeia 1985: 477–489.
Sexton, O. J., C. Phillips, and J. E. Bramble. 1990. The effects of temperature and precipitation on the breeding migration of the spotted salamander (Ambystoma maculatum) Copeia 1990: 781–787.
Taub, F.B., 1961. The distribution of the red-backed salamander, Plethodon c. cinereus, within the soil. Ecology, pp.681-698.
Experimental design: The research question is well defined and it is clearly stated how this research fills a knowledge gap. The methods are described with sufficient detail to replicate. My only suggestion would be to explore simpler models for the negative binomial count models, i.e., with just rainfall as a predictor. I feel there is justification to remove all other environmental covariates post-hoc.
Also there are some t-tests performed in the accompanying R script that are not described, and those results not included.
Validity of the findings: All underlying data have been provided, and an associated R script. However the code for the mark-recapture analysis is not included in the script. The R script also contains some t-tests which would be great to describe in the methods section and add to Table 1.

As I stated in ‘Basic Reporting’, the discussion could use some revision to improve clarity and flow. If this is addressed it will be much easier for readers to grasp the main findings and importance of the study.
Additional comments: I would include one or two sentences in the abstract stating the main findings (even if they are general)

Line 20-21: “are a range restricted” should be “is a range restricted”

Line 61-62: I would suggest moving this last sentence to the end of the next paragraph (~line 77), as it seems to fit better there, once you’ve explained the extent of urban structures in the CHUs.

Line 89-90: I would suggest moving this last sentence to come before the previous two (~line 86; i.e., so that if follows directly on from the sentence “Additionally, the site was drastically altered…”). I think this would improve the flow of the paragraph.

Line 92-93: I would name the site again for clarity. And I don’t think it’s necessary to state that your “goals evolved”. So the sentence would just be: “Here we report the findings of seven years of surveys at Brushy Creek Spring.“

Line 93-94: this first aim seems odd when in the previous paragraph you have stated that these salamanders can be “regularly detected” at this site. I think you either need to remove this aim, or revise the previous paragraph to make it clearer that the site status was unknown before this study (which is a cooler angle!).

Line 103-114: I think this overview of environmental conditions on salamander abundance/detectability should be its own paragraph, and come sooner in the introduction. You should try to limit the last paragraph to the aims/hypotheses/predictions of this study.

Line 123: “Texas” is misspelled.

Line 151-154: I would move the permit information to the end of this section (i.e., after you have described capture and measuring protocols)

Line 161: I’m not sure you ever used gravidity status in any of your analyses, so I don’t think it’s worth its own figure. I would rather see more results-based figures - at present there is only one.

Line 165-173: this environmental data could be a separate paragraph, or might be better attached to the end of the first paragraph in this section.

Line 244-245: this sentence belongs in the results

Line 248: when you found that day of year, DO, and conductivity did not influence salamander counts, why did you not drop them from the models? What is the AIC of simpler models (i.e. with only rainfall as a predictor)?

Line 286-287: this sentence belongs in the discussion

Line 289-290: This is incorrect – it is the difference in AIC values that is less than 2 (delta-AIC), not the AIC values themselves.

Line 307-329: I feel like these first two paragraphs belong in the introduction. This is background info that would help justify the present study. If you would like to keep it in the discussion, it definitely shouldn’t be the opening paragraphs. The first paragraph is typically a summary of your main findings and a statement about the relevance/importance of the study (i.e., how it will inform management/conservation).

Line 332: It seems like a missed opportunity to me that you emphasize the importance of “distance from spring outlet” for detecting salamanders, but you didn’t include anything related to this in your models… I feel this would be more informative than including the various rainfall models that all say roughly the same thing. Not sure if this is possible, but a figure showing the declining probability of detection with distance from spring outlets would be really cool!

Line 334: inconsistency between “spring-run” and “springrun”. Not sure which one is correct, but check document and stick to one of them.

Line 345-346: There is a huge body of literature on dealing with low detection probabilities in wildlife studies. This paragraph is fine to keep but should include lots of references.

Line 350-351: I know this is beyond the present study, but this type of uncertainty can be dealt with – either in a Bayesian framework where you place an informative prior on detection probability, or using a mark-recapture model that allows for temporary emigration. Below are some relevant citations:
Bailey, L.L., Simons, T.R. and Pollock, K.H., 2004. Estimating detection probability parameters for plethodon salamanders using the robust capture‐recapture design. The Journal of Wildlife Management, 68(1), pp.1-13.
Kendall, W.L. and Bjorkland, R., 2001. Using open robust design models to estimate temporary emigration from capture—recapture data. Biometrics, 57(4), pp.1113-1122.

