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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.

Title: DIFFERENTIAL RESPONSE TO STRESS IN OSTREA LURIDA AS MEASURED BY GENE EXPRESSION Review round: 1 Reviewer: 2

Basic reporting: This research compares patterns of expression of selected genes in response to acute heat and mechanical stress in 3 populations of Olympia oysters. These populations were selected based on a previous study showing differences in performance in the field, as reported in a different manuscript (a preprint in PeerJ). The research reported in this manuscript shows differences in gene expression in response to acute stress between populations, confirming that oysters from these locations show differences not only on performance, but also at the level of gene expression. These results have relevance for the restoration and aquaculture industries, indicating that more work should be done to evaluate the mechanisms behind those differences. Experimental design: The experimental design is adequate, although the stressors used were very acute, resulting in 100% mortality of exposed oysters. The authors should indicate if those are consistent with stress happening in the field. Validity of the findings: The research is well presented in general and the results are valid. I have attached a version of the manuscript with specific comments and suggestions. My major suggestion is to provide a more focused discussion that clearly differentiates between findings (differences in gene expression between populations in response to stress) and speculations regarding mechanisms behind those differences. The authors should also express that some caution should be taken into interpreting the relevance of these differences, since there was no difference between populations on their ability to survive these stressors (all oysters from all populations that were stressed died). Finally, I recommend that authors clearly indicate where the research should go next, and how that research will inform breeding in support of restoration efforts. Additional comments: No additional comments

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.

Title: DIFFERENTIAL RESPONSE TO STRESS IN OSTREA LURIDA AS MEASURED BY GENE EXPRESSION Review round: 2 Reviewer: 1

Basic reporting: Pass: The authors have addressed all my comments to review satisfactorily. I consider that the manuscript it acceptable for publication. Experimental design: Pass: The authors have addressed all my comments to review satisfactorily. I consider that the manuscript it acceptable for publication. Validity of the findings: Pass: The authors have addressed all my comments to review satisfactorily. I consider that the manuscript it acceptable for publication. Additional comments: The authors have addressed all my comments to review satisfactorily. I consider that the manuscript it acceptable for publication.

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.

Title: ­COMPARATIVE SPIGOT ONTOGENY ACROSS THE SPIDER TREE OF LIFE Review round: 1 Reviewer: 1

Basic reporting: The language is a bit verbose and very passive. The mss conveys a LOT of detail which is very important but diluted by the excesses. The following are examples: it is interesting that; e.g., we found this to be the case; we also found that the most likely state; it is possible that; we determined that; it is possible that; This means that; it would be helpful; we found that; we do know that; it is possible; etc. etc. Recheck spelling of all family names. The addition of the initials of a species author seemed excessive and if it is a journal demand then it is mostly unnecessary. In only few cases are the author's initial needed, eg. L. Koch vs C.L. Koch, O.P-Cambridge vbs F.O.P, Cambridge. In a number of cases, e.g. line 420, 525, references are needed. Unless the information is provided new here, cite a source. Also, ease off on the conjecture, e.g. line 524, 574. Experimental design: The science is EXCELLENT; the depth and rigour of the analyses is more than adequate and the questions posed/tested great! This is a very important work that MUST be published. Be careful with new "terms", like flankers and webless. Either put it in quotes are explain it. Please think about people for whom English is NOT their first language, e.g. l. 425 "hold steady" won't translate. line. 517, must end in ', respectively" Also, "sit and wait" works but "sit and pursue" seems contradictory, are these standard, if so, cite the original usage.l. 451. [We also observed snip!]... on PLS of both species" Which sexes? Validity of the findings: The findings are totally consistent, very valuable. However, the conclusions are useless, they should resemble something like the abstract. Additional comments: Really, it could do with a powerful tightening of language and probably a better structure of presentation of that amazing and valuable information you have.

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.

Title: ­COMPARATIVE SPIGOT ONTOGENY ACROSS THE SPIDER TREE OF LIFE Review round: 1 Reviewer: 2

Basic reporting: The article is well written in general, the references and context are well covered. I think the title is a bit “overscoping” - it is supposedly about ontogeny in spiders, but the ontogenetic content is presented on a specific clade of Araneomorphae (Entelegynae). I know that journals and authors usually benefit from that :) Experimental design: 295 al., 2016). We used a maximum likelihood method with a model of the weighted rate matrix of 296 substitutions for these spigots (Table 6). -- How was made the distinction between "absent" and "loss" in the scored species (data)? Rates in Table 6 need an explanation. (Same with Table s2). It seems very strange that only a few scattered and rather derived taxa lack any MS-Fl-PsF, and yet the ancestral state is inferred as "absent". This probably comes from the rate matrix, which needs a justification. (Otherwise the interpretation of lines 388-390 is dubious.) Validity of the findings: Figure 4: Where the identification of Pseudoflagelliform for Phyxelida comes from? I suspect it is arbitrary, since no histology was made (all were labeled as MS in Griswold et al. 2005). 384 gland spigots on the PLS (Table S1, Townley & Tillinghast, 2009). The predominately green 385 (Loss of MS spigot) clade was the RTA clade, -- but green = "MS" 386 blue/green (Pseudoflagelliform or Modified, respectively) -- but blue = "none" 340 ... In both species, a larger spigot was tentatively 341 identified as a ‘modified spigot’ (see Griswold et al., 2005: 61; character 96), with a potential 342 pre-modified spigot observed in the penultimate female stage of D. tenebrosus. These made no 343 other appearance in the ontogeny of both species spinning fields (Table 1, Figs. 7, 8). -- This is strange, as there is no nubbin of a modified spigot (MS) in the male. According to my experience, MS spigots leave a nubbin in male (Ramirez 2014: Table 5). The potential MS of Hogna is worth illustrating, since no MS was ever reported for lycosids. This finding is later downplayed in the Discussion. Additional comments: Minor edits: 73 2016). The former “Orbiculariae” Deinopoidea (cribellate orb builders) are now sister to the 74 RTA clade (includes wolf spiders and jumping spiders) rather than to the Araneoidea (sticky-silk 75 orb weavers) and Deinopoidea may not even be monophyletic (Garrison et al., 2016; Wheeler et 76 al., 2016). -- I would say "closer" rather than "sister", because these relationships are unstable, and the Deinopoiea are not monophyletic, according to those studies. Table 2 needs some explanation in the legend. -- revise English: 99 & Opell, 2002; Blackledge et al., 2009; Pechmann et al., 2010). It is possible the higher 100 fecundity trends observed in orb-weavers and non-web builders compared to cribellate silk users -- revise English: 163 taxa, particularly the singular, fiber producing spigot (MS, FL, PF) the on the PLS? 107 ... Most 108 of the Araneomorphae spiders possesses five types of spigots with another two appearing in ... 111 ... These are ... 116 ... 4) aciniform gland spigots (AC) on the PMS and 5) 117 aciniform gland spigots on the PLS that produce silk used in prey wrapping and lining egg sacs, 118 as well as the sheet portions in non-orb webs; and 6) cylindrical (=tubuliform) gland spigots 119 (CY) on the PMS and 7) cylindrical gland spigots on the PLS which are female specific and 120 produce fibers that form the egg sac (Fig.1, and see Fig. 1 in Garb, 2013). -- items (4-5) and (6-7) sound like of the same type. 130 paracribellar spigots on the PMS. However, in T. perfuga, the modified spigot is flanked by two 131 smaller, unknown spigots whose function is currently undetermined (R.E. Alfaro, unpublished 132 data). -- you can use Griswold et al. (2005) as a reference for this in T. radiata and many others. 291 N.I. Platnick (1987; Hypochilidae), Kukulcania hibernalis (N.M. Hentz, 1842; Filistadidae), -- Filistatidae 292 Thaida peculiaris F. Karsch (1880), -- ... (1880, Austrochilidae), 292 ... Megadictyna thilenii F. Dahl (1906; Nicodamidae) -- Megadictynidae 293 ... The Nicodamidae are sister -- Nicodamoidea

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.

Title: ­COMPARATIVE SPIGOT ONTOGENY ACROSS THE SPIDER TREE OF LIFE Review round: 2 Reviewer: 1

Basic reporting: The english is excellent, the references complete correct and through, and figures appropriately labelled. I had no complaint with the original submission but this is slightly improved. Experimental design: The scope and aims are well defined and answered. The methods are well designed and thoroughly documented. Excellent work! Validity of the findings: Although the results may not be earthshaking to some, to me they are really valuable and open new paths for future investigation. It is truly great work! Congratulations to the authors! Additional comments: No additional comments

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.

Title: PREDICTION OF LIFE STRESS ON ATHLETES’ BURNOUT: THE DUAL ROLE OF PERCEIVED STRESS Review round: 1 Reviewer: 1

Basic reporting: 1. The English language is communicable. However, there are many grammar mistakes and typos to be corrected. 2. The references are generally up-to-dated. There are several APA related mistakes to be corrected in your reference list. 3. The articles structure, figs, and tables are in a good shape. Raw data are provided. 4. The corresponding results are presented to address the proposed hypotheses. The proposed hypotheses should be back up with relevant literature. Experimental design: 1. This is not an intervention/experiment. A cross-sectional survey design was used. The cross-sectional data, as acknowledged by the authors, limit the inferences of their research findings. 2. I am not sure why it is necessary to examine the relationships among life stress, perceived stress and burnout. In particular, life stress and perceived stress are highly correlated or closely related to each other. Without a doubt, perceived stress will mediate the relationship between life stress and burnout. It may be also possible (to test the alternative model) that life stress is a mediator between perceived stress and burnout. 3. Some background information about athletes' levels (classifications) in Taiwan may be provided under the "Participants" section. 4. The overall internal reliability of ABQ can be included. 5. Baron and Kenny (1986)'s approach to examine mediation effects has been criticized (Zhao, X., Lynch Jr, J. G., & Chen, Q. (2010). Reconsidering Baron and Kenny: Myths and truths about mediation analysis. Journal of Consumer Research, 37(2), 197-206.). Please use either bootstrapping or Sobel test to check the mediation effects. 6. How the interaction terms were created in your moderation analyses? Validity of the findings: 1. If you take my comments about your mediation analyses, please update the findings of mediation analysis accordingly. 2. I am not sure why other demographic items were not controlled (entered as co-variates) in your regression analyses? Have you checked whether they were correlated to your major study variables? 3. Please state if your findings support your hypotheses or not. Additional comments: 1. Line 77-78: Is it necessary to include these references here? 2. Line 115-118: Include the page number if you used a direct quo here? 3. Line 167-182: Your literature review is weak in developing your hypotheses. Consider citing relevant research to back up your hypotheses. 4. Line 194: You don't need the "plus and minus" symbol here? 5. Line 207: Do you mean "measures"? Measurement refers to the data collection process. 6. Line 285: You need a reference here. 7. Line 292: Replace "Beta" with its symbol. 8. Line 342-383: I would suggest you to cut down this section as they are not directly related to your findings. 9. Line 385-390: It is good to go beyond your findings to make a conclusion. However, it does not read like a conclusion here. 10. Table 1: Demographic items can be included. Put your notes under the table.

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.

Title: PREDICTION OF LIFE STRESS ON ATHLETES’ BURNOUT: THE DUAL ROLE OF PERCEIVED STRESS Review round: 1 Reviewer: 2

Basic reporting: See my suggestions on the Discussion Section about some detailed comments. Other sections are fine. Experimental design: Research questions and hypotheses are well presented. Data analysis conducted in a good way. Validity of the findings: Data is robust and statistically sound. Additional comments: A well-conducted study and the steps of data analysis are conducted in a nice way. I have some suggestions for authors to consider that may improve their manuscript. 1. Lines 76-80: For the abstract, I would like to suggest the authors make it simple and only mention the aim of the study like “Building on the Smith (1986) cognitive-affective model of athletic burnout, the current study examine the relationship….” 2. Line 85: For the abstract, I would like to suggest not say BUT “but “counter-stress” component of perceived stress moderated”, use the word “while” or “and”. 3. Lines 188-189: Please remove the double quotation marks from “perceived distress” and “counter stress” or use the acronyms like PD and CS to represent the key words if not that many. 4. Line 325, I suggest authors remove the subheading of “Theoretical contributions/implications” from the manuscript. It is obvious in the discussion section. 5. Line 325, in the first paragraph of discussion, the authors mention: “The preliminary results provide several implications as follow…” Then paragraph starts like first second. My suggestion is that (a) in the first paragraph briefly revisit the main findings of the current study and remove the last sentence; (b) Do not say first, further, further. Just start with the topic sentence will be fine. 6. Lines 326-383: Switch the order of applied implications with limitations and future directions. 7. Lines 342-383: In the applied implications, I noticed that the findings of the current study is not been talked that much. However, I suggest the authors could think a little bit more to link each of the applied implication suggestions with the current findings. Just need an angle to cut in. 8. Lines 386-387: I suggest the conclusion can be rephrased, currently it reads like part of the suggestion rather than a conclusion of the whole study. It should be a highly summarized findings of the current study.

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.

Title: PREDICTION OF LIFE STRESS ON ATHLETES’ BURNOUT: THE DUAL ROLE OF PERCEIVED STRESS Review round: 1 Reviewer: 3

Basic reporting: Simple English, easy to understand. In the questionnaire uploaded, suggesting to add English version together with Mandarin language so that other researcher could read the questionnaires. Experimental design: Sufficient statistical analyses according to the research questions stated. Objectives achieved. Validity of the findings: The data is valid and statistically sound. Additional comments: I would like to congratulate the authors for conducting such an exciting study, looking on the other aspect of Smith's model on the perceived stress among college student athletes. I have attached some edits, and repeat them here: Abstract: Reference: Lundquist, & Wagnasson, 2013. Two separate hierarchical regression analyses found that the…. Line 90 – Delete “On the one hand”, suggesting “The college student-athletes are ordinary students;….. Line 93 – Delete “On the other hand”, suggesting use “Besides” Line 98 – arrange the reference alphabetically. Line 127 – suggest to use “the forth stage”… Line 129 – suggest to use “Many studies had adopted Smith (1986) … Line 160 – suggest it omitted some information… Line 171 – no researcher attempted to adopts … Line 184 – building on the above literature, Line 185 – the two components of Discussion, paragraph 2. – Further, we found several findings in the mediating and moderating …. For example, on the mediating effect of… Please add more information on the finding in the Discussion section.

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.

Title: PREDICTION OF LIFE STRESS ON ATHLETES’ BURNOUT: THE DUAL ROLE OF PERCEIVED STRESS Review round: 2 Reviewer: 1

Basic reporting: Line 171: incomplete citation (Barbosa-Leiker et al., 201?) Experimental design: NIL Validity of the findings: NIL Additional comments: NIL

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.

Title: PREDICTION OF LIFE STRESS ON ATHLETES’ BURNOUT: THE DUAL ROLE OF PERCEIVED STRESS Review round: 2 Reviewer: 2

Basic reporting: No comment. Experimental design: No comment. Validity of the findings: No comment. Additional comments: Thanks for your revision. I have no further comments.

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.

Title: THE RELATIONSHIPS BETWEEN EXERCISE AND AFFECTIVE STATES: A NATURALISTIC, LONGITUDINAL STUDY OF RECREATIONAL RUNNERS Review round: 1 Reviewer: 1

Basic reporting: The manuscript “The relationships between exercise and affective states: A naturalistic, longitudinal study of recreational runners is clear written, with relevant figures and tables, which are of high quality and well labelled and described. The structure conforms to PeerJ standards except the abstract because headings are missing. Nevertheless, there are some issues, which should be improved. My most important issue is with the theoretical background in the section introduction and some missing information about statistical analysis. Introduction: Experimental design: The introduction needs more detail (references and theoretical assumptions/backgrounds) and I would restructure it in putting some information in the method section. If you add these backgrounds, you could improve your argumentation and strengthen the relevance of your research question. Validity of the findings: data is robust and conclusion are well stated Additional comments: In this section, I listed some comments for each section (if necessary. Introduction: Line 29: These references are very old, try to find current evidence of the association between exercise and affective states. Line 53: Your intention is to analyse possible interactions. You mentioned this issue in line 53, for instance. Here, the argumentation would become stronger if you provide theoretical assumptions about these possible interactions (e.g. dual mode theory). Furthermore, you assume that valence and arousal reduce progressively as time passes. You also should add references for this assumption, for instance: Wichers et al. (2011). A time-lagged momentary assessment study on daily life physical activity and affect. Health Psychology Line 55 (et seq.): You mentioned that the association between exercise and affective states should be assessed in naturalistic studies. There are a lots of studies analysing the association between physical activity and affect in daily life and you should define in more detail your scientific contribution (see for instance the reviews of: Kanning, M., Ebner-Priemer, U., & Schlicht, W. (2013). How to investigate within-subject associations between physical activity and momentary affective states in everyday life: A position statement based on a literature overview. Frontiers in Psychology Movement Science and Sport Psychology; Liao, Y., Shonkoff, E. T., & Dunton, G. (2015). The acute relationships between affect, physical feeling states, and physical activity in daily life: A review of current evidence. Frontiers in Psychology Movement Science and Sport Psychology. Line 67 (et seq.): In my opinion you should explain in more detail the theoretical background of affective states (e.g. circumplex model, Russel, because you measure valence and energetic arousal) Line 70 (et seq.): This detailed information about your study belong in the section “Methods”, not in the section “introduction” Data analysis: Line 153: You should add information about the within- and between person variance (intraclass coefficient) and how you have centred the variables. To support understanding you could add the equations of the models. Results: How many missings do you have in your affect measures. You measured affect four times a day over six weeks (4measures x 42days = 168 measurement points). In average, how many affect measures did you achieve per person? Line 180 (et seq.): It is correct to abbreviate standard deviation with s.d.? Discussion: You should discuss in the limitation section that you assessed physical activity with self-reports. The activity data depends on what the participant enters into the platform. You are not able to control if the entry about time, distance and intensity of the run is correct.

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.

Title: THE RELATIONSHIPS BETWEEN EXERCISE AND AFFECTIVE STATES: A NATURALISTIC, LONGITUDINAL STUDY OF RECREATIONAL RUNNERS Review round: 1 Reviewer: 2

Basic reporting: No comment. Experimental design: Clear specification of inclusion and exclusion criteria (health status, motivations, etc.). Specify the average age of the female and male samples. Clear formulation of the main purpose of the research. Validity of the findings: Specify the limits, strengths and perspectives of the research. Additional comments: No additional comments

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.