Line 390: there is no mention in the discussion concerning the lack of importance of ‘day of year’, DO, and conductivity, but to me this is an important result, especially in the context of management implications. I would add a paragraph speculating on why these predictors were not important.

Table 1 title: “are” should be “at”

Table 1: The accompanying R script has various t-tests for these comparisons, so I think this table could be modified to include the results of these tests, e.g., separate columns for ‘Temp Present’ and ‘Temp Absent’ and a column for t-test result. Also the t-tests should be described in the methods.

Table 3 legend: just as an FYI, an equation is not displaying properly on the pdf

Figure 3: I’m not sure this figure is needed/relevant

Figure 5: I feel like you have a lot more non-detections in your dataset… are they not plotted or is there a lot of overlapping points? If there is an easy way to make this clearer that might be nice. Overlapping can maybe be fixed with geom_count() or jitter().


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: PREDICTING SURFACE ABUNDANCE OF FEDERALLY THREATENED JOLLYVILLE PLATEAU SALAMANDERS (EURYCEA TONKAWAE) TO INFORM MANAGEMENT ACTIVITIES AT A HIGHLY MODIFIED URBAN SPRING
Review round: 2
Reviewer: 1

Output:
Basic reporting: The revised manuscript has an improved article structure, includes all relevant raw data, and the figures are improved.
Experimental design: No comment.
Validity of the findings: All underlying data is now included. The conclusions are well stated in relation to original research question.
Additional comments: The authors did a great job revising the manuscript and have addressed all my previous major concerns and minor suggestions.

A few very minor suggestions:

L319: You can remove "(∆AICc)" because it is redundant.

L333: Change "The competing models mean" to "The competing model's mean".

L369-372: This is a valuable discussion on the lack of recaptures. I recommend at least mentioning that "survival" is estimated as "apparent survival", which cannot disentangle deaths from permanent emmigration. For example, individuals may be either swept downstream or permanently retreat to subsurface habitat.


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: SCAVENGING VS HUNTING AFFECTS BEHAVIORAL TRAITS OF AN OPPORTUNISTIC CARNIVORE
Review round: 1
Reviewer: 1

Output:
Basic reporting: a.	The language of this paper is clear and concise. The Introduction provides a broad overview of why carnivores may shift foraging strategies from hunting to scavenging, and how behavioral changes may accompany this shift. I think the authors should include more emphasis on how and why anthropogenic resources/urbanization leads to scavenging specifically while not also increasing small prey items to be hunted such as mice and birds.
b.	I am not convinced that an increase in scavenging would necessarily lead to a decrease in problem solving ability. If scavenging involves, for example, deliberate extractive foraging, this may provide selection pressure to enhance problem solving ability and innovation in scavenging individuals/species (Liker & Bokony, 2009; Auersperg et al., 2011; Griffin & Guez, 2014; Goodman, Hayward & Hunt, 2018; Barrett, Stanton & Benson-Amram, 2019; Daniels et al., 2019).
c.	The figures are simple, clear, and easily interpreted by the reader.
d.	The raw data and analysis code have been supplied on github. It appears to be complete, and has the appropriate accompanying metadata.

Auersperg AMI, von Bayern AMP, Gajdon GK, Huber L, Kacelnik A. 2011. Flexibility in Problem Solving and Tool Use of Kea and New Caledonian Crows in a Multi Access Box Paradigm. PLoS ONE 6:e20231. DOI: 10.1371/journal.pone.0020231.
Barrett LP, Stanton LA, Benson-Amram S. 2019. The cognition of ‘nuisance’ species. Animal Behaviour 147:167–177. DOI: 10.1016/j.anbehav.2018.05.005.
Daniels SE, Fanelli RE, Gilbert A, Benson-Amram S. 2019. Behavioral flexibility of a generalist carnivore. Animal Cognition 22:387–396. DOI: 10.1007/s10071-019-01252-7.
Goodman M, Hayward T, Hunt GR. 2018. Habitual tool use innovated by free-living New Zealand kea. Scientific Reports 8:13935. DOI: 10.1038/s41598-018-32363-9.
Griffin AS, Guez D. 2014. Innovation and problem solving: A review of common mechanisms. Behavioural Processes 109:121–134. DOI: 10.1016/j.beproc.2014.08.027.
Liker A, Bokony V. 2009. Larger groups are more successful in innovative problem solving in house sparrows. Proceedings of the National Academy of Sciences 106:7893–7898. DOI: 10.1073/pnas.0900042106.
Experimental design: The research questions are clearly defined and meaningful. There are a few areas where more detail should be provided in order to replicate this study:
a.	Please include more information on the coyotes participating in this study. Were they all captive reared? How many generations have been in captivity? What generation is this? Do the coyotes ever have the opportunity to hunt within their enclosures (i.e., prey accidentally wanders in)? This information would help readers interpret how much of the results of this study could be extrapolated to wild coyote or carnivore populations.
b.	I suggest the authors report how many total observations/observation time totals to summarize for the reader. It would also be helpful to include a table or timeline outlining when and in what order each trial was conducted, as this is a lot for the reader to synthesize on their own.
c.	Were any odor cues provided when the dummy coyote was presented? Please include whether odor was used as an additional stimulus in these trials.
d.	What defined a ‘capture’ of the car prey item?
Validity of the findings: a.	My major concern is the validity of time spent within 1m of the puzzle box as a measure of persistence. Most studies I am aware of that measure persistence in problem solving use a measure of time spent interacting with the puzzle box, or a rate of attempts at problem solving (Griffin & Guez, 2014; Chow, Lea & Leaver, 2016; Brubaker et al., 2017; Jacobson et al., 2021). I think the more likely explanation for the change in the persistence measure in this study is habituation to the puzzle box, which is supported by the fact that both groups increased the amount of time spent within 1m of the puzzle box over time and therefore number of presentations of the puzzle box. This is what I would predict would happen as the coyotes habituate to the novel object (puzzle box), and is what the authors discuss in regards to boldness in lines 354-356. I think this adds to your results of boldness differences between the two groups, since the hunting coyotes had a greater increase in this measure, but am skeptical that any valid conclusions can be made regarding persistence from this measure alone.

b.	Lines 394-397 should be included in the Results section, as this is quite a small sample that successfully adapted to the foraging device, and limits conclusions that can be inferred from these data. Results should be updated to include the total numbers of individuals that successfully participated in all trials.

c.	Could the change in activity budgets (lines 246 – 248) be attributed to natural seasonal changes in activity rather than the foraging treatment?

d.	I largely agree with the rest of the results and interpretation presented by the authors.

Brubaker L, Dasgupta S, Bhattacharjee D, Bhadra A, Udell MAR. 2017. Differences in problem-solving between canid populations: Do domestication and lifetime experience affect persistence? Animal Cognition 20:717–723. DOI: 10.1007/s10071-017-1093-7.
Chow PKY, Lea SEG, Leaver LA. 2016. How practice makes perfect: the role of persistence, flexibility and learning in problem-solving efficiency. Animal Behaviour 112:273–283. DOI: 10.1016/j.anbehav.2015.11.014.
Griffin AS, Guez D. 2014. Innovation and problem solving: A review of common mechanisms. Behavioural Processes 109:121–134. DOI: 10.1016/j.beproc.2014.08.027.
Jacobson SL, Puitiza A, Snyder RJ, Sheppard A, Plotnik JM. 2021. Persistence is key: investigating innovative problem solving by Asian elephants using a novel multi-access box. Animal Cognition. DOI: 10.1007/s10071-021-01576-3.
Additional comments: Please expand your ethics statement to include more information on the care and use of the study participants.


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: SCAVENGING VS HUNTING AFFECTS BEHAVIORAL TRAITS OF AN OPPORTUNISTIC CARNIVORE
Review round: 1
Reviewer: 2

Output:
Basic reporting: The manuscript is clearly written and unambiguous, and uses clear, technically correct text while conforming to professional standards of courtesy and expression.

The introduction and background are sufficient to determine how the work fits into the broader field of knowledge, and relevant literature is appropriately and extensively referenced.

The structure of the article conforms to an acceptable format of ‘standard sections’, and the figures presented are relevant to the content of the article, of sufficient resolution, and appropriately described and labeled. The raw data is posted to GitHUB.

The manuscript is self-contained with relevant results to hypotheses and thus is an appropriate ‘unit of publication’. The manuscript also effectively identifies directions for additional research and follow-up.

I do have some comments/questions related to the Methods and Discussion, which are detailed below.
Experimental design: The manuscript contains original primary research that falls within the Aims and Scope of the journal, and is relevant to both Biological Sciences and Environmental Sciences. The research question is well defined, relevant to the biological and environmental sciences, and meaningful. The submission identified the knowledge gap being investigated and makes clear statements on how the study contributes to filling that gap.