Title: WEEKEND EFFECT IN UPPER GASTROINTESTINAL BLEEDING: A SYSTEMATIC REVIEW AND META-ANALYSIS Review round: 1 Reviewer: 1

Basic reporting: Report written using a good standard of English. Concise and clear use of language to convey the intended message effectively. Use of professional help for manuscript editing noted and commendable. Seven figures and one table included in the article. Figure 1 shows flowchart of selecting eligible studies. Figure 2 shows Forest plot of OR for total mortality (30-day and in-hospital mortality) due to UGIB during weekend versus weekday. Figure 3 shows Forest plot of OR for in-hospital mortality only. Figure 4 shows Forest plot of OR for in-hospital mortality in a subset of patients with UGIB due to variceal bleed. Figure 5 shows Forest plot of OR for in-hospital mortality in a subset of patients with UGIB due to non-variceal bleed. Figure 6 shows Forest plot of mean time to endoscopy during weekday versus weekend. Figure 7 shows Funnel plot assessing bias in study. Table 1 summarises the pertinent points from the selected studies. All included figures and tables relevant to the reporting of the study and included appropriately. Of note, there is a mention of another table labelled S2 which is supposed to list the search strategy, which has been omitted from the reviewers copy of the article. Experimental design: Study was well designed and follows the expected standards for systematic review and meta-analyses. Study aims to assess the effect of weekend admissions on UGIB and the resulting outcome of mortality, which is a poorly understood phenomenon and is not widely reported. Study question framed clearly and subsequent study reporting clearly focuses on addressing the study question. Methodology reported well, in a clearly reproducible manner. However, ‘Table S2’ which supposedly lists the search strategy was not included in the manuscript for reviewer. The standard steps for performing systematic review was followed e.g. searching and screening by 2 different reviewers separately and the involvement of a third reviewer to resolve any disagreement. The major databases were comprehensively searched for without any language restrictions. Articles published in abstract only form or in non-peer review journals were excluded. There was also no mention of searches performed in the ‘grey literature’. However, although limiting the search to only peer reviewed journals from the major databases may restrict the number of studies that can be included, it may partly ensure that only higher quality studies with complete data are included. Validity of the findings: Data reported in a clear and relevant manner with selected Forest plots of interest included. Study findings were discussed in a fair manner, considering the effect of heterogeneity where relevant. The conclusion is well stated and addresses the study question. Given the small number of studies meeting the inclusion criteria for this systematic review and the specific issues highlighted by the authors e.g. use of non-standardized definition for weekend, use of different primary end-points and reporting of time to endoscopy only in some studies; this study raises more questions than answers. More robust well-designed prospective studies need to conducted in multiple centres worldwide to examine the weekend effect on UGIB admission to provide more conclusive findings. Overall the effort of the authors in conducting and reporting this study is commendable. Additional comments: Sound methodology and clear reporting of results. Good discussion with explanations for observed results backed by current evidence. Meets the required standard for publishing.

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.

Title: WEEKEND EFFECT IN UPPER GASTROINTESTINAL BLEEDING: A SYSTEMATIC REVIEW AND META-ANALYSIS Review round: 1 Reviewer: 2

Basic reporting: Comment 1. Please check grammatical error. For example, line 207 ” admission showed significantly shorted times” Experimental design: no comment Validity of the findings: Comment 2. There were only seven studies shown in figure 5 for the increased in-hospital mortality due to non-variceal bleeding in weekend. And with eight studies recruited in figure 6 showing time to endoscopy during weekday is shorter. Yet, only two studies overlapped in these two figures (Wu, 2014 and Youn, 2012). So, whether longer time to endoscopy (figure 6) may explain the increased in-hospital mortality due to non-variceal bleeding (figure 5) may be a question. Because the initial settings of patients were different. Ideally, It would be better if all the recruited studies are the same in figure 5 and figure 6. Additional comments: Comment 3. Line 227 to line 228, I don’t think the different definition of weekend from recruited studies will affect the result. As long as there is a mention that lacking of manpower during the day which was defined as weekend. Are there any explanation of the definition of their weekend (for instance, less manpower in the subspecialty during the defined weekend) in the quoted articles?

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.

Title: WEEKEND EFFECT IN UPPER GASTROINTESTINAL BLEEDING: A SYSTEMATIC REVIEW AND META-ANALYSIS Review round: 1 Reviewer: 3

Basic reporting: I would like to applaud the author for registering their protocol PROSPERO and following the PRISMA guideline. Many systematic reviews suffer from poor reporting and inadequate search methodologies. I was pleasantly surprised to see this systematic review was well reported and there was no major concern regarding the search strategies. a few minor suggestions: 1) When reporting the databases, it is important to report the interface and the coverage for each database [line 25 and 26]. Searching the same database via different interfaces can lead to different results. For example, EMBSE is available via OVID or embase.com. CINHAL is available via EBSCO, OVID, and ProQuest. 2) It is recommended to report the number of articles you retrieved per line of search query. 3) It is also a best practice to report any software you use for screening and/or citation management Experimental design: 1) In PubMed search, Line#6 and Line #17, there are typos: admission tim to admission time. I hope these were mistakes in reporting and not the actual search strategy. 2) In PubMed search, remove extra line (line #16) from the search. Otherwise, it might be misleading or confusing to the readers. A general comment: Databases are very structured and are meant to be searched by controlled vocabularies or index term. It might be important to use key mesh terms in your search. For example, Time-to-treatment or Time Factors could be very good mesh terms to add to your search. To increase the search sensitivity. Validity of the findings: No comment Additional comments: No comment

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.

Title: ISOLATION, IDENTIFICATION, AND PATHOLOGICAL EFFECTS OF BEACH SAND BACTERIAL EXTRACT ON HUMAN SKIN KERATINOCYTES IN VITRO Review round: 1 Reviewer: 1

Basic reporting: The manuscript provides sufficient information in introduction section. However, there are few suggestions for the improvement of the manuscript. I suggest the author to ask a native English speaker to revise the manuscript for spelling and grammar mistakes. The author needs to carefully recheck the manuscript, and the missing references should be added. Additionally, author should add the latest references. Regarding title of the manuscript, I recommend the author to change it. Title should clearly represent the inside message. Experimental design: The study is looking original with well-defined research questions. It fills a knowledge gap of isolation, identification, and health hazards of beach bacteria. It comprehensively and rigorously describes the pathological effects and underlying mechanisms of this strain. However, the manuscript lacks details in many methods used, which seems difficult to be repeated by other reserachers. How did the author control fungal growth? Because fungal spores can easily grow on growth medium. It is not clear whether each experiment was independently repeated three times, or the experiments were conducted once using three test samples.Please clarify it. Validity of the findings: Findings in the present manuscript are looking good. Figures should be saved in high resolution format, and figure legends should be improved by adding sufficient information. Additional comments: Results section should be written scientifically including the aim and outcomes of the experiments. If possible, add some references in results section too.

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.

Title: ISOLATION, IDENTIFICATION, AND PATHOLOGICAL EFFECTS OF BEACH SAND BACTERIAL EXTRACT ON HUMAN SKIN KERATINOCYTES IN VITRO Review round: 1 Reviewer: 2

Basic reporting: The aims and approach presented in the manuscript, entitled “Isolation, Identification, and Pathological Effects of Beach Sand Bacteria on Human Skin Keratinocytes” represent a relevant perspective for human skin infection on the beaches. The manuscript overall well written. Experimental design: Overall the experimental design is good enough Validity of the findings: Majority of the findings are novel and there is sufficient novelty in the current manuscript. Additional comments: However, the manuscript has some important revision that need to be addressed. I suggest minor revision 1. (Line 56-60) The following sentence should be changed with suitable one. “For example, Heaney and colleagues have reported that, following visits to both freshwater and marine beaches in different parts of the USA in 2003-2005, the incidence of diarrhea, vomiting, and nausea increased in visitors. 2. Figure 5. If possible mentioned the IC50 valve in the results. 3. (Line 61) The skin is the largest organ in body and provides the primary barrier against bacterial infections. Change to …..primary barrier against various infectious agents. 4. (Line 98-100) Change, the soil samples… to sand samples were collected from the seaside area. 5. (Line 103) Kang et al., 2015 change to italic Kang et al., 2015 6. (Line 131) 100 mg/ mL streptomycin change to 100 mg/mL streptomycin 7. (Line 206-208) An understanding of the pathological microbiome is necessary to understand microbes pathological to humans, and to enable the development of successful therapeutic approaches for their cure. Change this sentence to “An understanding of the human pathological microbiome is necessary to enable the development of successful therapeutic approaches for their cure”. 8. (Line 235) Change the “P. stutzeri” to’ FSRS” 9. Figures should be saved in high quality. 10. The legends should be improved for better understanding.

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.

Title: ISOLATION, IDENTIFICATION, AND PATHOLOGICAL EFFECTS OF BEACH SAND BACTERIAL EXTRACT ON HUMAN SKIN KERATINOCYTES IN VITRO Review round: 1 Reviewer: 3

Basic reporting: The English of the manuscript is enough clear throughout the text. Experimental design: The research is with in the scope of the journal and method adopted for investigation is technical and scientific. Validity of the findings: conclusion is supporting the results and hypothesis of the manuscript. Additional comments: This is an interesting work; the authors warn the people visiting beaches for luxury purposes to be harmful. Although there are interesting data presented in the manuscript, the article would need minor revisions before it could be accepted for publication. The authors may need to address the following suggestions for improving the quality of the paper 1. The authors did not mention the lethal dose or the IC50 concentration of the extract tested in cell proliferation data 2. (Line 57) remove “following” and visit to “visitors” 3. (Line 58) change “There have also been” to “There are several other similar reports” 4. Figure 2-1: cluster should be defined 5. Figure 4: IC50 value should be mentioned in the result if possible percentage of cell proliferation” 6. (Line 244) remove “as” after could 7. Please make sure that all the references are in the format of the journal.

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.

Title: ISOLATION, IDENTIFICATION, AND PATHOLOGICAL EFFECTS OF BEACH SAND BACTERIAL EXTRACT ON HUMAN SKIN KERATINOCYTES IN VITRO Review round: 2 Reviewer: 1

Basic reporting: Well explained; having brief background. Experimental design: Novel idea, with good outcomes. Validity of the findings: Authentic. Additional comments: No additional comments

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.

Title: ISOLATION, IDENTIFICATION, AND PATHOLOGICAL EFFECTS OF BEACH SAND BACTERIAL EXTRACT ON HUMAN SKIN KERATINOCYTES IN VITRO Review round: 2 Reviewer: 2

Basic reporting: The manuscript was revised appropriately and I would recommend for publication in PeerJ. Experimental design: The Experimental part was designed properly and written in a proper way. Validity of the findings: The manuscript was revised appropriately and I would recommend for publication in PeerJ. Additional comments: The manuscript was revised appropriately and I would recommend for publication in PeerJ.

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.

Title: EFFECTS OF THE VISUAL-FEEDBACK-BASED FORCE PLATFORM TRAINING WITH FUNCTIONAL ELECTRIC STIMULATION ON THE BALANCE AND PREVENTION OF FALLS IN OLDER ADULTS: A RANDOMIZED CONTROLLED TRIAL Review round: 1 Reviewer: 1

Basic reporting: While the manuscript adheres to a professional structure, there needs to be more precision with professional English, most notably in the introduction and discussion. For example – linking words related to a fall or falls: line 35 (insert A before fall), line 41 (change to balance deficits are one of the known risk factors for falls), line 45 (insert to before stand), line 46-48 (needs re-wording), line 229 (reached instead of reach). There is sufficient background within the introduction but further context could be included in the discussion regarding electrical stimulation. Experimental design: Methods explained in appropriate detail allowing for replication. It should be made clearer that the control group only undertook single leg balance training and did not receive further training or electrical stimulation. Pictures of standing position/visual feedback provided may help to illustrate intervention protocols. Effect sizes should also be included in table 2. Further information on falls and associated injuries would be also be valuable. Validity of the findings: Overall the data is robust and controlled. Were improvements in balance and reductions in falls due to the balance training or the electrical stimulation? Or a combination?I think the authors might be overstating the potential impact of electrical stimulation without strong links/justification. As improvements in strength were not measured it is difficult to say how much of an impact the electrical stimulation training had. It is more likely that the visual feedback balance training (together with the single leg balance training) provided most of the improvements in balance and associated reduction in falls. Whilst there are links to improvements in strength and possibly function with electrical stimulation, these associations have not been strongly argued.The authors could expand on the reasons for this improvement (associated with balance training and visual feedback) in more detail as it is a very interesting and important finding. In the conclusion – few adverse events (line 277). Does this mean there were some adverse events during training/testing – please clarify. Additional comments: I commend the authors on conducting this well powered balance intervention in older adults. The force platform based visual feedback is certainly an interesting area that has great potential for improving balance and potentially reducing falls in older adults.

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.

Title: EFFECTS OF THE VISUAL-FEEDBACK-BASED FORCE PLATFORM TRAINING WITH FUNCTIONAL ELECTRIC STIMULATION ON THE BALANCE AND PREVENTION OF FALLS IN OLDER ADULTS: A RANDOMIZED CONTROLLED TRIAL Review round: 1 Reviewer: 2

Basic reporting: • Manuscript was sent to me UNBLINDED. • Raw data was provided. • Manuscript was very clearly written and easy to follow Experimental design: • Research question was well defined, relevant and meaningful. The results fill the gap of how platform and electrical stimulus training can improved measures of balance and reduce falls. • Methods described in detail • Reported with rigorous RCT methods Validity of the findings: • This research seems to be valid for bridging the gap between platform and electrical stimulus training. On a side note, it would be interesting to see how effective each individual intervention (platform, stimulation & single leg balance) was. • Data is robust, statistically sound and controlled. • Conclusions were well stated Additional comments: • Overall the paper is very well written, easy to follow and methodologically sound. • I would speculate that perhaps a Repeated Measures ANOVA may be a more appropriate statistical method to use, but the description of the two-way factorial ANOVA sounds sufficient.

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.

Title: EFFECTS OF THE VISUAL-FEEDBACK-BASED FORCE PLATFORM TRAINING WITH FUNCTIONAL ELECTRIC STIMULATION ON THE BALANCE AND PREVENTION OF FALLS IN OLDER ADULTS: A RANDOMIZED CONTROLLED TRIAL Review round: 2 Reviewer: 1

Basic reporting: No comment Experimental design: No comment Validity of the findings: The authors have clarified their findings in the results and discussion. This clarification was aided by the edits made to the methods. Additional comments: Thank you for making the suggested changes to the manuscript. The paper now flows better and the discussion and conclusions are now better supported. Congratulations on completing such an interesting and well powered study.

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.

Title: A DIAGNOSTIC MODEL FOR MINIMAL CHANGE DISEASE BASED ON BIOLOGICAL PARAMETERS Review round: 1 Reviewer: 1

Basic reporting: While manuscript is written in easy-to-read English language, it can be made more structured and of professional quality by substantially improving syntax errors, e.g.... * Excessive use of abbreviations. Group them when applicable in abstract and under results section, e.g., type of proteins measured, immunoglobulins, etc. and suggest brief explanation on their relevance in MCD. * Provide specific examples instead of using keywords, e.g., "certain risk" (line 34), "widely used" or "mathematical model" (line 35), etc. Experimental design: 1. My first major issue is with rationale (line 56-66) for development of mathematical model which is to avert invasive nature of the renal biopsy in patients with NS. Definitive diagnosis of MCD requires renal biopsy but it warrants electron microscopic examination as renal parenchyma looks almost normal under light microscopy. However, patients, mostly children with NS secondary to MCD have very typical clinical presentation with marked dependent edema, weight gain with growth retardation along with marked albumin secretion in urine and decrease in serum albumin. Unlike other causes of NS, MCD disease have usually normal renal function and lacks autoimmune dysfunction. Hence, present clinical guidelines rarely requires renal biopsy for diagnostic purpose and biopsy is usually reserved for rare cases (1-5%) where patient do not respond to steroids (majority does) or MCD is seen in adults (rarely seen). Since authors aim to provide their method for diagnostic purpose, it would help to have strong rationale to promote its clinical application. Such non-invasive approach, if accurate enough can be of much value for smaller fraction (< 1-5%) of cases where diagnosis is challenging, e.g., adult cases with hypertension, autoimmune disorders, etc. However, for larger cohort of patients of MCD are children (80-90%) and I have concerns and seek authors view on how their method provide additional value to existing clinical guidelines. 2. Another important issue is with methodology for developing risk prediction model. While t-test and logistic regression based model should work in MCD given a typical (a few features with less variance) patient presentation as described earlier, most biochemical parameters that Zhu et al. used (table 1) are part of standard or extended panel of investigations carried out by a physician for a patient presenting with NS. In that case, it would be useful to see how authors' model perform with and without including baseline biochemical indicators, e.g., total protein, albumin, creatinine, and lipid profile. Also, unlike reported ROC statistics in training set of 798 cases, it is equally important to report ROC statistics in cross-validation set (split from training set of 798 cases) if not the gold-standard, test or held-out set which is independent from the training set. 3. Under ROC statistics results, it is useful to provide ROC statistics for combined model using all 13 parameters and perform stepwise forward or backward selection in estimating coefficients of each of 13 parameters to predict which of parameters are of high vs low predictive value. References: 1. Kumar, Abbas, Aster. Robbins Pathologic Basis of Disease. 9th edition (2015) 2. Mansur & Batuman. Minimal-Change Disease. 6/30/2017 http://emedicine.medscape.com/article/243348-overview Validity of the findings: 1. With biomarker based prediction models, it is reasonable to not expect high level of accuracy and hence, authors reporting of sensitivity and specificity in range of 70-80% seems fair. However, I suggest authors can improve discussion section by adding a note on prioritizing sensitivity or specificity depending of trade-off between diagnosis false positive cases versus omitting true positive cases, respectively. 2. In discussion section, authors show total cholesterol and thrombin time as risk factors with highest predicted probability. Although these are known risk factors and often used in secondary panel of investigation to evaluate patients with NS, it would be useful to know if predicted probability of these markers remains high also among cases (from their training set) where diagnosis of MCD was challenging. If so, this can be an important finding to let physician weigh more on values of these two indices over others for the challenging case. Additional comments: Zhu et al. have presented a logistic regression based diagnostic approach for minimal change disease (MCD), a common pathological feature of nephrotic syndrome (NS) in children. Authors have provided detailed supplemental data and description on their rationale, statistical methods and visualization of prediction results using standard ROC plots. After reading the manuscript in totality, I find following several issues (under three sections) which I suggest authors to address before it is being considered for second review.

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.

Title: A DIAGNOSTIC MODEL FOR MINIMAL CHANGE DISEASE BASED ON BIOLOGICAL PARAMETERS Review round: 1 Reviewer: 2

Basic reporting: The article is clearly presented Experimental design: the article is mathematical model for kidney disease in particular Minimal Change Disease. It is retrospective in nature. the data set is appropriate Validity of the findings: The validity of the findings may not transcend to other ethnic groups The authors note that HTN and diabetes were elevated in the MCD group. this does not appear to be the case in the table. It the best of my reading the ROC was not applied to a subset of individuals not included in the original analysis It is not clear if individuals entering the study were on other treatment for example therapy for HTN or hyperlipidemia Additional comments: I believe that the study is very interesting but would find it hard to generalize this to other population subsets

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.

Title: A DIAGNOSTIC MODEL FOR MINIMAL CHANGE DISEASE BASED ON BIOLOGICAL PARAMETERS Review round: 2 Reviewer: 1

Basic reporting: None Experimental design: None Validity of the findings: None Additional comments: I thank authors for having detailed look on their manuscript and addressing most of my comments. With respect to experimental design, given case population is all adults and issues with availability of renal biopsy setup in hospitals, it is reasonable to have computational based predictions to provide some level of support in addition to biochemical indices in cases with diagnostic dilemma. Accordingly, prediction model by authors may complement current diagnostic approach for MCD in adults. As prediction models are inherently prone for overfitting and resulting inaccuracy, I wish authors to follow up with at least cross-validated model if not the model performing better (arbitrary value of AUC ~>0.75 or more) based on actual validation set. Such statistical rigor on top of their approach to include biochemical indices in their existing prediction model should yield reliable non-invasive approach for adult patients with MCD.

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.

Title: A DIAGNOSTIC MODEL FOR MINIMAL CHANGE DISEASE BASED ON BIOLOGICAL PARAMETERS Review round: 2 Reviewer: 2

Basic reporting: appropriate Experimental design: the authors have addressed concerns Validity of the findings: the authors have addressed concerns Additional comments: the authors have addressed concerns

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.

Title: RISK FACTORS FOR OSTEOPOROSIS IN MALE PATIENTS WITH CHRONIC OBSTRUCTIVE PULMONARY DISEASE IN TAIWAN Review round: 1 Reviewer: 1

Basic reporting: no comment Experimental design: 1) In Table 1, basic characteristics of controls should also be presented for comparison with COPD patients. In order to conclude that osteoporosis is more prevalent in COPD patients, data should be adjusted for various confounders including smoking status, renal function, the presence or absence of diabetes, and so on. It is also important for the authors to exclude COPD in control subjects by spirometric test. 2) According to the authors, controls included family members of COPD patients. But exactly how did the authors recruit these subjects? And how many were family members of COPD patients? Validity of the findings: 3) In Table 2, mean values of CRP and proportion of subjects with CRP higher than 5.0 should be shown in each group. 4) In Table 2, eGFR should be included. If the authors considered creatinine as an indicator of muscle mass, how could they be sure that renal function was the same? 5) And in Table 5, important factors missing here, such as systemic steroid use, current smoking, respiratory function and so on, should also be included. Whether or not CRP contribution to osteoporosis was dependent on respiratory dysfunction is particularly important, because higher CRP is apparently associated with impaired respiratory function in Table 4. 6) In Table 4 smoking status in high CRP group (1/8, 1/8, 1/8?) seems wrong and should be corrected. And correct information should be used to analyze determinants of osteoporosis (See comment 4)). 7) Serum levels of 25-hydroxyvitamin D should be measured. Vitamin D deficiency is a well-known risk factor of COPD-associated osteoporosis. Additional comments: COPD-associated osteoporosis is common, but its precise prevalence as well as its mechanism remains largely unknown. Thus, the subject is important. The manuscript is generally well written. However, some critical information is lacking, and statistical analysis seems incomplete. Comparison between control and COPD subjects is far from conclusive because of the small sample size and the way of subject recruitment.

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.

Title: RISK FACTORS FOR OSTEOPOROSIS IN MALE PATIENTS WITH CHRONIC OBSTRUCTIVE PULMONARY DISEASE IN TAIWAN Review round: 1 Reviewer: 2

Basic reporting: Clear, unambiguous, professional English language used throughout - yes Discussion - Findings very well discussed Specific comments Lines 183-188 “However, the prevalence of osteoporosis in our study was lower than the 40% reported in a recent study also conducted in Chiayi, Taiwan (Lin 185 et al., 2015). This may be explained by differences in subject enrollment and osteoporosis. That study enrolled male and female COPD patients and defined osteoporosis as a BMD T-score <−2.5 or the presence of thoracolumbar vertebral compression fracture on radiography.” → Very good to put the difference in prevalence in perspective. Financial support and sponsorship, conflicts of interest and acknowledgments indicated. Intro & background to show context. Literature well referenced & relevant General comments Reasons for study well argued; referencing correct and up-to-date Specific comments Lines 50-52 “It affects >5% of the population (Centers for Disease Control and Prevention, 2012) and is predicted to become the third leading cause of death in the world by 2020 (Murray & Lopez, 1997).” → Which population, world-wide? 72-74 “Therefore, this study primarily aimed to investigate risk factors for osteoporosis, particularly whether increased CRP is a predictor for osteoporosis development in male Taiwanese COPD patients.” → Why did you specifically choose “high-sensitivity CRP”? Better predicator of osteoporosis than IL-6? Structure conforms to PeerJ standards, discipline norm → yes Figures and relevant, high quality, well labeled & described Specific comments Table 2 a, b, and c (indicating the different statistical tests) are confusing, one expects that they would indicate significance → Suggest that the description (or indication) of what test was used for which analysis being put in the “Statistical analysis” section – lines 133-135, and significance being highlighted in the table. Table 3 See comment on Table 2 Table 4 See comment on Table 2 Are these still the COPD patients? Raw data supplied, but as I don’t use SPSS, I can’t read the file. Can you please provide the data in an Excel file? Experimental design: Original primary research within Scope of the journal → yes Research question well defined, relevant & meaningful. It is stated how the research fills an identified knowledge gap → yes Rigorous investigation performed to a high technical & ethical standard General comments • In- and exclusion criteria provided • Written informed consent o Possible to see an English version of the consent form? • IRB# provided Specific comments Lines 79 “It enrolled male COPD patients aged ≥45 years from……” AND 83-85 “Subjects who were willing to undergo anthropometric measurements ….” → How and by whom were the COPD patients recruited? 93-107 Description of the “Questionnaire interview and medical records review” → Would recommend putting the information in a table – would make reading it much easier. 120-121 “Lung examination: Chest radiography and spirometry data were interpreted by a chest physician.” → Not possible to get more than one chest physician to interpret the CxR? Was he/she a radiologist? Methods described with sufficient detail & information to replicate → yes Validity of the findings: Validity of findings Rationale & benefit to literature is clearly stated Data is robust, statistically sound, & controlled Data analysis – correct Results – well described Specific comments Lines 161-163 “The high hs-CRP group tended to have lower BMI, although there were no significant differences in age, smoking habits, and history of acute exacerbation (Table 4).” → Suggest moving this up to the previous paragraph – keep the hs-CRP information together. 159-160 “CRP showed strong associations with COPD severity and pulmonary function parameters (including COPD GOLD stage, FEV1, FEV1% predicted, FVC, FVC% predicted, FEV1/FVC, and FEF 25–75%) and steroid use.” → Can you differentiate the significance according to the different GOLD stages? Additional comments: Conclusions are well stated, linked to original research question & limited to supporting results → yes General comments → Very well written manuscript

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.

Title: RISK FACTORS FOR OSTEOPOROSIS IN MALE PATIENTS WITH CHRONIC OBSTRUCTIVE PULMONARY DISEASE IN TAIWAN Review round: 2 Reviewer: 1

Basic reporting: good Experimental design: no comment Validity of the findings: Conclusions are now well stated and supported by the results. Additional comments: Revision has been well done.

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.

Title: RISK FACTORS FOR OSTEOPOROSIS IN MALE PATIENTS WITH CHRONIC OBSTRUCTIVE PULMONARY DISEASE IN TAIWAN Review round: 2 Reviewer: 2

Basic reporting: Lines 50-52 “It affects >5% of the population (Centers for Disease Control and Prevention, 2012) and is predicted to become the third leading cause of death in the world by 2020 (Murray & Lopez, 1997).” → Which population, world-wide? Reply: Much thanks for the reviewer’s carefully reading. The population meant the people in the United States. It seemed not suitable to put this statement here since it was not world representative. We have deleted it in the text and in the reference. (Page 4 # line 51-2; Page 20 # line 343-4) Comment from reviewer: Addressed√ Lines 72-74 “Therefore, this study primarily aimed to investigate risk factors for osteoporosis, particularly whether increased CRP is a predictor for osteoporosis development in male Taiwanese COPD patients.” → Why did you specifically choose “high-sensitivity CRP”? Better predicator of osteoporosis than IL-6? Reply: We have chosen CRP because it was a common, cheap and quite available test in our hospital. To clarify this, we have addressed it in Page 5 # line 75-6. Comment from reviewer: I don’t see the clarification. Experimental design: Raw data supplied, but as I don’t use SPSS, I can’t read the file. Can you please provide the data in an Excel file? Reply: OK, we will upload the raw data in Excel file. Comment from reviewer: Addressed√. The study enrolled Taiwanese people, therefore, there was no English version of informed consent. We have translated the inform consent form into English for the reviewer. Comment from reviewer: Addressed√. Lines 79 “It enrolled male COPD patients aged ≥45 years from……” AND Lines 83-85 “Subjects who were willing to undergo anthropometric measurements ….” → How and by whom were the COPD patients recruited? Reply: I ask my several colleagues, who are also chest physicians to help us to enroll the out-patient COPD patients in chest clinic in Chiayi Chang Gung Memorial Hospital. If the patient had the interests to know more about the project, the chest physicians including me, would call the research assistant to help to explain the project more completely. If the patients agreed to participate the project, they would be asked to read and sign the inform consents. Comment from reviewer: Addressed√. Lines 120-121 “Lung examination: Chest radiography and spirometry data were interpreted by a chest physician.” → Not possible to get more than one chest physician to interpret the CxR? Was he/she a radiologist? Reply: Chest radiography and spirometry data were interpreted only by me. I am a chest physician but not a radiologist. However, in Taiwan, interpretation of chest X-ray is essential for becoming a qualified chest physician. Comment from reviewer: Addressed√. Validity of the findings: Tables Table 1 Looking good Table 2 Control (36) COPD (59) L-spine BMD (35) L-spine BMD (58) Hip BMD (57) Chronic hepatitis (58) If you weren’t able to measure these, mention that at the bottom of the table Table 3 Variables: “B”? Table 4 hs-CRP – put in either the mean ± SD or the %, it doesn’t read well with both in Table 5 Looking good Table 6 Looking good Table 7 Variables: “B”? Specific comments Table 2 a, b, and c (indicating the different statistical tests) are confusing, one expects that they would indicate significance → Suggest that the description (or indication) of what test was used for which analysis being put in the “Statistical analysis” section – lines 133-135, and significance being highlighted in the table. Reply: We have highlighted all significant value in bold in all tables according to the reviewer’s suggestion. We have explained the rules of statistical analysis in the “Data Analysis” section (Page 9 # line 135-42).. But we think the analysis process is complicated. It is very difficult to descript it without such labeling in each table. Therefore, we still kept them in each table and we think the reader would not be confused after highlighting each significant value in “bold”. Table 3 See comment on Table 2 Reply: The same reply as in Table 2. Table 4 See comment on Table 2 Are these still the COPD patients? Reply: The same reply as in Table 2. Table 4 was the comparison of “COPD patients” with low or high CRP. To avoid be confused by the reader, we have added the word “COPD” in the title of the table. (see Table 5, changed from Table 4) Comment from reviewer: Addressed√. Lines 159-160 “CRP showed strong associations with COPD severity and pulmonary function parameters (including COPD GOLD stage, FEV1, FEV1% predicted, FVC, FVC% predicted, FEV1/FVC, and FEF 25–75%) and steroid use.” → Can you differentiate the significance according to the different GOLD stages? Reply: Because the case number in this study is small. Further analysis with cases divided into GOLD stage I-IV to compare the pulmonary function parameters difference between low and high hs-CRP revealed loss of most of the significance possibly due to insufficient statistical power. However, the tendency towards lower pulmonary function parameters associated with increased hs-CRP seemed still existed. The results were as following (Tables provided). Comment from reviewer: Addressed√. Additional comments: Lines 161-163 “The high hs-CRP group tended to have lower BMI, although there were no significant differences in age, smoking habits, and history of acute exacerbation (Table 4).” → Suggest moving this up to the previous paragraph – keep the hs-CRP information together. Reply: We have moved it up to the previous paragraph according to the reviewer’s suggestions. (Page 11 # line 169-73) Comment from reviewer: Addressed√.

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.

Title: FASTVIROMEEXPLORER: A PIPELINE FOR VIRUS AND PHAGE IDENTIFICATION AND ABUNDANCE PROFILING IN METAGENOMICS DATA Review round: 1 Reviewer: 1

Basic reporting: I found the language in the manuscript to be sometimes a bit inaccurate and/or awkward, and I believe this should be improved so that the whole manuscript is entirely clear even for non-specialist readers (see examples below) Experimental design: no comment Validity of the findings: no comment Additional comments: In this manuscript, the authors present a new tool (named FastViromeExplorer) destined to help detect and quantify known viruses in metagenome dataset. This tool is using a relatively recent approach (kallisto) to map metagenomic reads to a viral database, and then add a set of cutoffs to identify false positives (usually viral genomes which recruit reads only on a portion of the genome due to a repeat / shared region). The whole pipeline is provided as a stand-alone tool. The main caveat of this tool is that it relies on databases so can’t provide any virus discovery. Given the state of the viral databases, this limitation should be made very clear as early as in the abstract (note that this can be presented as a trade-off with speed though, since any pipeline involving de novo assembly and identification of novel viral genomes would likely be much more computationally demanding and longer). As of now, the only mention of this bias is in the Conclusion section l. 354 “, the limitation of FastViromeExplorer is that it cannot identify a virus or phage if a similar sequence is not present in the reference database.”, which I consider is not enough for a non-specialist reader to understand how big/small of a problem this reliance on databases can be for different use case scenarios. The authors could also test / illustrate this database bias by generating simulated metagenomes including new viral genomes published since they created their database, and observe the maximum “distance” at which FastViromeExplorer is able to identify these viruses (i.e. how different from the database genomes a new virus genome can be), although I don’t see this additional benchmark as absolutely essential. More generally however, I would ask the authors to provide a much more detail description of which type of study would benefit from FastViromeExplorer (e.g. known pathogen detection) and which should use the more “standard” pipeline relying on de novo assembly (e.g. viral diversity exploration). My other main concern is with the expected coverage computed as part of the mapping cutoffs, and calculated using a Poisson law. This doesn’t take into account biases linked to the PCR amplification of sequencing libraries, usually required for all/most “low input” cases, i.e. ng of input material, which is still relatively frequent for example in clinical setups (see e.g. PMID 23663384). In these PCR-amplified libraries, the coverage of a genome is not uniform (and less so when the number of PCR cycles increases), which means that the expected coverage cutoff used could wrongly exclude a number of “true” viral detections. Although I suspect there is no magic trick to solve this, I believe the authors should discuss these cases, and provide guidelines for how to use FastViromeExplorer when the user would not expect a uniform coverage. Finally, I found the language in the manuscript to be sometimes a bit inaccurate and/or awkward, and I believe this should be improved so that the whole manuscript is entirely clear even for non-specialist readers (see examples below). Additional comments: l.23: “In a healthy human body there are estimated to be” feels a bit awkward to me, please rephrase. l. 25-26: “diabetes […], and depression […] and cancer” Please remove the first “and” or reword the sentence. l. 31: “composition and behavior of environmental microbiomes”. I am not sure a microbiome can have a “behavior”, maybe “metabolism” or “host interaction” could be more appropriate ? l. 41: “annotating the taxonomy” should be “annotating genes for taxonomy” or “taxonomically affiliating”, but I don’t believe it is correct to say one “annotate” a “taxonomy”. l. 56-58: “However, read assembly can be […] annotation.” Although I agree with the authors about the current challenges of assembling metagenomes, it would be great it they could add one or two references to back this claim. l. 95: “30 million paired-end RNA-seq reads for the human transcriptome” shouldn’t it be “from a” human transcriptome “sample” rather than “for the human transcriptome” ? l. 207: “through analyzing metagenomics data” should be “through the analysis of metagenomic data”. l. 254: “is only one time process” should probably be modified to something like “is computed only once”. l. 270: “As viruses are known to have fast mutation rates, Blastn and its variants (e.g., Blastp) are considered the “gold standard” approach to annotate viral sequences in metagenomic data but very time-consuming, having similar performance yet running much faster is highly desired for an annotation tool.” I disagree with this statement, and am not convinced it needs to be made at this point in the manuscript. First, the authors are comparing read mapping method, and correctly using blastn as an alternative to their tool, which has no real link to the high mutation rate observed in some viruses. On the other hand, blastp is used to annotate novel viruses (i.e. de novo assembly of viral genomes, usually only distantly related to viruses in the databases, and for which the only level at which sequence similarity can sometimes be observed is on the amino acid sequences). This is an entirely other approach, hence I believe referencing blastp in the context of this manuscript is misleading. Overall, I would suggest the authors remove this sentence entirely. l. 285-287: The number of viruses identified seems very low, and I believe it would be helpful if the authors could add one sentence with the expected number of viruses in these samples, so that a reader can appreciate how well a database-relying approach using NCBI RefSeq worked in this case. l. 310: Should provide information about the number of viral contigs identified with the larger DB (as it can be seen as a proxy for the number of viruses detected). l. 311: “Therefore, the host information of the mVCs can be used to examine the annotation result.” I believe the authors should specify here how many sequences are associated with a host in this larger database and how these were derived. As it stands now, a reader could think that the two databases (RefSeq and IMG/VR) are equivalent in terms of host information, which would be misleading. l. 317: “or or” should be “or” l. 339: “Blastn” should be “blastn” l. 350: “can annotate both” I disagree with the use of “annotate”, FastViromeExplorer does not provide any annotation, but instead can detect / quantify known viruses. This comment apply throughout the manuscript, hence variations of “annotate / annotation” should be modified elsewhere too when relevant (e.g. 352).

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.

Title: FASTVIROMEEXPLORER: A PIPELINE FOR VIRUS AND PHAGE IDENTIFICATION AND ABUNDANCE PROFILING IN METAGENOMICS DATA Review round: 1 Reviewer: 2

Basic reporting: I am happy with the writing style. Experimental design: Good. I am happy with most of it other that the fact that this should have been tested with the full genbank viral database and not just the RefSeq as a database. Validity of the findings: This method will be useful for people looking at known viruses in metagenomic datasets Additional comments: The MS by Tithi et al describes a new rapid approach to identifying known viruses from metagenomics datasets. The approach relies on a reference viral database that is well curated e.g. GenBank or even the JGI viral metagenomics dataset and kallisto for rapid mapping of the reads. The output from the pipeline developed for FastViromeExplore provides a list of viruses that is sorted based on abundance. The latter is calculated based on number of reads. I think this is a great tool for people who want to look for ‘known’ viruses in their dataset. However, I do see some major issues with the use of the word virome in the context of this software. 1) The tool described here is not a virome explore but a virus explore. The pipeline is heavily reliant on a nucleotide database. Over and over various studies in the last 10 years have shown that we have barely scratched the surface of viral nucleotide sequence space. Hence most of the virus discovery relives on protein sequence space. 2) The abstract “Identifying viruses and phages in metagenomic data has important implications in improving human health” is a bit odd. First of all phages are viruses and second identifying known viruses is not necessarily going to improve human health – it will trigger a response for clinicians and epidemiologists. Also with the approach you’ll only identify known viruses that may be emerging. 3) I am not sure refseq on it’s own is a good enough set of sequence for testing this tool. I would have tested in on the whole GenBank viral DB which includes variant. Roughly the RefSeq sequences represent a centroid in a 10% sequence variantion sequence space at a genome level. So it is likely that some reads may not be assigned to the correct “taxa” as it is missing from the database. Also you make a comment about in silico chimaeras during de novo assemblies. Read mapping to reference genomes also suppers from the same issue – that is assigned of reads to a wrong taxa. So you may want to address this. 4) Line 24-25: Please check this “Studies have shown that there are connections between human gut microbiome (viruses and bacteria) and diseases such as diabetes…” but as far as I am aware these studies have only looked at the role of bacteria not viruses. I like this paper and think it has merit for publication in PeerJ. I also think that the authors may want to take a step back note that this tool is only for identifying viruses based on nucleotide sequences in the compiled database. I would change the name of the tool to FastVirusExplore a the word virome is totally misleading and this tool does not at all enable virome exploration.