The experiments and investigation were rigorously conducted in conformity with prevailing ethical standards for animal care, and the methods have been described with sufficient information to be reproducible by other investigators.
Validity of the findings: The manuscript does not report on research that is redundant or derivative of existing work.

The authors have provided all underlying data in an acceptable discipline-specific repository (https://github.com/pars2997/scavenging-hunting-coyotes).

Conclusions are well stated, linked to original research question & limited to supporting results. I do have some requests for some additional details and context.
Additional comments: The manuscript is comprehensive, well-written, appropriately referenced, and uses appropriate methods for data collection and analyses. I have mostly minor comments.

Introduction

The Introduction does an excellent job framing the research, and hypotheses and predictions are clearly stated

- lines 68-70 - while I agree with this statement and think it's accurate, can any supporting references be added?

- line 75 - different citation format, what reference supports this statement?

- line 76 - I would change "carnivores" here to "predators", since bottlenose dolphins are used as an example (they are carnivorous but not "carnivores" per se)

Materials & Methods

lines 122-127 - The study used 16 coyotes (8 pairs) out of approximately 90 adult coyotes that are maintained as male-female pairs in outdoor enclosures ranging from 0.1 – 1.0 ha. How close are the enclosures to one another, and could adjacent pairs influence the behaviour of the coyotes used in the study? If yes, how was this addressed?

lines 125-126 - "Coyotes in this facility display similar behavioral budgets to wild coyotes (Shivik et al., 2009)"
- did the behavioral budgets in Shivik et al. (2009) use similar methods as the ones developed in this study? (timing, bout length, etc.)

lines 128-130 - The study overlapped the coyote mating and pup rearing seasons from February to June, and half the coyote pairs (two treatment pairs, two control pairs) bred during the study period. I am curious as to whether breeding behavior could have influenced other coyote behaviors, and whether adjacent animals are close enough to one another to react in any significant way. (see additional comments on this below)

- lines 148-149 - "Initially, we left the prey model stationary and scattered additional food around it for coyotes to become comfortable with the prey model and associate it with food."
- how long was this initial period?

lines 153-155 - "If coyotes did not successfully hunt on a given day, we removed the prey model and returned at least two hours later and fed the pair using standard procedure for the facility."
- how often did this happen, and was it considered in the analyses?

162-165 - "We modified the ethogram from Shivik et al. (2009; Table 1) and conducted continuous observations for 15 minutes for each individual coyote. Each month, we observed each coyote during four non-feeding times, four feedings without the prey model, and four feedings with the prey model."
- did the original study use the same methods?
- is their support in the literature for the use of 15 minute observation bouts? Any data on sensitivity of ethogram results to longer or shorter observation times?

line 202-204 - the coyote dummy was visual only, correct? No coyote urine, etc. was used? (see additional comments on this below)

Statistical Analyses
211-213 - For behavioral budget data, we combined behaviors into four categories: resting, active (locomoting and stereotyping), feeding (foraging and eating), and social (aggression, play, howling).
- Table 1 says "Moving" and not "active" for the broad category
- how was proportion of time spend standing or scent marking considered in analyses, since these behaviors are included in the ethogram but not listed here?
- the sentence should also include the interactions with the prey model (as noted in Table 1)

Results

lines 254-256 - "During feeding observations, both hunting and nonhunting coyotes increased the proportion of time they spent feeding (z = 3.025, p = 0.002) which was paired with a decrease in time spent active but not feeding (z = -8.611, p < 0.001; Figure 2)."
- we would expect an increase in time spent feeding when they are being fed, what about other behavior categories besides "active"?

Discussion

lines 379-380 - "The subjects were insulated from the effects of intra- and interspecific interactions, dynamic environments, and other aspects of natural systems."
- some additional context is needed here on how subjects were insulated from other coyotes
- for example, were study pairs kept separate from other animals at the facility, and if so, how?

line 382 - change "approached" to "approach"

lines 394-396 - "In this study, only two of eight coyotes in the hunting treatment fully adapted to the foraging task and regularly fed from the prey model."
- one pair, or two individuals from different pairs?

lines 408-414 - what about adding scent (i.e., coyote urine) to the dummy?

Conclusions

- line 425 - is Parsons et al. 2021 or 2022? 2021 is used here vs 2022 elsewhere

- line 426 - remove period after "conflict"

Some of the biggest differences between hunting and nonhunting coyotes occurred in May and June (e.g., proportion of time spent resting in Figure 1, proportion of time spent interacting with the prey model in Figure 3). What effects, if any, might the coyote's breeding status (e.g., rearing pups) have had on these findings?