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.

Title: DNA BARCODE-BASED SURVEY OF TRICHOPTERA IN THE CROOKED RIVER REVEALS THREE NEW SPECIES RECORDS FOR BRITISH COLUMBIA Review round: 1 Reviewer: 1

Basic reporting: Introduction Main Issues. This work could be an important baseline for future monitoring and impact assessment. However, the way is written is more like a report and not a scientific article. There is not a clear question or hypothesis presented to be evaluated. There has been growing literature on this topic presenting great information of species list of Ephemeroptera, Trichoptera, and Plecoptera in Northern Canada and this study can be used to compare the species richness with other regions in Canada (look for Yukon and Northwest Territories EPT records in Cordero et al. 2016). Based on previous species distributions studies: how are your contributions relevant? Are the new records you present unexpected? What is the novelty: altitude, latitude? Put your study in a scientific context and not only in a report format. As well, more support in your introduction is necessary, there are many concepts or facts that are not cited minor issues: line 29: what portion of the COI? be concise, refer to the subunit I. Also this information needs a citation. line 42: Support this statement. How do you know this area is true Arctic? mind that in Northern Canada, latitude alone is not an indicator of a geographic area. Recent studies have addressed this regional classification. Experimental design: Main Issue: The paper does not follow the format of a scientific article but looks more like a report. The research question is not stated and hence no gap in the knowledge is addressed. The contribution is not relevant the way it is presented. It needs revision about the goal of the article. specific issues: Methods. What is the importance of using both morphological and molecular identification analysis? Could you solve any problem presented by morphological analysis? Did you apply a sampling protocol in the different sites? Validity of the findings: Main Issues: The use of BOLD or other database is very useful as an initial reference. However, assumptions or conclusion made from direct comparison or the match percentage is not very strong evidence. There are many mistakes in the registered species names in the databases and the only way to confirm your findings is to put your individuals in a phylogenetic tree and see real differences. Your initial idea is good but the way how you support your findings is not enough, even to claim for first records, much less for claiming new species records. Be careful with conclusions based on arbitrary 2% line 140: If there are no other studies how you found only three new records if all the other records should be new too? You need to dig deeper in the literature since there are good records from the 1980's for places like Yukon that are far more Isolated, I can't imagine a good amount of literature for aquatic insects in British Columbia, considering the number of studies made on altitudinal gradients in the Rocky Mountains. Additional comments: I think the authors should be encouraged to re-think this paper. I always find very valuable information about diversity but this information should be put in a scientific context to increase the relevance of the paper as well as the journal quality. I would recommend to accept the manuscript if they address two main issues: 1. Question or hypothesis. 2. Comparison of data by the use of a phylogenetic analysis.

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.

Title: DNA BARCODE-BASED SURVEY OF TRICHOPTERA IN THE CROOKED RIVER REVEALS THREE NEW SPECIES RECORDS FOR BRITISH COLUMBIA Review round: 1 Reviewer: 2

Basic reporting: This paper makes a very good contribution to the knowledge of the caddisfly fauna of an interesting river in an interesting ecosystem. The methods of analysis are relatively new to trichopterogy and represent one of the first attempts to use established DNA databases as a means of identifying local caddisfly biodiversity. However, I think the paper needs better justification as to why this method is better than the “traditional” method of collecting adult caddisflies with light traps or by netting or other means. Trichoptera species taxonomy is based on male genitalia (almost all holotypes described in the 20th century and now are males). Larvae are secondarily associated with males through rearing, use of pharate adults, or by COI sequence match. The authors should explain why they collected larvae only, or at least mention the value of adults in caddisfly taxonomy. They may have valid reasons - adult taxonomy is specialized, adults do not fly to light in cool weather, adults may fly in from other watersheds, etc. If the objective was to show that collecting larvae only was sufficient to assess the biodiversity of a stream, they should state that. It would be interesting to compare the results of a strictly larval survey with adult collecting only although I realize that this is beyond the scope of the paper. However, as presented the objectives of the paper are not expressed in context to standard caddisfly survey methods nor is it indicated their relative value. In addition, the three new records are not placed in any context of the known distribution of the species. For example, C. harwoodi is largely an eastern North American species, so its presence in BC is surprising. This should be at least mentioned, if not discussed. Without knowing otherwise, one might conclude that the vouchers of these sequences in BOLD are misidentified. I found only a few typos grammatical issues in the paper. I am still under the assumption that data are plural, but in some places it is used as a singular noun. Also, the authors use dashes quite a bit to separate clauses. Perhaps it is more appropriate to use commas or semicolons. I did not find the larval photographs to be informative, although I think photographs of vouchers are required for submission to BOLD. Do these photographs match known, published descriptions of these species> I especially find the photographs of the adult Lepidostoma to be uninformative. I can only tell that it is a Lepidostoma and it appears to be a female. There is no indication of how this specimen was obtained. Experimental design: see above Validity of the findings: see above Additional comments: see above

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.

Title: DNA BARCODE-BASED SURVEY OF TRICHOPTERA IN THE CROOKED RIVER REVEALS THREE NEW SPECIES RECORDS FOR BRITISH COLUMBIA Review round: 2 Reviewer: 1

Basic reporting: 1. The initial paragraphs of the introduction lead the reader to understand the importance of determining the biodiversity in an impacted area. However, when describing the studies area, I consider that the information from line 53 until line 74 could be summarized in a couple of lines. This information, although interesting in general, lead to ambiguity. I would recommend condensing this information. 2. In line 78, The objective number 2, states that they want to explore the biodiversity of Trichoptera. However, biodiversity has several components (alpha, beta, gamma diversity, abundance indices, ecological diversity, functional diversity, etc) that were not analyzed in this study. They only analyzed species richness and that was stated in objective 1. The objective 3 is redundant since that is expected in objective 1. I would recommend being concise with the aims and the reach of this study. 3. the sentence on line 148-150 is very confusing. The idea between DNA and morphological species identification is not clear. Please clarify this sentence. Experimental design: The corrections suggested were done and the methods look more clear and concise. There are objectives, they were developed and the results were totally within their framework. Validity of the findings: This study as a baseline will be very important for future references, especially in isolated areas that are impacted by human activities. The data collected and analyzed provide support to his conclusions and can be used as a reference in future studies. Additional comments: The corrections made by the authors were key to give clarity and relevance to this study. There are minor corrections that should be done and I mentioned above, but I recommend this paper for publication.

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.

Title: DNA BARCODE-BASED SURVEY OF TRICHOPTERA IN THE CROOKED RIVER REVEALS THREE NEW SPECIES RECORDS FOR BRITISH COLUMBIA Review round: 2 Reviewer: 2

Basic reporting: Ok Experimental design: OK Validity of the findings: OK, although I still think the record of C. hardwoodi should be presented with some degree of uncertainty. Additional comments: Several typographical and other errors that should be corrected and the whole paper proofread very carefully. Note comment on use of parentheses for species authors' names. Ensure bibliography is properly formatted to journal style.

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.

Title: ACHILLES HEEL OF A POWERFUL INVADER: RESTRICTIONS ON DISTRIBUTION AND DISAPPEARANCE OF FERAL PIGS FROM A PROTECTED AREA IN NORTHERN PANTANAL, WESTERN BRAZIL Review round: 1 Reviewer: 1

Basic reporting: The study deals with an important issue in Brazil (feral pig invasion) and with an interesting ecological question (why did the specie disappear in a Protected Area?) with considered applicability to conservation strategies (invasive species control). However, the study failed to properly describe the situation (Specie disappearance), there were misleading use of concepts (Potential niche x occurrence of specie), the methods lack more details for evaluation (RDA and SDM), and the analysis and results could be substantially improved: 1 –Specie disappearance: The manuscript is ambiguous and unclear for the situation about the disappearance of the species in the PA. It is plausible the concentration of S.scrofa population has moved from PA to the adjacent cattle ranch due to the land use change. This phenomenon is really interesting, novel and useful for decision making regards to specie’s control. However, the specie’s condition was not clear to the readers till the Discussion section. Is strongly recommend to be fair with the readers since the beginning. Actually, the specie didn´t disappear from PA as pointed out along the text (Title, Abstract and Introduction). At "Study area" section, it was clear the species still there: "Park rangers report that visual records of feral pigs were extremely rare and the species is seldom recorded". The Results section unfortunately failed to inform information about the presence/absence. By the other hand, the Discussion section started with "The low number of records of feral pigs in the PA..." and gave the impression the specie’s abundance in PA was low but it has not disappeared. The specie´s disappearance is a key situation for the study and it must be properly describe for the readers and for better evaluation. Moreover, the apparent specie´s abundance reduction was based on the informal observation in the past in the two areas (PA and the adjacent cattle ranch). However, there were no measure of abundance for spatial and temporal comparison between them and the population concentration could be superficial, especially by considering the distance between the areas. The two areas are very close for the biology of the specie and it is likely there is only one population with the same variation in both areas. The population reduction in PA could happen in both areas and the study was unable to detect and demonstrate it. In regards of high sampling effort with few specie’s records, it is plausible to considered the current low S.scrofa abundance in PA, but there is no such information to the other area for proper comparison. The so called disappearance could be an overall phenomenon within the region, the municipality of Barão de Melgaço, without relationship with the PA effect. 2 - Potential niche x occurrence of specie The effect of PA in occurrence of specie is the key issue for the study goals: “(i) identifying the spatial distribution patterns of feral pigs and (ii) inferring about the effect of landscape change, due to the implantation of a Protected Area (PA), in the occurrence of the species”. To these goals, the occurrence must be somehow measured in the field like occupancy (e.g., Mackenzie et al., 2006 - “Occupancy Estimation and Modeling” ). However, the study used the potential niche for this goal instead. The occurrence was then an extrapolation of the niche theory and not the proper measuring in nature. The study considered potential niche as “probability of presence” (Figure 4), i.e., a kind of “potential presence”. Therefore, the study was reduce to a merely description of the potential niche variation in the study area and it had limitations to the inference about the true PA effect on the specie occurrence. Furthermore, the potential niche was dependent on 69 current records of the specie and it can results in biased estimation to the present and past niche/occurrence. The method has the assumption of unreal perfect detectability and it can easily be violated by false absence (e.g., Mackenzie et al., 2006 - “Occupancy Estimation and Modeling” ). The data base for the potential niche modeling also need more details for better evaluation of this method as described below. For this case and goals, I suggest using proper methods for occurrence other than potential niche, like occupancy models. 3 – More detail need for RDA and SDM Multivariate analysis like RDA are proper evaluated with the complete results of axis and not only percentage of explained variation by each axis (Fig. 2). The complete results can be provided as table in supplementary material but it is also interesting to plot the results against the explanatory variable and ordering them to highlight the main effect. Species Distribution Models (SDM) used in this study was based in 69 records of the species without more details. It fundamental to provide more details for better of evaluation of this method as such its geography distribution (e.g., in the map, Fig. 1) and how they were collected (camtraps only or all data of field effort like the monitored natural licks and artificial pounds). I suggest providing this information on the text and, if it was the case, I discourage the use of data based on the monitored natural licks and artificial pounds because they can work as attractive site and biased the presence and then the potential niche estimation. 4 – Analysis and results improvements As many other invasion biology case, the human dimension is an important issue. However, the study was limited by considering only ecological variables. The results can be very biased because the variable comparison is based on relative importance. I suggest including in the analysis a human variable like the distance of settlement or other one that represent the influence of human mediated invasion. For PA effect, the cattle was removed as well as the human presence with their culture (wild pig management) and livestock (free range system of domesticated pigs). The feral pigs in Pantanal is reported and known as an abundant and old population, but it still unclear how much the real human presence conserve this population by local management with constant introduction (free range of domesticated pigs) and cultural hunting (Desbiez et al., 2011). This alternative interpretation does not reject the Authors´ one, but the article could have interesting improvement by considering human influence in this invasion biology. So far, the analysis have the assumption this invasion is strong dependent of biology of the species (dependent of "changes in the landscape and vegetation structure"), but the indirect human influence can much stronger. The management of cattle changes the landscape, but it also means constant human presence with their externalities as such conserving the wild pig population in higher abundance with protection (captive pigs close to the house) followed by constant restocking (scapeing pigs) and biased harvest (preferable male hunting and some female care). The Achilles heel of S.scrofa can be the amount of propagule and restocking. An extra variable considering the human influence can improve the analysis. Experimental design: The Index of Use (IU) has several limitations for the analysis and it could be replaced by other index or analysis. The first limitation is the spatial dependence of the camtrap sites. The minimum of 600 m is not enough for S.scrofa, puma and jaguar because the species has wider home range in 15-28 days. The same animal can use several sites and overestimate the IU. An alternative is to test the dependence for each species reducing the intervals as it was done for shots intervals. I suggest at least justifying clear the reason to use the sampling interval in each site. The other limitation of IU was the assumption of the perfect and constant detection within and among species. The Authors should consider this index (IU) has the naïve assumption of perfect detection, i.e. when the species are presence at the site, it is always recorded and it scores the index; otherwise when the specie are absent the specie is not recorded. Thus, there is no false absence. The detection is also equal for all sites and all species has the same detection probability. However, it is expected different detection probability among sites, i.e. difference between forest and open area, for example. In the same manner, jaguar sounds more difficult to detect than puma and S.scrofa. It is very important then to control the detectability for this analysis. For this issue, I suggest the use of occupancy models (Mackenzie et al., 2006 - “Occupancy Estimation and Modeling” ). Validity of the findings: The findings were very interesting but dependent on the kind of variables (only ecological variable) and spatial dimension for the species. It is important to consider human variable to balance the relative importance among ecological variable. Such variables can be justified by the conflict of interest and it has important implications for controlling the specie in other parts of Pantanal, as the Author justify in the Introduction section ("direct conservation decision-making"). The conflict of interest was an important reason to consider the S. scrofa among the 100 of the world’s worst invasive alien species (Lowe et al., 2004) and its control depend on the overcome of this feature (Choqnot et al., 1996, Lowe et al., 2004). Without controlling the human influence, it is difficult to eradicate feral population. The findings (“strong relationship with pasture”) can also be biased by the spatial dimension of the study because it was very narrow for the species. The results can just be a local effect of abundance variation or reduction of the abundance in the periphery of the local population´s range. In wider geography scale, the findings was not expected because the invasion is very old (>200 year old) and the species had been restricted to the Pantanal biome for centuries even with land use change around to open area like pasture-like system. For some reason, the species have not invaded the rest of Brazil even with enough time and favorable land use change. These considerations don’t compete with the original propose. They are just a complement. The suggestion can change the analysis but improve the interpretation, match the real invasion situation of the species in this country and provide more insights for decision making. Additional comments: No additional comments

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.

Title: ACHILLES HEEL OF A POWERFUL INVADER: RESTRICTIONS ON DISTRIBUTION AND DISAPPEARANCE OF FERAL PIGS FROM A PROTECTED AREA IN NORTHERN PANTANAL, WESTERN BRAZIL Review round: 1 Reviewer: 2

Basic reporting: The study deals with feral pig invasion, a relevant problem worldwide and an important issue in Brazil . The authors, properly describe the past and current situation at the study area, and try to answer very interesting ecological questions applying proper research methods and analysis. Adittionaly, the study is very important for conservation strategies and invasive species control. Experimental design: Like in most biological invasions, in the studied case, the human dimension is an important topic. However, the study was focused only in ecological variables. The importance of invasion history is also underestimated. It would be relevant to include in the analysis historical data about the micro invation within Pantanal, and a human variable like the distance of settlement or other one that represent the influence of human assisted invasions in the modelling. Also, I recommend that, whenever feasible, similar macro-scale approaches to the species distribution modeling are pursued to capture the extent of conditions that support the species populations and generate hypotheses about species limitations or invasion potential that can be tested in combination with this type of finer-scale research. Validity of the findings: Although long-term movement data for wild pigs is lacking in South America, social factors such as value of wild pigs as a recreational hunting resource or as farmed species are at least as important as natural dispersal in driving the current distribution of wild pigs. As such, we recommend that future research investigating the distribution and invasiveness of wild pigs should include social factors in addition to biological factors (what biotic and abiotic factors limit wild pigs populations?) to address competing hypotheses and generate effective management solutions. Despite of this, I strongly recommend the publication of this very interesting manuscript. Additional comments: No additional comments

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.

Title: CRUSTACEAN AMPHIPODS FROM MARSH PONDS: A NUTRITIOUS FEED RESOURCE WITH POTENTIAL FOR APPLICATION IN INTEGRATED MULTI-TROPHIC AQUACULTURE Review round: 1 Reviewer: 1

Basic reporting: - The English language should be improved throughout the manuscript to ensure a clear understanding of the information by an international audience. Some examples where the language could be improved include lines 20, 24, 28, 35-36, 49, 51, 60-70, 74,78, 103, 105, 123, 324, 327, 332, 339, 345, 385, 433, 437,443, 682, 689, 696, 703, 708. Phrasing employed and sentence construction in the following lines makes the comprehension difficult. 16-18, 51-53, 80-82, 336-337, 341-343,367-372, 389-391, 422-425,443-445. - The structure of the introduction as well as the background description does not clearly demonstrate how the work fits into the broader field of knowledge. Moreover the literature referring specifically to the field of study is limited and should be further developed to include references related to various aquaculture species nutritional requirements. - The structure of the article is conform to an acceptable format of ‘standard sections’ - Figures are relevant to the content of the article, of sufficient resolution, and appropriately described and labeled. - The raw data provided does not appear as machine-readable format as it appears as table and figure. Experimental design: - The manuscripts describes original primary research within Aims and Scope of the journal. - The research hypothesis is relevant & meaningful for the development of sustainable aquaculture practices, however it is not clearly defined and does not clearly state the aims to fill a given knowledge gap. - Ethic statement is not provided and might not be applicable considering the field of study. - The biochemical analysis and statistical analyses methods are clearly described, however the general experimental design is questionable to reach the proposed conclusions taking into account the restricted sampling period (1 week) and the number of replicates (n=2). Validity of the findings: - The data on which the conclusions are based is robust and statistically sound within the restricted limits of the study, however it can be considered incomplete to reach the proposed conclusions given the limited time during which the samples were collected. Considering the recognized effects that environmental parameters and seasons have on the characteristics and specificity of the species studied, analysis of various replicates at different sampling periods would be required to support discussion, conclusions and recommendations about the species to be intensively cultures as alternative feed sources. - Part of the discussion focusing on the general field of study is limited and should be further developed to illustrate diverse potentials of alternative sustainable feed sources for aquaculture production. -Generally the discussion is referring to studies performed out of the scope of the field of study of the research undertaken making the global comprehension difficult. - Part of the discussion referring to IMTA is not plainly justified considering that it is not clearly stated that the experimental samples were collected within IMTA systems. - L 311: Please indicate the larval species the text is referring to. - L 362-363: Please provide the references. Additional comments: The manuscript is of interest considering the quest to provide sustainable alternative feed sources for the further development of aquaculture, however it requires significant major revisions and various points (indicated in the review) which should be improved upon before acceptance.

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.

Title: CRUSTACEAN AMPHIPODS FROM MARSH PONDS: A NUTRITIOUS FEED RESOURCE WITH POTENTIAL FOR APPLICATION IN INTEGRATED MULTI-TROPHIC AQUACULTURE Review round: 1 Reviewer: 2

Basic reporting: The article is in general well written. Experimental design: The mean values are a result of only two measurements (n=2). However since there are no experimental treatments (this is just a decription of the composition), and each sample consists of many individuals I consider this acceptable. Validity of the findings: The authors present the idea as a novelty, however this is not new. In pond culture (often freshwater), this is an often used technique in extensive culture systems. Whether this will be a realistic scenario for large scale production remains to be seen however that is not in the scope of this article. The data seems to be robust and is discussed in the light of previous studies. The PCA plots are mentioned in the results, however do not come back in the discussion. I do not see the clear purpose of these plots and suggest to only include the tables. Additional comments: Major comments: The authors use the terms ‘trace/major metals’, ‘trace elements and metals’ trace and major elements’, etc. I would recommend to use elements for both the trace and the major ones. Many of the elements mentioned are not metals. The authors describe that the prey collected in these ponds are lower in heavy metals than prey caught in other locations. The abundance of Ulva sp. could be a reason for this as this green algae is known to readily accumulate heavy metals and thus might lower the concentration in the water. (This is just a comment and does not have to be included in the article). Minor comments: Keywords I am not sure what ‘major metal’ as a keyword means Abstract Line 33: change ‘alternatives preys’ into ‘alternative prey’ Line 38: Calcium should not be written with a capital, just ‘calcium’ Introduction Line 51: change ‘Sothern’ into ‘Southern’ Line 62: change ‘optimum’ into ‘optimal’ Line 62: remove ‘high’ Line 75: ‘depends on limited and overexploited fisheries’. The fisheries is finite (limited) but it is not reasonable to call all the fisheries overexploited. Moreover, a substantial part of the fish meal and fish oil is now coming from fish slaughter waste. Line 94-96: Furthermore, ….in aquaculture. In this senctence ‘elements’ would be a better word than ‘metals’. Also, the suitability of the prey is not evaluated, it is their nutritional composition that is evaluated. Please change. M&M Line 123: I presume the samples were weighed before and after burning so include ‘gravimetrically’ before determined. Statistical analysis: Lines 190-191: Sentence can be taken away Discussion The discussion starts with the argument of overfishing, but that FAO report does not distinguish between fisheries for human consumption and for fish meal production. The argument of fisheries having (over)reached its limits and fish meal being a finite resource, meaning that if we want to increase the fish feed production we have to find alternatives makes a scientifically more correct argument. Lastly, the authors suggest this as a possibility for extensive fish farming. It would be nice to see a few lines on the possible environmental impacts of this type of farming when it will find a market since it will put more pressure on the marshlands (or not?).

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.

Title: A NEW SMALL-BODIED ORNITHOPOD (DINOSAURIA, ORNITHISCHIA) FROM A DEEP, HIGH-ENERGY EARLY CRETACEOUS RIVER OF THE AUSTRALIAN–ANTARCTIC RIFT SYSTEM Review round: 1 Reviewer: 1

Basic reporting: The ms is well writen and provides good new and relevant information on both the geology of the site and anatomical characteristics of the specimens. The ms is supported by relevant and complete references from the available literature. I would recommend further elaboration and discussion about the phylogeny of the new taxon. Figures of osteological elements should have the same standarized scale bars in all of them. I recommend using a single bar, without subdivisions, indicating the scale (1 mm, 1 cm, 10 cm, etc). Experimental design: The new taxon should be supported within a phylogenetic context. There are several data matrices that the authors can use. The diagnosis based on authapomorpies is ok but it is important that combination of other characters be supported by a phylogenetical analysis. Validity of the findings: As said above, a phylogenetic analysis of the new form within the Ornithopoda clade is mandatory. Some of the putative autapomorphies proposed by the authors could be unveiled as homoplasies or ambiguously distributed features. Authors need to provide data sets of characters, with the new taxon properly scored, and a posible phylogenetical position of it. Additional comments: The ms is an important contribution about the poorly known ornithopod diversity from Australia. I encourage the authors to work a little deeper on the phylogeny of the new taxon.

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.

Title: A NEW SMALL-BODIED ORNITHOPOD (DINOSAURIA, ORNITHISCHIA) FROM A DEEP, HIGH-ENERGY EARLY CRETACEOUS RIVER OF THE AUSTRALIAN–ANTARCTIC RIFT SYSTEM Review round: 1 Reviewer: 2

Basic reporting: The article is clearly written, professional and unambiguous it its language. The introduction and context for the article are sufficiently developed, and the appropriate literature is cited. The body of the description is very thorough, and the quality and number of figures exceeds normal requirements. The measurement raw data are shared (when possible), with exceptions where ratios are provided without the raw data. The CT scan data/3D models do not appear to be included in the submission. The paper is self-contained and does not contain extraneous information. Hypotheses are not clearly laid out or tested, but given that it is a taxonomic description this is not expected. Experimental design: This represented original research, and falls within the Aims and Scope of the journal. The research questions are defined and of interest to the pertinent scientific community. The investigations performed were thorough and rigorous. The anatomical description is very detailed and of high technical standard. There are minor issues with the limited quantitative analyses performed. It would have been helpful to see the specimen/taxon placed in a cladistic context, and while this is not required its absence should at least be explained/justified. For the most part, documentation of the mythologies is sufficient for replication (and I was able to replicate several of the results), but more details on CT scanning an measurements would be useful. Validity of the findings: The conclusions are clearly stated and backed up by the body of the paper. The data presented are accurate (with a few minor exceptions). Speculations do not over extend past those supported by the data. Additional comments: General comments: This is a very thorough anatomical treatment of interesting small-bodied ornithischian material from the Early Cretaceous of Australia. The anatomical description is very thorough, and I was surprised by the amount of description given the limited nature of the specimen. I am very happy with the separation of the description from the comparison (although this slips up in a few places - especially the end). The number and quality of the illustrations is excellent. It is a good contribution to the field and will be a very useful paper and resource. While I do not doubt that this is a distinct and diagnosable taxon, I do have some concerns over the diagnosis. There are a large number of proposed autapomorphies for such limited material, and for material that is often not regarded are being overly diagnostic for related taxa. While some of these are autapomorphies, and many of them are strong and likely to be robust in future analyses (e.g., 2, 3, 7), the bulk of these features are not apomorphic. For these other features, a list of taxa that share this feature should be provided, and would be greatly beneficial. Some proposed autapomorphies (e.g., 4) are actually several features that are grouped together to form and ‘autapomorphy’. Rather than one large list, it would be more useful to break it down into two categories: firstly proposed autapomorphies and secondly other characters which can be used to differentiate from taxon XX. I do prefer the discrete autapomorphies over those based on relative proportions, especially when considering the effect of size/growth on these features. While I appreciate the use of quantitative data to show differences in proportions between taxa (Figure 28, S5, S7, S8) I do not believe the simple comparisons of ratios is the most useful test. A ratio has two components, the numerator and denominator, but when two ratios are different you do not know if that is due to a difference in the numerator or denominator (or both). Likewise when two ratios are the same, you have no context of the absolute size of the elements. Since you need both the numerator and denominator to calculate a ratio, it is far more informative to simply plot these against each other in the graph. If you log both axes, any line with a slope is 1 represents a constant ratio. To illustrate this, I have taken the raw data (where available) from Table S3, and quickly plotted it up as series of ratios, and also in a bloxplot (colour-coded for taxa) (see attached). This allows for an examination of relative size (position along a line of slope = -1) and absolute size (position along a line of slope = 1) to be observed at the same time(I realized I switched the axes, but the point is the same). All points falling along the diagonal lines (slope of 1) in the figure will have the same ratio. Boiling down a biplot of numerator/denominator sets into a series of ratios is the mathematical equivalent of projecting the 2D data of a graph onto a 1D line (specifically a line with a slope of -1). While I appreciate the need to reduce the number of axes when that number is greater than three, our minds are very good at teasing out relationship in 2D space, and there is no reason to simplify these data into 1D space. Doing so removes a lot of the useful data, specifically absolute size, and which of the ratio components is changing. That being said, I do not this that this is a huge issue with the paper, it is just a better way to present the data, and can hopefully be incorporated. The original data for the described specimen are provided (Tables 2-5) which is very good. Some of the data for the other taxa used for comparisons are presented (Table S2, S3, S4) which is great, but the majority of the data presented are not the raw data, but the ratio between two metrics. The raw data cannot be back-calculated from these ratios, and (as argued above) ratios have limited use compared to plotting up the raw data. Including the raw would both allow for better current analyses, and will serve the science better moving forward. There are several errors in the tables, which are either math errors or typos (I can’t tell which). For example, the ratios for two of the seven taxa (Mattaburasaurus and Parksosaurus) in table S2 cannot be obtained from the supplied raw data. I only really checked out this table (because it has the most raw data), but a thorough check of the remaining math should be done. The lack of cladistics analysis is confusing. I am not suggesting that one should always be required when a new taxon is described - there are several reasons why this may not be the case. However, if one is discussing potential relationships of the new animal in both the abstract and conclusion, it is reasonable to expect a cladistics framework, or a justification as to why this was not done. In this case I suspect the analysis will result in a low resolution, large polytomy, due to the nature of the material and poor character scoring in these anatomic regions in related taxa. If this is the case, explain why and it may help convince others to look for characters in these areas. If one is planned, indicate that as well. As far as I can tell the CT data and/or resulting 3D digital models are not included in the supplementary materials. Inclusion of these data may be required to be consistent with PeerJ data sharing policies. I will leave that decision to the editor. All in all, a great paper about an exciting specimen/taxon. I am looking forward to seeing it published. Specific comments: Throughout the document “zygopophysis”/”zygapophysis” are used seemingly interchangeably. There may a reason for this, but if not, use a consistent spelling. Line 85 – “hyperextended” is this mean to indicate ‘elongate’? Line 97 – the first usage of “Eric the Red West” should include the abbreviation ‘ETRW’ that is used in the rest of the paper Line 130 - One minor omission to the methods is the details as to how the measurements were obtained. Where they obtained by calipers, tape, or digitally from the CT scans? Line 135 – What voltage and amperage were the CT scans performed at? Line 147 – the 11% intervertebral gap that was added needs more explanation. Presumably this was added to more accurate reconstruct the length lost due to intervertebral disks? Since the tail is still largely articulated, I have a hard time understanding how the disk lengths could be lost while retaining articulation (since the soft tissue is what is holding it all together). Line 397 – It is unclear what is meant by a “linear” spinal process. Are these not all linear? Does this mean thin? Straight? Of constant thickness? Line 445-446 – There is an apparent conflict. The first line suggests that the sutures are difficult to distinguish past Ca8, yet the second line discusses the position of the sutures on Ca10 and ca13? Line 471 – “The centrum OF the referred…” Line 487 – “… the anterior and posterior margins of the spinal processes are parallel AND OF CONSTANT ANTEROPOSTERIOR WIDTH…..” This this is intended meaning? If so it should be clarified more. Line 488 – I do not understand the intended meaning of “The dorsal tips of these spinal processes are convex and their ventral tips, angular” Line 490 – “Proximately narrow” do you mean “proximally constricted” Line 501 – “linear” is this meant to indicate straight? Line 512 – I think the incorrect figure is referred to here. Do you mean Figure 12? Line 514/5 – Here is the first comparison in a so far comparison free description. Line 565/5 – Other comparisons. I am not overly bothered by this, but is does break down the nice discrete sections you have established. Line 599 – The term “restoration” is unclear. Would “digital 3D model” or similar work better? Line 630 – “The distal (‘dorsal’) margin is obtuse” I am unclear what this is trying to say. How can the distal margin of the tarsal element also be the dorsal margin? Line 681 – It is unclear what is meant by “less-derived ornithopods”. This is a vague and imprecise term. Do you mean ‘non-iguanodontian ornithopods”? Line 819 - “length centrum” should be “centrum length” Line 857 and 861 – “Parksosaurus warreni” Line 865 – “C. browni”should be italicised. Line 984 – two successive periods Line 1193 – It is unclear what is meant by “linear along their length”. Does this mean straight? Line 1275 – It may be helpful to end this section with a statement similar to… “Future cladistic analyses may help to test these hypotheses of relationships. Line 1334 – The Anderson et al 1985 is a bit out of date. It may be good to also refer to the updated Campione et al 2014 (Meth in Ecol Evol) paper. Figures 9, 10, 20, 21, 22, 23, 24, 25, 26, 27, 31A , 33 – Rather than simply describe these as “virtual” I would suggest “Digital 3D models” or similar. It is just a more specific description of what they are. Figure 16 – Label “C” is in the wrong position. I think most people will be able to figure out that it pertains to the image on the left, but an arrow may help.

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.

Title: A NEW SMALL-BODIED ORNITHOPOD (DINOSAURIA, ORNITHISCHIA) FROM A DEEP, HIGH-ENERGY EARLY CRETACEOUS RIVER OF THE AUSTRALIAN–ANTARCTIC RIFT SYSTEM Review round: 1 Reviewer: 3