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: SCAVENGING VS HUNTING AFFECTS BEHAVIORAL TRAITS OF AN OPPORTUNISTIC CARNIVORE
Review round: 1
Reviewer: 3

Output:
Basic reporting: The manuscript is well written, formatted clearly, and easy to follow. The authors have provided significant background for the context of the study and cited the relevant carnivore literature well. I have a few detailed comments for parts of the introduction that could be clarified or further expanded. In particular, I think there should be more acknowledgment of the variety of ways that some traits have been defined in the previous literature to distinguish exactly how they are being defined here. This is relevant for boldness which has often been separated from responses to novelty (see Carter et al., 2012). Since the terminology has varied widely in the literature, I think it is ok to use the term boldness in this study but it must be defined clearly and how it may differ from other studies using the same term should be outlined.
Experimental design: Some methodology needs clarification or further definition, but otherwise methodology and analysis appears appropriate.
It could be helpful to have a table with the traits studied, how you are defining each trait and the measures used specifically so the reader can keep track of the various measures for each trait. An images of the coyote dummy would also be helpful to include in supplementary materials. An important missing part of the methodology is more detail about who was conducting the behavioral observations or coding the video and measures of coding reliability for these observers. For the focal sampling, were the behaviors from the ethogram coded live? By one or multiple observers? How was this observer determined to be reliable? And for the video coding, there should be a portion of the video doubly coded for a measuring reliability of the trained observer to record latencies and durations.
Validity of the findings: The results and conclusions of this study are valid. The only piece I think may need to be supported more thoroughly or potentially reframed is whether the coyotes changed their foraging behavior or were provided with the opportunity to hunt but perhaps did not truly change a lot of their behavior. I know you present data on their proportion of interaction with the prey model over the course of the study but I still think it's not really clear how many of the coyotes were hunting. Potentially adding data about the number of times coyotes had to have food scattered after the prey model was provided could help support whether the behavior was changing. If you agree that there was not a lot of change in behavior, as it appears based on lines 394-6, then I think this needs to be discussed. For example in lines 303-304, you could address that perhaps the coyote hunting behavior didn't change enough to affect other behavioral changes, explaining some of the results. I still think even if the hunting wasn't adapted by all coyotes these results are still valuable, especially since you see the interaction effects for some measures. Perhaps language about the change that is happening should also be altered to say that the coyotes were presented with the opportunity to hunt, such as in line 298. I think with this issue discussed further, the conclusions are appropriate for the results.
Additional comments: Overall I commend the authors on this longitudinal multifaceted investigation of coyote behavioral traits and changes based on coyotes being provided with the opportunity to engage in hunting behavior to obtain food. I think the study has a strong between and within subjects design with appropriate controls for the non-hunting group.
Line by line comments
51-52- It would be helpful here to expand on the non-urban effects or provide a couple examples on how humans effect in non-urban ecosystems
56- predators “use of” alternative resources
60- I think you need to briefly introduce animal personality here with a definition before jumping into the traits
64-66- I would argue that persistence doesn’t necessarily need to be with new stimuli and could be applied to interactions with any foraging
70- I think you should clarify the trait changes for these particular traits here. I’m assuming you mean population-level changes with bolder, more persistent and more innovative individuals.
75- citation should be changed for Peer J format instead of number
123-4- It may be helpful to compare this housing to the species-typical social grouping of coyotes for those readers that may not be familiar with their behavioral ecology
128- overlapped “with” the
129-30 – this would be a good point to address in the Discussion, I would imagine that having pups around could have a significant affect on the behavioral responses to novelty and the adults’ interest in the puzzle box. If differences in the behavior of these pairs or lack of differences were observed please briefly describe in the Discussion
148-148- About how long did this phase last? Was it the same for every coyote or dependent on their behavior?
167-9- It may be more clear to move this information to before the detail about the persistence measure after introducing that you observe 4 non-feeding times, as on my first read I thought you were describing a set of observations other than those you outline in the earlier sentence.
174- the latency to approach the puzzle box was only included as the novel object test for the coyotes’ first exposure to the puzzle box correct? Should add some detail here.
177- What was their initial distance from the object? Was the object placed before coyotes were let into the enclosure from a door a certain distance away? Or was this placed while they were in the enclosure and they typically would retreat to an approximate location? Especially since the enclosures seem to be quite variable in size (0.1-1 ha) the latency could be highly variable based on enclosure size unless there’s a defined “start” time. I think this is an important methodological detail to outline for any latency measure in this study.
186- When you write that the box remained in the enclosure throughout the period, do you mean it was not removed between trials?
187- So is this a pre-test familiarization period not included in the 10 trials? It would be helpful to clarify this.
189- The peanut butter and extra food were added consistently outside of the box for every trial?
192-3 Why was the box left open on days 6 &7?
198-200 I think it’s important to point out that an object with food could elicit a different response than objects without (see Greggor et al., 2015). Your definition of boldness could include both these different responses but this may be something to clarify and potentially discuss later in relation to your results.
202- Does the dummy have a scent or is it just a visual stimulus? I’m curious if you think olfactory information could influence the response of the coyotes.
211-13 - Why combine the behaviors into categories? Perhaps due to low counts for some behaviors
220- How was time measured here? Months or days?
221- Were the outcome variables latency to approach and to touch modeled separately? Throughout this section it would be helpful to specify if outcomes are modeled separately or if any combination is happening
237- Is the proportion of time interacting and within 1 m combined or separate variables?
Throughout the Results section, please cite the tables in your supplementary materials with your model results.
268- It would be better to report the results here as no significant effects in the model of the treatment, time or interaction on the latencies.
283- I didn’t realize there was a pretrial phase for dummy, it should be described in the methods for the conspecific aggression trials. Or are you referencing a separate pilot with a coyote that was not included in this sample? It may be the terminology that is confusing, I am not sure whether the “pretrial” comprises baseline measures in October 2019 that are mentioned in the focal sampling sessions or a separate phase for these tests.
288- I think it would be helpful to describe the component loadings for the PCA, what behaviors made up the first two components of the PCA or have a supplemental figure showing the PCA results
300- Maybe add “over the course of this study” to clarify that the increase is reflecting the temporal aspect.
349-350 I would emphasize the process of habituation to the object/decreasing neophobia along with the process of learning
352-3- add Johnson-Ulrich et al., 2018 and Daniels et al., 2019 as citations for the link between persistence and problem solving
382- “approach”
426 delete period before citation
Table 1. How was directing attention defined in the investigation behavior? By the orientation of the head or body?


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: SCAVENGING VS HUNTING AFFECTS BEHAVIORAL TRAITS OF AN OPPORTUNISTIC CARNIVORE
Review round: 2
Reviewer: 1

Output:
Basic reporting: No comment
Experimental design: No comment
Validity of the findings: Inter-observer reliability needs clarification. See comments below for more detail.
Additional comments: The authors have submitted a responsive, well-revised manuscript. I commend the authors for laying the groundwork for future studies investigating how changes in foraging behavior may impact other behavioral shifts in wild predators. The additional figures and tables provide clarity for the reader and support for the presented results. The revisions in the text provide the reader with additional information that allows for deeper interpretation of results, and the authors have successfully clarified many areas of the text. I have a few minor suggestions and questions discussed below. Line numbers refer to the manuscript PDF.

Line 67: This is a bit unclear. I’m assuming you mean predators incorporating more scavenged foods may alter their own behavior. Or are you referring to interactions with other predators?

Line 111: Unclear. Suggest changing to ‘and reliance on anthropogenic food may increase the prevalence of human-wildlife conflicts.’

Line 140: Would be helpful to specify what species/taxa you are referring to here.

Line 143: Change “compared” to compare.

Line 154: Cite the previous literature here.

Lines 294-296: What defined “interaction” with the puzzle box? How was this different from touching/approaching the puzzle box? Puzzle box was only a ‘novel object’ in the first presentation, correct?

Line 317: What statistical test was used to evaluate inter-observer reliability? Are you reporting 10% agreement here (or 90%)? If so, 10% is quite low agreement.

Lines 324-325: Same question as above. Reliability of simultaneous coding only 1 video is also quite limited.

Line 380: What were the sexes of the successful hunters?

Lines 412-420: I understand the necessity to report representative results here. However, the next section discussing the puzzle box suggests there were notable differences between novel object vs. puzzle box approach, which is unsurprising given the food/time associated with multiple presentations of the puzzle box. To clarify, briefly re-state that the novel object trials with the puzzle box were only the first presentation of the puzzle box.

Line 422: A clear definition of an interaction with the puzzle box is also useful here.

Line 464: Suggest adding in ‘respectively’ after “hunting trials” as persistence only applied to hunting trials.