Basic reporting: no comment Experimental design: no comment Validity of the findings: A few speculative statements in the anatomical descriptions should be moved to the discussion or removed. These are discussed in my comments below. Additional comments: This paper presents a thorough description of a specimen that is important in understanding the taxonomy of Australian ornithopods. I particularly appreciate the thorough description of the geological context (at both a macro scale and at the quarry level), and the many figures showing the details of the anatomy of this specimen. The descriptions of the anatomy would be improved by remaining focused on features that can be directly observed and refraining from speculation about things like the location of muscles. This can be included in the discussion section of the paper, but clutters the description section. There are also some features that are discussed, notably the tprl and the abductor groove on MT II, that are not clear in the figures. The models created from CT scans have low resolution, and while these are still useful in showing certain features not visible in the photographs, small features are not clear in these images. There are still clear autapormorphies of this specimen that can be discussed: I would advise emphasizing these to clean up the description section and make it more focused. Additionally, lists of proportions can clutter a description with superfluous information. An example here, from the description of pedal digit I-1: “The proximodistal length of pd I-1 is 47% that of mt I and 27% that of mt III. The transverse width of pd I-1 at its proximal end is 56% that of proximal pd II-1 and the dorsoplantar depth, 50% that of proximal pd II-1.” These measurements are all available in Table 5—unless a particular proportion is something that is diagnostic or will be highlighted in the discussion in comparison to other taxa, it need not be listed in the written description. Based on the evidence presented, I’m unconvinced by the pathology section. I would want to see either close-up photos or histological sections of the affected areas—this is something where surface texture is important, so CT imagery is not a useful figure to demonstrate this. In short, I would like to see a more convincing figure demonstrating the pathology. Otherwise, I would simply remove this section. In terms of phylogeny, it would be worth mentioning or considering the analysis of Boyd (2015), in which many of the taxa referred to in this text as basal ornithopods are in fact recovered as basal neornithischians outside of Ornithopoda. While you can certainly disagree with those findings and continue referring to these taxa as basal ornithopods, this alternative phylogeny should at least be mentioned. On the topic of terminology, there is a mixture in this manuscript of the terms anterior/posterior and cranial/caudal. While either set of terms is fine, please pick one and stick with it for consistency and clarity. There also seems to be no abbreviation of genus names—after the first use of a binomial, the genus can be abbreviated. When multiple references are cited, they should be put in chronological order. Again, I applaud the authors’ thoroughness in describing and illustrating this specimen, and I look forward to the publication of the manuscript. Following is a list of specific comments and suggestions for improving the manuscript. Attached is a pdf containing further edits—these are simply minor grammar/spelling/punctuation errors.   Line 97: insert (ETRW) after first use of the full name “Eric the Red West” Line 269-78: The Interpretation of the Anchor Sandstone says paleosol deposits overlie the lake deposits, but the caption of figure 5A says the Anchor Ss has “shallow lacustrine at top”. Line 384: I realize this is a diagnosis, not a phylogenetic systematic analysis, but I think that breaking features down in a systematic way can be instructive. Features 4 and 5 are each three different features, with some overlap between them. Feature 4 deals with (a) shape of the spinal process, (b) length of the spinal process, and (c) orientation of the spinal process. If each of these features are unique and useful in diagnosing Diluvicursor, they should be listed separately. Feature 5 is about the height of the neural arch, which is tightly tied to length of the neural spine already covered in feature 4. You compare it here to three different measurements, but each of these can also vary between species (thus, they are separate characters). I would stick to one reference point, probably centrum height, as a comparison. Comparing the height of the neural arch to the length of the transverse process is a different feature, since that number is highly dependent on the length of the transverse processes (which is covered in feature 6). There are similar issues with feature 11. Line 398-9: You need to be more specific in your shape descriptions. What, precisely, is the difference between “boot-shaped” and “hatchet-shaped”? Line 578-82: The terms “hatchet-shaped” and “boot-shaped” aren’t defined here. It would be helpful to describe the discrete features here: are the proximal and distal edges of the hemal arch parallel? Does either edge diverge from the other? Is it symmetric or asymmetric? These are much more useful descriptions. Things like hatched-shaped and boot-shaped are fine as short-hand descriptions for continued discussion of the features, but they need to be defined in a more rigorous way at some point in the manuscript. Line 613: “The fibula is anterolaterally positioned relative to the tibia.” But you note later that the fibula is displaced. Because of that, I would avoid discussing the relative positions of the tibia and fibula, unless there is other evidence of their anatomical relationship. Line 614: You refer to a condyle on the fibula, but previously discussed malleoli on the tibia. If using the term malleolus, you should use this for the lateral malleolus of the fibula as well. Line 630: “The distal (‘dorsal’) margin…” I don’t understand how a feature on the astragalus is both distal and dorsal. Inserting a sentence at the beginning of the description explaining the orientation you will use for your description of tarsal/metatarsal/phalangeal elements would be useful. Line 656-7: “The metatarsus is compact, elongate and roughly cylindrical in shape.” I would describe this as an elliptical cylinder, as the cross section is oblong. Line 660-5: This could all be shortened to something like: “Despite diagenetically imposed imbrication of the metatarsals, all elements are here described in life position.” Line 675-7: “The M. extensor hallicus longus (e.g., White et al., 2016) that would have located within the extensor groove on the distal condyle, likely extended proximally along the medial margin of mt I and mt II.” This seems entirely speculative—I’m not sure what value it adds to the description. Line 681-2: “…as in less-derived ornithopods”. Less-derived than what? I would use the term “basal ornithopods” here, or more technically, “basally-branching ornithopods”. But also see Boyd 2015: some of these taxa may not be ornithopods, and are better referred to as basally-branching neornithischians. Line 728: “Adductor and abductor tendons could also have located within the medial and lateral grooves” (of mt III). This is rather speculative. In addition, interior digits are abducted and adducted by interosseous muscles and lumbricals. While these could be termed “abductors” and “adductors” based on function, the muscles themselves are not generally referred to this way. Line 754-5: “The plantar portion of the cotyle is split in the axial direction (Figs 19A, 23B), which could be pathological.” Why not taphonomic? Is there any evidence either way? Line 769-70: “Placement of Diluvicursor pickeringi in the Ornithischia is supported by the combination of a distally tapering mt IV…” Is this supposed to be distally curving? I don’t really see (nor is there a description of) it tapering. Line 796-7: “The triangular intervertebral processes developed on the posterior-most caudal vertebrae of Diluvicursor pickeringi (Fig. 17) are similarly unique.” I’ve actually seen something similar in Gasparinisaura, specimen MCS-Pv 001 at Museo Cinco Saltos, which has a nearly complete and articulated tail. The intervertebral process is only present on two or three vertebrae, but they are quite similar to what is shown in this paper. To my knowledge, this specimen hasn’t yet been described, so I’ll leave it to the authors’ discretion whether to make this comparison or not, but I thought you might like to know about it—it certainly strengthens the case for a close relationship between Australian and South American ornithopods. (The ventral grooves on posterior caudal vertebrae are also present in this specimen.) 831,3,5: Spell out the full names of the lamina, at least the first place they are mentioned in the description, rather than sprl, tprl, and prsl. 839-41: “A prominent prsl on the caudal vertebrae of theropods, such as the abelisaurid Majungasaurus crenatissimus (O'Connor, 2007), suggests this feature is also plesiomorphic in dinosaurs, although variably expressed among taxa.” Or it arises frequently due to parallelism. Either way, it’s not diagnostic. 847-55: Again, I’ll refer to the undescribed specimen of Gasparinisaura MCS-Pv 001. While it has some hemal arches with rounded distal ends, just two vertebrae distal to this and still within the middle caudals are hemal arches with asymmetric triangular ends similar to those seen in NMV P185992/NMV P185993. It is important to remember in these discussions that the shape of hemal arches changes quite drastically based on position even within middle caudal vertebrae of one individual. Drawing conclusions about taxonomy or systematics based on these bones is dangerous unless comparisons can be made between fairly complete series. 885-8: “…suggesting that the position designated Ca 3 in Diluvicursor pickeringi is close to correct. It is reasonable to suggest that up to four caudal vertebrae could have been present on the Diluvicursor pickeringi holotype anterior to that designated Ca 1.” Based on what? How did you arrive at the number four? 968-70: “Similar features of the hallux in the early ornithischian Lesothosaurus diagnosticus (Thulborn, 1972), suggests this condition may be plesiomorphic for ornithopods.” While it’s true that Lesothosaurus has an “early” temporal range, in the context of describing its features as plesiomorphic, the better term here would be “basal” or “basally branching”. 970: “However, the halluces of the early ornithischians…” see above. 973-4: “indicating that the plesiomorphic condition of the hallux in ornithischians is presently not understood.” Well, what about outgroups? What would they indicate? 996-7: “…within a dorsolaterally oriented extensor groove (Figs 31A–B, S6E–F).” Figure 31 does not show an extensor groove on any of the specimens—or at least nothing is labelled as such. Figure 24A and 24F show the extensor groove clearly for D. pickeringi. 1020: “coerzeei” Should be coetzeei 1028: “Anti-mortem”: I think you mean antemortem? 1152-4: “a protuberance is also developed on each of the paired spinoprezygapophyseal lamina (sprl), between which, the transprezygapophyseal lamina (tprl) extends dorsally to both the neural canal and the prezygapophyses”. I don’t understand this description. The protuberances on the sprls appear to be dorsal to the prezygopophyses. How does the tprl then extend dorsally from the protuberance of the sprl to the prezygopophyses? 1267: “coerzeei” Should be coetzeei 1384-9: “Investigations on disparity in dental and cranial features between both co-occurring dinosaurian and ancient mammalian herbivores have addressed questions of habitat preferences within these groups…. However, the palaeoecological implications of cranial, dental and postcranial disparity between small-bodied ornithopods have yet to be investigated.” You say these disparities have been investigated in dinosaurs previously, but not in ornithopods. Maybe the first sentence should specify “some dinosaur groups” or “non-ornithopod dinosaurs”. Figure 1: The color of water in figure 1C is very similar to that of land in figure 1A, and the background land color in 1C matches the water in 1B. This makes it difficult, at first glance, to discern where the coastline is in 1C. It’s overall a lovely figure, but consider making the color schemes consistent across all parts. Figure 5A: I’m a little confused by the parenthetical “(top only)” in the description of the Anchor Sandstone. The description says it is a channel deposit with shallow lacustrine sediments at the top. But you have more than the shallow lacustrine sediments in your section. If you are trying to indicate that the site doesn’t include the full thickness of the Anchor Sandstone, this could be indicated by dashed arrows continuing downward in the schematic cross section. 5B: Just for the sake of clarity, it might be useful to change “intertidal zone” to “modern intertidal zone”, and “shore platform” to “modern shore platform”. Figs 9, 10, 20: Perhaps this is a difference in Australian vs. American English, but describing a vertebra as “virtual” sounds to me as if there is something reconstructed or not quite real about it. I would describe this as a three-dimensional model or maybe “CT imaging of anterior caudal vertebra”. Figure 10: “tprl, intraprezygapophyseal lamina” should be transprezygopophyseal lamina to match descriptions in the text. Figure 19: The description of the views would be better if the elements were indicated, e.g., 19A: Tibia, fibula, and tarsals in anterior view, pes in plantomedial view. I would also argue that the tarsals seen in 19B are in something closer to a distal view than anterior. Figure 20: “pd #, pedal digit number and phalanx position”—since you only point out metatarsals in this figure, why not use something like “mt” (or at least shorten the description to “pedal digit number”). Figure 24: It would be nice if the parts of this figure were realigned so that MT I and its phalanges were aligned. They can remain as separately labelled parts of the figure, but aligning them such that a lateral view of MT I is next to a lateral view of the phalanges would make their relationship easier to understand. Figure 30C: why is the schematic reflected while the photograph is not? If the purpose is to compare with the right limb of Diluvicursor, just reflect both the photo and schematic. Figure 31: what view are these seen in? Figure 33: As mentioned above, the CT imaging is not the best way to demonstrate pathologies. Consider a close-up photograph here instead. Figure 35: “Extent of viscera (brown shading) ventral to hypaxial musculature (dashed lines) not shown.” If the cloaca extends this far posteriorly, it would be located more medially then ventrally (for a nice illustration of this in Alligator, see Mallison et al. 2015). Honestly, I think you could ignore viscera, since this is such a schematic view, and since this isn’t relevant for most of the tail. However, if you want to include viscera, they should be properly placed medial to the left and right hypaxial muscles. Table 1: What sources are you following for this nomenclature? What do the asterisks mean? Refernces Boyd, C. A. (2015). The systematic relationships and biogeographic history of ornithischian dinosaurs. PeerJ, 3, e1523. Mallison, Heinrich & Pittman, Michael & Schwarz, Daniela. (2015). PrePrint Version: Using crocodilian tails as models for dinosaur tails. PeerJ PrePrints. 3. e1653. 10.7287/peerj.preprints.1339v1.

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.

Title: POSITIVE SELECTION ON HUMAN GAMETE-RECOGNITION GENES Review round: 1 Reviewer: 1

Basic reporting: . Experimental design: . Validity of the findings: . Additional comments: I have read the paper by Hart et al. The authors have tried to identify specific sites under selection that could account for previously discovered linkage disequilibrium (LD) between human C4BPA and ZP3 and extend the analyses to include human ZP2. I find that the paper is interesting and it is generally well-written. However, I have several comments which can help improve the paper. Why rs2286428 and rs200481427 are no in Table 1? Page 14, line 293, middle, Table 2 should be Table 1. Page 15, bottom paragraph, the authors list the test methods for selection, but only Vitti, Grossman & Sebati 2013 is referred. To me, the references of Tajima’s D, the composite likelihood ratio test (CLR), and Fay and Wu’s H, as well as xp-EHH should be given too. Although Vitti, Grossman & Sebati 2013 was published in 2013, it missed the latest method EHHST and xp-EHHST developed in 1. Zhong M, Zhang YW, Lange K, and Fan RZ (2011) A cross-population extended haplotypebased homozygosity score test to detect positive selection in genome-wide scans. Statistics and Its Interface 4:51-63. 2. Zhong M, Lange K, Papp JC, and Fan RZ (2010) Extended homozygosity score tests to detect positive selection in genome-wide scans. European Journal of Human Genetics 18:1148-1159. EHHST and xp-EHHST has a better feature of easy to calculate and easy to interpret results than xpEHH. Hence, the authors should add the results of these better methods to Table 3. The related software can be found on https://sites.google.com/a/georgetown.edu/ruzong-fan/about The plots of Figure 3 are not clear to me. I wonder if the mean or median allele numbers can be provided to make it easy to understand. In Table 3, the p-values should be provided in addition to the test statistic values. Minor issues: in references, there are some problems. For example, the tauthors of following ref Wang H, Choi Y, Bamidele T, Xuefeng W, Morris N, Zhang X, Broeckel U, Hanis C, Iardia S, Redline S, Cooper RS, Tang H, Zhu X. 2017. Genome-wide survey in African Americans demonstrates potential epistasis of fitness in the human genome. Genetic Epidemiology 958 41:122–135. should be: Wang H, Choi Y, Tayo B, Wang X, Morris N, Zhang X, Broeckel U, Hanis C, Kardia S, …

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.

Title: POSITIVE SELECTION ON HUMAN GAMETE-RECOGNITION GENES Review round: 1 Reviewer: 2

Basic reporting: Overall, this report reads well with professional English, well thought out hypothesis and supporting evidence for them. Although I am not entirely familiar with the field, the included references appear to be correct and overall attribution seems appropriate. My only suggestion is to include significance values in Fig. 3 and to more clearly delineate their findings from previous reports, especially in the discussion. Experimental design: No comment Validity of the findings: No comment Additional comments: No additional comments

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.

Title: POSITIVE SELECTION ON HUMAN GAMETE-RECOGNITION GENES Review round: 1 Reviewer: 3

Basic reporting: Overall well written and referenced, see general comments for some specific suggestions for improvement. It would be helpful to provide a link in the fertility data supplementary file to the location of the previously published values for individual families, on which the summary stats are based. Experimental design: Questions are clearly defined and methods appropriate. See suggestions for some additional statistical tests in general comments. I particularly appreciate the acknowledgment of 'researcher degrees of freedom' and possibility for inflated false positives. The authors do a nice job clearly showing the 'story' of their study. Validity of the findings: The authors are clear about the relative robustness of their findings and which should be treated with caution. Additional comments: Hart and colleagues investigate evolution of egg and sperm binding proteins in humans. They test for evidence of positive selection on two egg coat proteins ZP2 and ZP3, consistent with findings reported in other mammals. Furthermore, they test for positive selection on C4BPA the sperm protein which is the putative human homolog of mouse Zp3r, which binds to the egg coat. They find evidence for positive selection on one or more amino acid sites in each of these proteins, but not in the egg coat protein ZP1. Moreover, they report associations between the candidate selected sites in ZP2 and C4BPA with fertility in a Hutterite population. This is an interesting study testing implications of the pattern of rapid adaptive evolution of fertilization proteins in mammals (and other taxa) - usually viewed in the context of reproductive isolation and speciation - for reproductive compatibility within humans. The authors do a nice job explaining the limitations of the methods employed for within species data, and are clear that their findings are suggestive and should be treated with caution - but are exciting leads for future research. 1. I found the manuscript well written and easy to follow, with the exception of the abstract - the framing of the question was somewhat muddled. I suggest rewriting the beginning of the abstract and streamlining the description of results. For example the intro focuses on co-evolution between egg and sperm proteins, but that's not clear from the abstract - which starts focused on egg proteins. 2. population genetics analysis A. it is unclear to me why it was not possible to perform analyses on the ZP3 site using downloaded variable sites. Most/all of the test statistics can by computed using other programs or calculated directly. Was the author/administrator of the 1000 genomes selection browser contacted to correct the error in order to run analysis? Please include analysis for ZP3 using other software (at least Fst, TajD, Fay & Wu's H, CLR) or explain in more detail why this wasn't possible. B. I suggest adding McDonald-Kreitman (Nature 1991) tests. While conservative, this test is commonly used to distinguish high amino acid polymorphism due to balancing selection vs. relaxed purifying selection. As other great ape sequences were used to identify ancestral vs. derived mutations, appropriate outgroup sequences are available for MK. πR and πS (replacement and silent diversity) values should be reported for each gene. C. It would be useful to add popgen tests for the combined sample, in addition to individual populations, to evaluate the hypothesis that divergent alleles are maintained over time in humans. (although I agree with argument from L379 that this may be problematic for LD-based tests) D. Fst is not a test for selection - change in description of tests and results (beginning L348) to indicate evidence is only for population differentiation not selection 3. positive vs. diversifying vs. balancing selection - Unfortunately, this terminology is not used consistently among different people in the field and some clarification is needed here. It seems in the MEME paper, "diversifying" and "positive" selection are used interchangeably (and for interspecific comparisons). Diversifying sensu Murrell et al 2012 is not the same as balancing selection, but it seems in this manuscript the authors suggest that significant results in MEME are evidence for balancing selection. Be careful to distinguish exactly what the test shows - i.e. rapid & adaptive amino acid substitution at a site - particularly for this intraspecific analysis. - Title, L13, L16 - change 'selection' to 'positive selection' Add haplotype tables including variable amino acid sites for each gene as supplementary. Table 2 - add #s of nonsynonymous and syn polymorphisms for each gene. Minor comments: L16 - there have been many papers on sperm proteins too - e.g. reviewed in Turner & Hoekstra IJDB 2008 L18 - change to "selection the human egg-coat genes ZP3 and ZP2. We also found evidence for positive selection on C4BPA, which encodes a repetitive..." L22 "Several additional lines..." this sentence is confusing right after saying no selection on ZP1 - re-word L55 - is this the right reference? title still calls it sp56 L140 "one candidate site under diversifying selection" to "one candidate site each under" L178 - caveats about applying these models within species are explained clearly later in the manuscript. It would be helpful to either move them here or add "(but see caveats below)" or similar. L706 could add mention of parallel amino acid changes at same sites in ZP3 in multiple rodent taxa and mammals, reviewed in Turner & Hoekstra IJDB 2008 Fig 1 - add x axis with aa positions Fig 2 - add colors to indicate % alleles from different populations Fig 3 - smaller, jittered points; add P vals to graph or legend

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.

Title: POSITIVE SELECTION ON HUMAN GAMETE-RECOGNITION GENES Review round: 2 Reviewer: 1

Basic reporting: good Experimental design: good Validity of the findings: good Additional comments: I am satisfied by the changes made in response to my comments and those of other reviewers, or the clear and thorough explanations why they were not possible/advisable. I appreciate in particular the extra work necessary to run selscan to run haplotype tests for ZP3.

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.

Title: A STREPTOMYCIN RESISTANCE MARKER IN H. PARASUIS BASED ON SITE-DIRECTED MUTATIONS IN RPSL GENE TO PERFORM UNMARKED IN-FRAME MUTATIONS AND TO VERIFY NATURAL TRANSFORMATION Review round: 1 Reviewer: 1

Basic reporting: The paper is not organized well. For example, there is no connection between section "3.4" and "3.3". Experimental design: The experimental design is strange. I don't know why the author using EMS to induce mutation. In addition, the author did not tell us how many mutant they have gotten and what are they. Validity of the findings: The authors claimed that they have developed a new novel development of EMS-induced SM-resistant method. But I do not think so. Additional comments: Some experiment details should be described in the methods section instead of in the results section.

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.

Title: A STREPTOMYCIN RESISTANCE MARKER IN H. PARASUIS BASED ON SITE-DIRECTED MUTATIONS IN RPSL GENE TO PERFORM UNMARKED IN-FRAME MUTATIONS AND TO VERIFY NATURAL TRANSFORMATION Review round: 1 Reviewer: 2

Basic reporting: 1 Writing of a scientific article should be concise and clear. However, the logic of this paper is vague. Numerous grammatical errors also hinder understanding of the paper. 2 Lines 50-52, “A prerequisite for this process includes a marker-containing strain (Reyrat et al., 1998); a streptomycin resistance marker based on rpsL or rrs mutations is one of the most frequently used phenotypes (Tsai et al., 2014).” The authors mixed up the concepts of positive selection marker and counterselectable marker. Streptomycin sensitivity is one of the most-used counterselectable markers in allelic exchange as described in the papers that the authors cited. To this end, a streptomycin-resistant strain and a suicide plasmid containing a wild-type rpsL gene are required. When the suicide plasmid is used to deliver an inactivated allele of the target gene in the chromosome, expression of the wild-type rpsL gene on the integrated plasmid confers a streptomycin-sensitive phenotype upon streptomycin-resistant strains and allows for selection with streptomycin to detect loss of the vector. However, in this study, the authors used sucrose sensitivity, which is conferred by the sacB gene on the suicide plasmid pK18mobSacB, instead of streptomycin sensitivity in the selection of the deletion mutant (lines184-201). Streptomycin-resistance only served as a positive selection marker for the wild type strain. In fact, any antibiotic to which the SC1401 strain is resistant but E. coli S17-1 is sensitive can serve as a positive selection. Did the authors analyze and compare drug resistance profiles of both strains before starting the study? Experimental design: 1 Lines 168-170, “ The cells were screened by TSA++ containing 25 μg ml-1 of SM. The visible single colonies were propagated and the DNA extracted and sequenced for further identification.” Did the authors identify the mutants with genomic DNA sequencing?!! The corresponding data were not presented in the section of Result. In this experiment, streptomycin selection is not enough for allelic exchange because the frequency of double crossover events may be low and illegitimate recombination may occur. Addition of 10% sucrose to the selection medium is appropriate. 2 Lines 221-222, “The visible single colonies were identified by PCR and western blotting.” Why and how did the authors identify the colonies with western blotting assay? Validity of the findings: 1 Lines 320-323, please describe the assay of Western blot in the Section of Material and Methods. 2 Lines 326-327, “Point mutations in rpsL which don't confer negative effect on growth were assumed to be more effective in verifying natural competence.” Did the authors compare the growth rates of the wild type and the mutants? 3 Lines 334-336, “Genomic DNA of HPS32 and HPS32 couldn’t confer SM-resistance to SC1401, which also in support of our elucidation highlighted in “Introduction” that spontaneous mutations in this species occur at a fairly low level.” Why? Is transformation the cause of spontaneous mutation? Additional comments: No additional comments

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.