Line 520: By “different means” do you mean innovation/problem solving? Suggest clarifying and adding supporting citation here.

Line 534: Missing ‘and’ between “reward habituated,” make “novel object” plural.

Line 587: Citations for heritability of problem solving/persistence?


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: SCAVENGING VS HUNTING AFFECTS BEHAVIORAL TRAITS OF AN OPPORTUNISTIC CARNIVORE
Review round: 2
Reviewer: 2

Output:
Basic reporting: Comments on original MS all still applicable. The revised version is of similar quality.
Experimental design: Broad comments on original MS still applicable, and revisions have addressed any of my concerns, requests for additional context, etc.
Validity of the findings: No comment.
Additional comments: The authors have extensively revised the manuscript and addressed all of my concerns. They appear to have also addressed the comprehensive comments from the other reviewers and the editor. The manuscript is much improved.


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

Input:
Title: SCAVENGING VS HUNTING AFFECTS BEHAVIORAL TRAITS OF AN OPPORTUNISTIC CARNIVORE
Review round: 2
Reviewer: 3

Output:
Basic reporting: The authors have improved the manuscript with additional literature and background information as requested.
Experimental design: The authors have clarified their methods as requested by myself and the other reviewers.
Validity of the findings: The results are valid and the authors addressed reviewer concerns well. The information about coding reliability was a good addition, but a standard measure should be used rather than a statement about durations being within a particular percent. I would suggest reporting intraclass correlations (ICCs) as this has been a standard measure in behavioral studies.
Additional comments: The authors did an excellent job addressing all of the reviewer’s concerns including my own. I think with the previous revisions and a few more minor changes, this paper is ready for publication in Peer J.
Line by line comments based on the revised PDF:
166-169 Because you added the section about subjects, I think you can move and combine these statements with what is written in the subject section to avoid repetition
243- To help clarify that this baseline is the same as pretrial described later could rephrase to “baseline measures in a pretrial phase..”
288- Is this a reprieve or just encouraging interaction? I see how it’s a reprieve from having to work for food, but it seems like the overall purpose was to get the coyotes to interact more with the box.
Figure 1. This figure is very helpful! I think you could just point out directly where the pretrial phase was at the bottom of the figure for increased clarity.


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: HOW BEAVERS AFFECT RIVERINE AQUATIC MACROINVERTEBRATES: A REVIEW
Review round: 1
Reviewer: 1

Output:
Basic reporting: Very neat and pleasant introduction, giving good background in a nutshell. The theme is interesting, and has not been recently reviewed.
Experimental design: The study questions are clear and relevant, and the search process well enough described. Limiting the review to lotic habitats on one hand leaves out some interesting aspects, on the other makes the review clear and concise. Moreover, the authors wisely still discuss some aspects, which have not been studied in lotic setting but are relevant, on the basis of studies made in lentic wetlands.
Validity of the findings: The review well summaries the existing knowledge of the effects of beavers on invertebrates on lotic habitats. And, it identifies a gap in that the lentic habitats are much less studies in this respect. Connected to this, it would be interesting to see a pondering about what s the share of the beaver ponds made in lentic and lotic habitats. I know some studies in limited areas of this kind, but I wonder if there would be a possibility to make a crude large scale estimate of this matter?
Additional comments: Individual points:

Patterns and Community Shifts

Richness and other biodiversity
Here you could consider mentioning an experimental study from Finland (Nummi 1989) which shows the shift in invertebrate community in a creek section upon a damming imitating beaver, both in the littoral and creek bed as well as showing the change in number of emerging insects. It also demonstrates the substantial flow of organic terrestrial matter to the water in the from tree leaves in the early phase of inundation.
Maybe here you could mention also the considerable within patch heterogeneity, including deep water behind the dam, flooded swamps, shallow marshes etc., noted by e.g. Bush & Wissinger 2016 and  Willby et al. 2018. This also related to the discussion later, in lines 356-363, about “low-gradient streams may be turned into large wetland complexes”.
l. 150-151. Missing a word? These types of changes will cause shifts IN aquatic macroinvertebrate density and biomass.

Feeding groups
In the discussion of the abundance of different feeding groups it could be relevant to shortly deal with effect succession on these; e.g. shredders may be abundant in new beaver ponds where there is plenty of dead coarse organic matter in the form of dying plants, or predatory inverts might be abundant in new flowages because of little competition/predation from fishes.

Research Gaps
Ecosystem processes
l. 313. Along with birds, maybe bats as a group, or e.g. near threatened species Myotis dasycneme could be mentioned here.