Title: A STREPTOMYCIN RESISTANCE MARKER IN H. PARASUIS BASED ON SITE-DIRECTED MUTATIONS IN RPSL GENE TO PERFORM UNMARKED IN-FRAME MUTATIONS AND TO VERIFY NATURAL TRANSFORMATION Review round: 1 Reviewer: 3

Basic reporting: The level of English is acceptable. I found a few spelling errors and non-idiomatic expressions. The Introduction and background sections will have to be improved. The references from line 41,42 below should be substituted for the relevant original sources. 40 producing significant mortality and morbidity in pig farms and leading to serious economic losses 41 in the pork industry throughout the world (Yue et al., 2009;Zhang et al., 2016b). Structure conforms to PeerJ standards as far as I can judge. Abstract is within the specified limits. The manuscript has six figures which seem a bit excessive as I think the manuscript lacks an important figure. This figure should be a scheme that shows how the so called "in-frame" deletions happen. This figure should contain relevant plasmids and primers. I think figure 2 could be put among the suplemental figures to make room for this new figure. All relevant raw data was supplied. Experimental design is adequate and the findings are valid. # Specific line by line comments 27 Combined with natural transformation-based knockout system and this 28 genetic technique, multiple deletion mutants or attenuated strains of H. parasuis can be easily 29 constructed correct to: Combined with a natural transformation-based... --- 38 pleomorphic, NAD-dependent opportunistic bacterium Please explain what a NAD dependent bacteria is? Is it auxotrophic? --- 58 2011). To generate SM mutants by using correct to: 58 2011). To generate SM resistant mutants by using --- 61 et al., 2014). However, spontaneous or UV-induced mutants of streptomycin resistance in H. 62 parasuis probably occurs at a fairly low rate in H. parasuis; SM mutant generation in this organism 63 is very inefficient using these strategies (data not shown). correct to: 61 et al., 2014). However, spontaneous or UV-induced mutations leading to streptomycin resistance in H. 62 parasuis probably occurs at a fairly low rate; SM mutant generation in this organism 63 was very inefficient in our hands using these strategies (data not shown). The statement is too strong give the lack of experimental detail. --- The lines 65 - 85 do not belong in the materials section. Some should go to introduction and some to results. --- 90 S2. Plasmids were propagated in E. coli DH5α or S17-1 (λpir) and grown in liquid Luria-Bertani LB means Lysogeny Broth and NOT Luria-Bertani. --- 118 growth. Moreover, the MIC-S of EMS induced SC1401 derivatives were also determined using 119 above method. correct to: 118 growth. Moreover, the MIC-S of EMS induced SC1401 derivatives were also determined using 119 the above method. --- 138 Wild type SC1401 (deposited in GenBank under the accession NO. NZ_CP015099.1), which The strain was not deposited, what was depositied was the genomic sequence. --- The sections 2.7. Construction of Plasmids pkTLR and pkALR and 2.8. Construction of Unmarked In-Frame Targeted Mutant 1401D88ΔtfoxΔarcA of H. parasuis Would benefit from the extra figure that I mentioned previously. It is very hard to follow the details of the genetic designs. --- 210 The genomic DNAs of H. parasuis SC1401 derivatives 1401D88 and 1401D43 via site- 211 directed point mutation were used to verify natural transformation capacity of H. parasuis strain This is a mix of concepts that is hard to understand. What was done vis site-directed mutagenesis? Rephrase to clarify. --- 255 we used a discontinuous induction method in our experiments. The culture in a 10mM of EMS- Please correct spaces between dimension and units here and possibly throughout the manuscript. --- The section between lines 271 - 289 needs to be split up to introduction and results or discussion --- 357 In this study, we found that EMS has a characteristic dual function in H. parasuis. High 358 concentration of EMS inhibits growth (20mM) or even exhibits a bactericidal effect (30mM), 359 whereas low concentration introduces random somatic hypermutations in genome. Although, this is not directly my field this must surely be something that is observed every time EMS is used. I remember performing experiments as an undergraduate to demonstrate that the a cmpound would be bacteriostatic at low concentration and bactericidal at higher. Please consider removing this. --- Experimental design: Experimental design is adequate. See above for more details. Validity of the findings: Findings are valid. See above for more details. Additional comments: No additional comments

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.

Title: A STREPTOMYCIN RESISTANCE MARKER IN H. PARASUIS BASED ON SITE-DIRECTED MUTATIONS IN RPSL GENE TO PERFORM UNMARKED IN-FRAME MUTATIONS AND TO VERIFY NATURAL TRANSFORMATION Review round: 2 Reviewer: 1

Basic reporting: The manuscript still needs a thorough proofreading to improve the English language used. Experimental design: In my comment that “In this experiment, streptomycin selection is not enough for allelic exchange because the frequency of double crossover events may be low and illegitimate recombination may occur. Addition of 10% sucrose to the selection medium is appropriate.” I mean the experiment of “2.6. Site-Directed Mutagenesis”, not the experiment of “2.8. Construction of Unmarked In-Frame Targeted Mutant 1401D88ΔtfoxΔarcA of H. parasuis 1401D88”. In 2.8, 10% sucrose was added into the selection medium. However, In 2.6, the sucrose was not used in the selection medium. Why? Validity of the findings: No comment Additional comments: No additional comments

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.

Title: STAT-TRACKS AND MEDIOTYPES: POWERFUL TOOLS FOR MODERN ICHNOLOGY BASED ON 3D MODELS Review round: 1 Reviewer: 1

Basic reporting: This paper introduces the term 'mediotype’, which incorporates a multitude of digital 3D data files relevant to the field of (fossil) track research. The word is enclosed by inverted commas in the title and is a practice that should be continued throughout the body of work. I have no further comments to add to their Basic Reporting as are very well structured and of publication quality. Experimental design: The authors identify a significant gap in palaeoichnology, that is how to obtain track data that can be utilised in quantitative comparative studies (albeit in a series of paired analyses). They succeed in their approach by utilising the powerful, innovative, and newly developed DigTrace software. The authors provide a series of case studies that are relevant for the application of the method, by which the findings are very clearly documented. This approach is of very high significance for this research field (particularly dinosaur tracks, and is one that I am confident that progressive palaeoichnologist are/will be eager to employ. As clearly stated by the authors, this is not a methods paper. The aim of the paper is to introduce ‘mediotype’ as a term for use by the wider palaeoichnological community to incorporate as range of ‘statistically-generated three-dimensional track models [SG3D]’. While ’mediotype’ is a pleasant term that is simply to use, I do not think the term will be received unfavourably by palaeoichnologists for the following reasons : • Etymology implies a type specimen: ‘Mediotype’ adds to now a growing list of ICZN non-formalised terms (e.g., ‘digitype’, ‘plastotype’). Issues of repositories and accessibility to ‘type’ specimens will also come into play. Line 423 "unlike conventional palaeontology finding a 'morphologically-perfect' specimen is almost impossible" I am of the opinion that there will be strong resistance for the digital construction of an 'ideal' by any means. The authors "argue that the use of 3D data may assist in correcting this omission" which may be appropriate in for analyses but I disagree that type specimens should be a chimera of track data, nor would this be acceptable for type specimens for other research fields. • Etymology implies ‘averaging’ of data: In practice, ‘average’ data sets (mean, median) appear to be the preferred documentation source for use in this paper (supplied figures, raw data). While I am in agreement that such track data are perhaps the most useful data sets for palaeoichnologists, the fact that their term encapsulates non-averaging statistical data makes use of the term non-inclusive and potentially misleading. • Implies a single specimen: Even ‘digitype’ and ‘plastotype’ inferred specimens limited themselves to single representation. ‘Mediotype’ include six csv file types are generated with the DigTrace software (mean, median, maximum differences, minimum differences, standard deviation, point-to-point comparisons) most of which are not elaborated further after their mention in the paper. • Implies that only type specimens can be used in the generation of SG3D track data whereby limiting application (e.g., just as ‘digitype’ is limited to type specimens), a restriction that is almost certainly not the intention of the authors. Validity of the findings: The placement of landmarks can be problematic for tracks that are, by their very nature, highly variable abiotic structures. Although While the software the authors show preferences for using (Digtrace) displays significant robustness around this issue. One aspects that is confusing it the greyscale visualisation and interpretations of the standard deviation ‘mediotypes’ shown in figures 1D, F; 5C; 6B, D. Here, white indicates low deviation and black indicates high deviation. Yet in each example that the authors state in-text that there is low deviation the plantar impressions are darker than the surrounding surface. In the example that the authors state in-text as high deviation (e.g., figure 6 B) most of the plantar impression is white and surrounding surface is darker. In these cases, the data contradicts the authors claim. This is particularly important to resolve as readers that become users of the Digtrace software may face the same issues. Otherwise very well structured. Additional comments: This is an important, well-written paper for dinosaur palaeoichnology and has the potential to be a very positive and broad influence for future research in this field. The use of the term 'mediotype' is with significant problems that can be resolved, for the most part, by substituting the non-ambiguous and more accurate term 'statistically-generated three-dimensional track model' (SG 3D) of 'statistically-generated digital surface model' (SG DSM). For further comments see attached annotated pdf.

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.

Title: STAT-TRACKS AND MEDIOTYPES: POWERFUL TOOLS FOR MODERN ICHNOLOGY BASED ON 3D MODELS Review round: 1 Reviewer: 2

Basic reporting: No comment. Experimental design: No comment. Validity of the findings: No comment. Additional comments: Dear all, I should emphasize that I am conservative regarding the use of 3D imaging in vertebrate ichnology. Therefore I would probably be categorized a ‚traditionalist‘ following the terminology of the authors. I do not refuse the application of 3D imaging in vertebrate ichnology, but I just do not see any use of hitherto suggested methods in order to solve the main problems of studying fossil tetrapod footprints. The morphology of tetrapod tracks is the result of the superimposition of anatomy, gait and substrate. A majority of vertebrate ichnologists prefers an ichnotaxonomy highlights anatomically-based characters of the imprint morphology and trackway pattern. Therefore, the main challenge to these researchers is to distinguish anatomical input of tracks and trackways from extramorphological characters (= influence of gait and substrate conditions). In which way a ‚mediotype‘ sensu Belvedere et al. may help anatomically-focused vertebrate ichnologists? As far as I understand the authors, the ‚mediotype‘ is mainly a tool to quantify the differences between two or more tracks. The result is a number which nothing helps to interpret the reason for the calculated difference(s). I definitely see that I could introduce tresholds such as a similarity index, e.g. two tracks should be similar by 90% in order to assign them to the same ichnospecies. However, I do not know how much of the similarity/dissimilarity is based on anatomy, gait, and substrate. Thus, where is the progress? I totally accept that the ‚mediotype‘ is one out of several tools for vertebrate ichnologists but I doubt that it has the potential to revolutionize this paleontological discipline. What to do with this manuscript? It may be worth to describe the ‚mediotype‘ method and its application as examplified by the authors. But I feel that the importance of the paper is overstated. My suggestions: I would go along with the proposal to introduce tetrapod ichnotaxa at the base of long and well-preserved trackways, only. Thus, the introduction of ichnotaxa (diagnosis, description, measurements of track and trackway parameters) could be completed by data from 3D imaging in a way that the similarity of tracks within each specimen of the type series is calculated separately. This might result in useful values for other workers who want to quantify the similarity of their own specimens to the type material. By calculating discrete specimens the authors avoid the accusation of arbitrary selection as they base their models (‚mediotype‘) on type specimens exclusively (that are used by ‚traditionalists‘ as well). I do not see any use to extend the data base of the ‚mediotype‘ beyond type material (according to the requirements on quality and quantity of new ichnotaxa). I think it would be a good paper to describe and illustrate the process of introduction a new ichnotaxa including a ‚mediotype‘ in most detail (documentation of the type material, traditional diagnosis, measurements, description, 3D model, ‚mediotype(s)‘ production). In addition, it would be helpful to apply the ‚mediotype‘ to similar specimens from another locality to examplify the tool. Certainly, my suggestions are not so far from the content of the author’s manuscript. However, I feel that the manuscript and its ideas would probably find more acceptance when the methods are demonstrated at a single example and in most detail. A good example would be Robledopus macdonaldi Voigt et al., 2013, small reptile tracks from the Early Permian oft he New Mexico as the ichnotaxon is based on a type series and there is ambiguous material from other localities of the same or similar age in New Mexcio. Finally, I welcome the idea of the ‚mediotype‘ if it will be restricted to type material. The authors should think about my suggestions to focus on a discrete and fully elaborated example. At the present stage, I do not see that the manuscript will be of much use to the vertebrate ichnology community. Sebastian Voigt 2017-08-17

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.

Title: STAT-TRACKS AND MEDIOTYPES: POWERFUL TOOLS FOR MODERN ICHNOLOGY BASED ON 3D MODELS Review round: 2 Reviewer: 1

Basic reporting: no comment Experimental design: no comment Validity of the findings: I am a supporter of the use digital methods to evaluate the 3D topography of track surfaces, and view such approaches as providing much needed quantifiable data to a field of science that is prone to much subjectivity. One of the main aims pf this paper is to provide information of a recently developed tool for quantifiable comparative track analyses. I feel biased in my desire to have methods, like those employed by these authors, to be widely used by dinosaur ichnologists, and while the paper provides some new results from previous published examples, it is unclear as to its novelty. The authors explain the use and strengths of the approach very well, but this is not methods paper that would be of benefit to a wider audience and facilitate the adoption of this method by researchers. Shortcomings. The quantifiable aspects of this approach can be made much clearer in the examples the authors use. For example, the values of standard deviation should be stated within the text for the respective ‘stat-tracks’, as well as shown as a calculated range of standard deviation along the figured grey-scaled 'stat-track'. Inclusion in track figures of digit impression number are features currently absent and would facilitate ichnological assessments. Currently the statistical variants are comparative via grey-scale colouring of tracks yet lack numerical data. My earlier appraisal of this work expressed confusion associated with author interpretation of grey-scaled 'stat-tracks’. While the authors have now elaborated their explanation further the point I wished to raise has been lost, that I hope to clarify here. In regard to the grey-scaled 'stat-tracks’, it is repeatedly stated that lighter-to-darker colours indicate less-to-more deviation (respectively), yet appear to not adhere to this criterion in interpreting the data. For example: in assessing sauropod tracks the are “pronounced similarities amongst the tracks. The resemblance is so similar that, …all these tracks could fall in the range of intraspecific or even intra-trackway variation”. Yet the ‘stat-track’ (Fig. 1D) shows very light colouration outside the track region and dark grey-black within the track region, the latter indicating areas very highly variants and can contradicts the statement within the text. But this is not in isolation, occurring in figures 5 and 6. In regard to the figure 6 theropodan tracks, “the standard deviation stat-track (Fig. 6B) shows only few similarities” which contradicts the very light colouring of the impressions of the digits II and IV and metatarsophangeal pad. The authors explanation of these similarities as “highly biased by the completely different preservation of the two specimens”. It is not made clear to the reader whom may wish to employ the tool outlined in the paper how to distinguish topographical similarities/differences as ichnologically significant/insignificant. A more comprehensive evaluation is wanting. The second aim of this paper is to introduce new terms for potential future use in ichnotaxonomy. In considering neologisms for a very specific discipline of science, I question if it would be preferable to seek a more specific journal for the publication of this paper. This may be within either a palaeoichnological journal, or preferably a bulletin specific for nomenclature, where the validity of their proposal can be more definitively evaluated. Additional comments: No additional comments

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.

Title: STAT-TRACKS AND MEDIOTYPES: POWERFUL TOOLS FOR MODERN ICHNOLOGY BASED ON 3D MODELS Review round: 2 Reviewer: 2

Basic reporting: No complaints. Experimental design: No comment. Validity of the findings: No comment. Additional comments: Dear all, the revised version of PJ_18800 addresses most of my former cautions with this manuscript. I especially welcome the separation of „stat-track“ and „mediotype“. It is still my opinion that the importance of paleoichnological studies is first of all limited by the quality of studied tracks and traces and only subsequently affected by the applied techniques. In the end, however, application in other studies will be the best way to decide about the utility of the author’s concept. Therefore I recommend this ms for publication. Sebastian Voigt 2017-11-27

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.

Title: REFINING AMINO ACID HYDROPHOBICITY FOR DYNAMICS SIMULATION OF MEMBRANE PROTEINS Review round: 1 Reviewer: 1

Basic reporting: Figure 3. PMF insertion profiles for mass centers of sidechain analogs relative to the bilayer center. The letters (w, g, o, c, etc.) at the plot are very confusing, please use a different mark. Figure 5. Linear regression, please show a small mark, and then the letter next to the mark, otherwise is hard to understand the data presented here. Figure 6. CgProt PMFs for one and two-residue backbone moieties. Again, the letters overlap and they are difficult to see. Please add a different mark. Experimental design: Methods are well described, the results and findings are relevant and interesting. Validity of the findings: It would be interesting if the authors present how their hydrophobicity force field refinement address the protein-protein interaction in a transmembrane helix dimer. Additional comments: No additional comments

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.

Title: REFINING AMINO ACID HYDROPHOBICITY FOR DYNAMICS SIMULATION OF MEMBRANE PROTEINS Review round: 1 Reviewer: 2

Basic reporting: While the literature cited is failry complete, for the reader to get a complete picture of the field, I suggest to adding the reference to the recent paper by Pawlowski et al who developed a continuous lipid model for coarse-grained simulations of membrane proteins (W Pulawski, M Jamroz, M Kolinski, A Kolinski, S Kmiecik Journal of chemical information and modeling 56 (11), 2207-2215) Experimental design: No comment Validity of the findings: No comment Additional comments: This is a very good paper that sunstantially advances the development of coarse-grained simulations of membrane proteins. The parameters of the CgProt force field were refined to reproduce the peptide-insertion PMFs calculated by all-atom simulations and experimental hydrophobicities. The resulting force field was shown to perform significantly better regarding the insertion of peptides into lipid membranes.

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.

Title: REFINING AMINO ACID HYDROPHOBICITY FOR DYNAMICS SIMULATION OF MEMBRANE PROTEINS Review round: 1 Reviewer: 3

Basic reporting: The manuscript by Hills reports on the refinement of the CgProt 2.4 coarse-grained force field for proteins. This is one of several force fields for protein simulations. The text and the figures are reasonably clear, with a few exceptions. The graphical quality of Figure 1 is rather poor, I suggest using molecular models instead of chemical formulas. Literature references are not always appropriate; for example, in the introduction, when writing about other coarse-grained force fields for proteins, the author mentions the MARTINI force field but provides incorrect citations. At line 92, the author refers to himself the idea of temperature coupling to separate heat baths, and to Arnarez the idea of implicit solvent for CG simulations. Bizarre. The text is far from self-contained, particularly in the methods section: - the description of the force fields is completely absent; one would need to dig out one or more previous papers to understand the meaning of acronyms for different particle types and the shape of the potential energy function. - I did not find a description of bonded interactions (except for the restraints on helices). - How was the position of the CG particles calculated? - which structural properties of proteins are reproduced? which dynamic properties? - what is the speedup compared to an atomistic simulation? Experimental design: The goal of the research is well defined: to correct the flaws present in previous versions of the force field. Based on the results presented, the goal seems to have been achieved. At the same time, it is not very clear what is the general goal of the force field; what are the pros and cons compared to other force fields available in the literature? The design of the simulations has no original point. All of the simulations seen here have been done before by others, using other methods and/or force fields. The description of the methodology is insufficient to reproduce any of the calculations: no mention of the algorithms used for thermostats and barostats, nor the cutoffs (if present). Validity of the findings: The technical quality is difficult to assess because a description of the force field is missing (see above for details) and the simulation parameters are rather incomplete: no mention of the algorithms used for thermostats and barostats, nor the potential energy functions used, nor the cutoffs (if present). Methods and figure 3: the AA data has been calculated or extracted from the figures by MacCallum et al.? Line 218, “The favorable attraction of basic residues for the ester bilayer region has been dubbed snorkeling.” Why would it be valid only for basic residues? In fact, in a real system, snorkeling does not have much to do with the supposed attraction for the ester group, but rather with the tendency of a charged side chain to remain hydrated. The ester region in a lipid bilayer is generally hydrated, hence the proximity of charged side chains with lipid ester groups. The real physics of the system is hidden by the implicit solvent. Additional comments: The general philosophy of the force field is not described: what are the target properties? what kind of problems should be tackled by this force field? Does this force field make progress with respect to other similar, available methodologies?