Aquatic insect quatification
Here it was interesting to note the high number of predators in spite of the fact that quite much e.g. corers were used which hardly trap many of the very mobile predatory insects. I m not sure if this needs any comment.

Freshwater biodiversity crisis
l. 341. Maybe Bush &Wissinger 2016 could be added as reference here.
l. 347-349. Here, in the connection of the mere structural things, you might consider mentioning dead trees, especially trunks fallen to the water.


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: HOW BEAVERS AFFECT RIVERINE AQUATIC MACROINVERTEBRATES: A REVIEW
Review round: 1
Reviewer: 2

Output:
Basic reporting: 1/ -The authors conduct an interesting literature review on how beaver activity affects aquatic macroinvertebrates. I applaud the idea and its broad scale, but have reservations about the manuscript. However, I think that refinement of the article is required to improve clarity, and I provided some suggestions to achieve this.
Experimental design: 2/ -I commend the authors for the level of detail with some of their literature reviews, however some important references are missing.
Validity of the findings: 3/ -The authors should revise/clarify the research questions.
4/ -More explanations and interpretations must be added for the results, which are not enough. To sum up - I think this could be a very interesting review, but is hard to follow the argumentation as it stands.
Additional comments: Title.
5/ -Instead of "A synthesis of global data", just "A review" (it is more adequate).

Abstract.
6/ -Headings in a structured abstract would increase its clarity. Please remember, that headings in structured abstracts (i.e., Background. Methodology. Results. Conclusions) should be bold and followed by a period.

Introduction.
7/ -In general, the Introduction doesn’t really summarise the whole manuscript. I would recommend that the authors re-design to succinctly describe in more detail the work that they set out to do. Some important references are missing.
8/ -Line 46-47. ‘...becoming increasingly interested in how beaver engineering activities alter stream ecosystems within a modern landscape context’ - see Brazier et al., 2021
9/ -Line 48. ‘…can improve salmonid fish habitat’ - see also Kukuła & Bylak, 2011
10/ -Line 69. ‘…can support different aquatic macroinvertebrate taxa’ – see also Bylak et al., 2021 - support for endangered mussels.

Research aims.
11/ -Line 81-85 - Point 1 and 2: These are the objectives of the research paper, not the review paper.
12/ -My recommendation is to, (A), revise/clarify the research questions. For example the second research question presented is: “Determine if the aquatic macroinvertebrate communities differ in composition between beaver ponds and lotic stream segments” I could not see how this research questions was addressed by the analyses conducted. Three research questions stand out (based on the data collected): (1) What is the state of knowledge in understanding of taxonomic and functional changes in benthic macroinvertebrate assemblages of the beaver-altered streams? How has the field of knowledge evolved over time? (2) Which types of associations between beaver activity and aquatic invertebrate taxa have been described to date? What are the most relevant trends or patterns? (3) Knowledge gaps (which are good as they are). Please modify also the Abstract section accordingly.

RICHNESS AND OTHER ...
13/ -Line 139 – ‘a metacommunity effect’ – metacommunities in the beaver dam-and-pond complexes have been previously described in detail in fish populations - see Bylak & Kukuła, 2018.

INVERTEBRATES OF BEAVER-ALTERED LAKES AND WETLANDS
14/ -Line 383-397 - Is this whole paragraph really needed ??? (see - Title and Survey methodology)

References.
15/ Following, you will find some new related references which should be added to literature set:
-Brazier et al. 2021. Beaver: Nature's ecosystem engineers. doi: 10.1002/wat2.1494
-Bylak et al., 2021. Potential use of beaver Castor fiber L., 1758 dams by the thick-shelled river mussel Unio crassus Philipsson, 1788. doi:10.1080/13235818.2019.1664371
-Bylak & Kukuła, 2018. Living with an engineer: Fish metacommunities in dynamic patchy environments. doi:10.1071/MF17255
-Kukuła & Bylak, 2011. Ichthyofauna of a mountain stream dammed by beaver. doi: 10.2478/v10086-010-0004-1

Figures.
16/ -A flowchart should be added to the article to show the research methodology.
17/ -Please also increase the font size of numbers in Figure 1.


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

Input:
Title: HOW BEAVERS AFFECT RIVERINE AQUATIC MACROINVERTEBRATES: A REVIEW
Review round: 2
Reviewer: 1

Output:
Basic reporting: ok
Experimental design: ok
Validity of the findings: ok
Additional comments: The authors have well replied to the points I raised.