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.

Title: REFINING AMINO ACID HYDROPHOBICITY FOR DYNAMICS SIMULATION OF MEMBRANE PROTEINS Review round: 2 Reviewer: 1

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

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.

Title: USING UNDERWATER VIDEO TO EVALUATE THE PERFORMANCE OF THE FUKUI TRAP AS A MITIGATION TOOL FOR THE INVASIVE EUROPEAN GREEN CRAB (CARCINUS MAENAS) IN NEWFOUNDLAND, CANADA Review round: 1 Reviewer: 1

Basic reporting: This article, addressing the trapping efficiency of the Fukuii fish trap, is a very important study that should be of great interest to those biologists who use this trap to monitor and control the European green crab. Green crab researchers in Australia and on both coasts of North America are using this trap because it is light and can be folded. However, no one has ever questioned its trapping efficiency. Knowing that the efficiency is only 16% is especially important to programs designed to “eradicate” newly discovered satellite populations. It is important to note that poor catches may not mean that a population has been reduced to a low enough level to prevent self-seeding. The article is well written and easy to follow. The introduction includes all the relevant references and clearly states the context of this study. The figures are clear and well described. I wonder if Figure 6, showing catches with and without the camera can be left out. All that would be needed is a statement that there was no significant difference in the catches. Experimental design: The experimental design is rigorous and well described. The fact that the authors used of a no-camera control treatment and deployed the traps at various sites and various conditions is very impressive. Validity of the findings: The data are robust and the analysis is sound. From this rigorous study we learned that most meaningful data can be obtained at sites with high crab densities, during daylight and with good visibility. Knowing these parameters will make it easier to test modifications to the Fukuii trap that will increase catch rate. The number 1 modification for increasing catch rate would be to work on “difficulty in completing entry”. I am suggesting that the authors describe some modifications that could be tested to achieve this goal. Even though the authors did not test any of these modification, they are in the best position to advise fellow green crab biologists for getting better catch rates. Additional comments: This is a very needed piece of information for all researchers who use the Fukuii trap. Knowing the traps limitation needs to be incorporated in any monitoring and control studies. It would help if the authors made some suggestions for improving the design of the Fukuii traps. Researchers using new traps with tight fitting openings may not trap as many crabs as researchers using older traps with sagging mesh openings. That could lead to a difference in CPUE.

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.

Title: USING UNDERWATER VIDEO TO EVALUATE THE PERFORMANCE OF THE FUKUI TRAP AS A MITIGATION TOOL FOR THE INVASIVE EUROPEAN GREEN CRAB (CARCINUS MAENAS) IN NEWFOUNDLAND, CANADA Review round: 1 Reviewer: 2

Basic reporting: Writing is generally clear and easy to follow, but some sentences should be more concise to improve flow (specific changes annotated in PDF). The discussion could be shortened by removing some redundant sentences. Some important references are missing (annotated in PDF). Experimental design: This study addresses an important gap in our understanding of the efficacy of a widely-used tool. The authors do a good job of clearly specifying their objectives, which are to 1. Evaluate performance and efficiency in terms of catchability (CPUE of Fukui traps) and 2. Understand patterns in catchability between different sites. To this end, they are only evaluating steps 3-6 of catching a crab in a Fukui trap: the approach, locating the entrance/attempting entry, and entering the trap. Importantly, they are not evaluating catch relative to abundance of crabs around the trap, nor are they considering the retention of target catch until the gear is hauled. There is some confusion about the Methods: which videos were included in the analysis. The authors use an “elimination threshold” of a mean of 10 green crabs caught per deployment (Line 170) because “effective trapping requires that green crab be present in sufficient numbers within the area being fished” (line 359). But, in section 2.4.1 the authors say that videos were eliminated based on no or low “activity,” determined by counting the approximate number of green crab present in the FOV (Line 186). Please clarify whether videos were eliminated based on low catch or on low abundance around the trap, so that the text matches with SI Table 2. Validity of the findings: The dataset is small in terms of number of videos (8 out of 37 deployments used in analysis) but the study is focused only on the approach, attempted entry, and entrance into the trap. To that end, there were 2,373 approaches, which is a sufficiently large dataset to make conclusions about the performance of the Fukui trap. Conclusions are well-stated, and speculation regarding the causes of the results are clearly stated as such. However, the potential importance of additional factors should be mentioned (e.g. sex and year, annotated in PDF) as well as the day-night effect, which is interesting and not given enough attention in the discussion. Be careful to specify where you are referring to the effect of the camera on the trap, versus environmental factors or the trap itself (annotated in PDF). Additional comments: This is a good study that is beneficial to the literature and applicable to management of invasive green crab. The manuscript structure is easy to follow (the 6 steps are clearly described). I have made the following suggestions to improve the validity of the findings and the readability of the manuscript: (also annotated in yellow comment boxes on PDF) 1. Line 117: Add more recent reference. Have any studies specifically been done on C. maenas and lights? See annotation 2. Line 122: Citing Kahle & Wickham 2013 doesn’t make sense unless you add that you made figures with ggplot2 3. Line 127: Should mention habitat type as this could have an effect on crab abundance, behavior, and therefore catchability (e.g. Bellchambers 2013, “Assessing the effectiveness of two methods of habitat characterisation for understanding species habitat relationships, using the western rock lobster (Panulirus cygnus George)”) 4. Line 130: reword suggested 5. Line 132: scientific name of herring first time you mention it 6. Line 170: Did you test the effects of day/night? 7. Line 189: Explain that this is the “elimination threshold.” See general comments about the confusion with the video selection criteria/elimination threshold 8. Line 238: state potential relevance of genetic differences for catchability 9. Line 272: Which sample subset are you referring to? Only used 8 videos in analysis? 10. Line 306: Present in order (most common – least common, or visa versa) 11. Line 318: Include model results (p values for study site effect) 12. Line 326: How is saturation defined in the literature? 13. Line 378: Inconsistent with your later speculation about trap construction. 14. Line 386: Clarify – all bycatch present in the trap upon retrieval were released alive 15. Line 390 – rephrase, “within seconds” is misleading 16. Line 451 and 454: Rephrase these sentences: “often” and “not uncommon” are misleading as this was the “least common reason for failure” 17. Line 463: suggested changing word order 18. Line 478: The disconnect is not necessarily between abundance and final catch, it’s between attraction to the trap and final catch. 19. Line 479: Suggested reword 20. Line 494: Also individual traps as a factor in future studies 21. Line 497: Should mention other studies with this same finding: Watson & Jury 2013 (“The relationship between American lobster catch, entry rate into traps and density”), Sturdivant & Clark 2011 (“An evaluation of the effects of blue crab (Callinectes sapidus) behavior on the efficacy of crab pots as a tool for estimating population abundance”) 22. Lines 515 and 518 belong in the introduction 23. Lines 526 – 535: most of this should be moved to the introduction 24. Line 545: What is CHM? 25. Line 585: Specify of C. maenas (see comment) 26. Line 586: Should also consider the effect of year (Tremblay et al. 2006) and sex – mention other relevant studies 27. Line 590: consider the peer-reviewed literature regarding the efficacy of sampling techniques (e.g. Stobart et al. 2015, "Performance of Baited Underwater Video: Does It Underestimate Abundance at High Population Densities?", Roberson et al. 2017, "Potential application of baited remote underwater video to survey abundance of west coast rock lobster Jasus lalandii"). 28. Line 604: This sentence is redundant, consider removing 29. Line 609: Should also consider testing lights to improve CPUE (See comment for line 117 – Nguyen 2017). 30. Line 610: redundant sentence, consider removing. 31. Line 615: Should mention future studies on tradeoffs in trap design, also potential effects on bycatch rates 32. Figure 11: Hard to read, change red color scale

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.

Title: THE OLD AND THE NEW PLANKTON: ECOLOGICAL REPLACEMENT OF ASSOCIATIONS OF MOLLUSC PLANKTON AND GIANT FILTER FEEDERS AFTER THE CRETACEOUS? Review round: 1 Reviewer: 1

Basic reporting: Literature references incomplete, important and relevant references missing (see pt. 3 and 4) Experimental design: The manuscript is mainly speculation with relatively little new information, of which I cannot fully judge the importance (pt. 3 and 4). Validity of the findings: The authors present several conclusions: 1. Ammonites changed ecological role during their life time, as indicated by their extreme increase in size during ontogeny. They do not seem to bring up evidence for this statement, but it seems to be quite obvious – few organisms could eat an ammonite with diameter >1 m, many could eat ammonitella of about 1 mm. 2. Large female ammonites produced very numerous offspring (r-selected), so juvenile ammonites likely were an important food source for planktivores. 3. After the end Cretaceous extinction, juvenile ammonites were replaced by holoplanktonic gastropods, large pachycormid fish by filter-feeding chondrichthyans and cetaceans (sharks and whales). The authors present newly generated information only as a part of to pt. 2, i.e., estimating fecundity of large ammonites. I know very little about ammonites, and leave the evaluation of this (important) part of the manuscript to other reviewers or the editor. I am able to evaluate pt. 3, and in my opinion the authors do not show convincing evidence that holoplanktonic gastropods diversified shortly after the end Cretaceous extinction, thus can be said to replace juvenile ammonites – there is considerable evidence that they radiated after the start of the Eocene, at least 10 million years later. The authors do not cite references showing the radiation in the Eocene. In my opinion the paper should thus be rejected, or (if the information on the ammonite fecundity is by itself sufficiently novel) be rewritten without linking extinction of ammonites at the end of the Cretaceous and expansion of Thecosomata more than 10 million years later. Additional comments: Remarks by line number: 42-44: ‘..organisms…were erased by the consequences of an impact in Mexico and flood-basalt eruptions in India..’, followed by references. This sentence reads as if there is agreement in the community that the main cause of the extinctions were the combined effects of impact and volcanic eruption, but nothing is further from the real state of affairs: I would argue that the references show that hardly anyone would say this, but most people would argue that either the impact (Schulte et al) or the flood basalt eruptions (Keller et al) were the almost exclusive cause of the extinctions. Replacing ‘and’ by ‘or would improve the sentence.. 47: only planktonic foraminifera, benthic foraminifera do not suffer significant extinction (Culver, Alegret et al.). 48: the bivalve statement is too simplistic; their pattern of extinction can in my opinion not be described as being ‘partial or total disappearance’ – see e.g. Vilhena et al 2013 Nature Sci Repts. 51-52: Did the Puzosiidae live during the late Maastrichtian, i.e. did they become extinct at the end of the Cretaceous? How common were they (and potentially other large ammonites) in the ecosystem? (something about how common they were appears in line 125, but ‘quite common’ is not very informative). 90: please define ‘derived ammonites; - are these heteromorphs? Were Puzosiidae derived? 113: how big is the lectotype of P. seppenradensis? 119: anything know about semelparous-iteroparous lifestyles of large ammonites? Is it probable that such large organisms are semelparous?? 136: why discuss belemnites if they were already in decline (from about 35 million years before the K/Pg extinction)?? Do we know anything about coleoids replacing them? 153: do we know enough to make such a sweeping statement ‘these early psot-hatching developmental stages’ – all ammonites? Only derived ones? 154-155: refer to buoyancy – Shigeta 1993, Lethaia? 162: reasonable argument, but did not larger ammonites eat smaller ones as well? (I see- said in lines 169-170) – so how important were these large planktivorous fish? Common enough to have major impact? 167: I do not see that coeval extinction of two groups confirms that one ate the other- especially not if they both became extinct at the end of the Cretaceous (ammonites did)- see also below (189) 178-185: I do not really see why the discussion whether extinction of belemnites is linked to that of ichthyosaurs is relevant to the main story in this manuscript, and also do not quite see how extinction of that predator would lead to extinction of its prey only in the Pacific. 182: CTB? Cretaceous Tertiary Boundary (rather than KPg)? 189: once again, I do not see that coeval extinction of groups during a major mass extinction (when many things went extinct) has anything suggestion to make as to their trophic relations. 191-206: In my opinion it just is not true according to most evidence that the associated extinction (during the KPg) coincided with (i.e., occurred at the same time) as the rise of holoplanktonic gastropods, specifically Thecosomata. If we look at some recent papers (as well as those cited in lines 202-204), then it becomes clear that the ‘rise of the holoplanktonic gastropods’ (if equated with the increase in diversity of thecosomata) considerably post dated the KPg (and the extinction of ammonites): we see a single taxon in the Campanian and the Paleocene, with increased diversity only by the earliest Eocene, i.e., more than 10 million years after the KPg extinction. I realize that the cited Corse et al. say that there was a diversifying event ‘just after the Cretaceous/Tertiary mass extinction’, but the paper that they cite for this statement in fact only describes occurrence of pteropods in the Eocene, which in my opinion cannot possibly be called ‘just after the Cretaceous Tertiary mass extinction’ – even in geology I do not call 10 myr later ‘just after’. I suggest that the authors look up recent articles by Janssen & Peijnenburg, 2017, An overview of the fossil record of Pteropoda (Mollusca, Gastropoda, Heterobranchia)Cenozoic Research 17(1), 3-10, and look at their nice compilation figure, Burridge et al., 2017, Time-calibrated molecular phylogeny of pteropods. PlosOne, 12(6): e0177325, who state ‘Using a fossil-calibrated phylogeny that sets the first occurrence of coiled euthecosomes at 79±66 mya, we estimate that uncoiled euthecosomes evolved 51±42 mya and that most extant uncoiled genera originated 40±15 mya.’ , and Janssen, A. W., Sessa, J. A., and Thomas E., 2016. Pteropoda (Mollusca, Gastropoda, Thecosomata) from the Paleocene-Eocene Thermal Maximum of the United States Atlantic Coastal Plain., Palaeontologia Electronica, Article 19 (3), 1-26. These all show diversification of holoplanktonic gastropods in the Eocene (as did the paper cited in Corse et al). I guess one could still compare the ecological role of holoplanktonic gastropods and compare to juvenile ammonites, but the authors argue that the latter replaced the former shortly after their extinction, and that is not supported by the literature cited above - see below. 213-214, 216-217: yes, quite a few pteropods have been reported from the Eocene, but that is NOT ‘just after the K/Pg extinction. 221-223: the baleen whales may have replaced giant Cretaceous planktivorous fish as to ther ecological role, but as shown in fig. 3, they only evolved in the earliest Oligocene (during the cooling of the Earth and establishment of Antarctic ice sheets, commonly (though not universally accepted) linked to diversification and increased abundance of diatoms; see e.g., Norris et al., 2013, Science, 341, 492; Marx & Uhen, 2010, Science 327, 993). So it seems to me not valid to link the origin/diversification of whales to extinction of large Cretaceous planktivorous fish more than 30 million years earlier. 228: there were no early Paleogene (=Paleocene-Eocene) whales. Please document early Paleogene chondrichthyans- provide reference (also 255: which 3 lineages? Provide reference). 252-253: there was no Paleocene expansion of Thecosomata (at least, we have no evidence for that), there was an Eocene (10 million years later) expansion.

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.

Title: THE OLD AND THE NEW PLANKTON: ECOLOGICAL REPLACEMENT OF ASSOCIATIONS OF MOLLUSC PLANKTON AND GIANT FILTER FEEDERS AFTER THE CRETACEOUS? Review round: 1 Reviewer: 2

Basic reporting: English is generally good; I have marked up some suggested word changes and typographical errors in the pdf of the manuscript. References are very thorough and capture the context of the paper's topic well. They do need to be proofread more carefully, as there are inconsistencies in citation format, e.g., abbreviating vs. not abbreviating journal titles, capitalizing each word in book titles vs. not, including "Topics in Geobiology" and volume number vs. not. The figures are beautifully constructed, but are not described or discussed in any detail in the text. They really need to be explained more both in the text and in the figure captions. For example, Fig. 2 does not provide units for the zooplankton plots. Are these numbers of species? Numbers of genera? A percentage? What is the reader supposed to notice from looking at these plots? In Fig. 3, what are the colored dots and black lines in the plot supposed to represent? What is the reader supposed to learn from this figure? How does this figure support the author's argument? It is worth the authors' time to describe the figures and their message within the text. Experimental design: The topic of what role hatchling ammonoids and belemnoid played in Cretaceous planktonic food webs, and how they and their potential predators were replaced after the end-Cretaceous extinction, is an interesting one that has not been well-explored in the past. The research questions are not presented very clearly in the introduction. The authors state that they consider two research questions (lines 54-59), but the first item is not phrased as a question at all. It would be helpful to rewrite this paragraph, making the questions as explicit as possible. The methods used to estimate the fecundity of large Cretaceous ammonoids are clearly explained and make sense; these are the primary new contribution. Much of the rest of the paper involves synthesis of various observations derived from the published literature. Validity of the findings: The authors primarily use valid existing published data and observations, plus reasonable new calculations of the likely gonad volume and egg production of the largest known Cretaceous ammonoid Parapuzosia seppenradense, to argue for the importance of ammonoid hatchlings in the zooplankton of Late Cretaceous oceans. They then discuss some likely predators on these hatchlings, as well as replacement taxa for both hatchlings and their predators after the end-Cretaceous extinction. While somewhat speculative, these discussions are supported by reference to observations in the existing literature. One concern I have is that the belemnoids seem "tacked on" to the paper, with just brief discussion of their importance to Cretaceous planktonic food webs. It would be helpful if the authors could add a little more information and/or data about belemnoids to support their claims. Additional comments: This paper makes an interesting and well-supported case for the importance of ammonoids and belemnoids in Cretaceous planktonic food webs. I do note a few key issues to address in a revision (in order of importance): 1) Reframe the paragraph that presents your research questions. Make the research questions actual questions, and ideally phrase them to go beyond the descriptive (e.g., "what groups might have...?") to test a hypothesis or prediction. 2) Make better use of the figures by describing them in the text, making clear what the reader should be learning from each one, and adding more information to the captions explaining figure components. 3) Amplify the discussion of belemnoids, which seems like an afterthought in this version of the manuscript. 4) Ensure references are consistent in their format.

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.

Title: THE OLD AND THE NEW PLANKTON: ECOLOGICAL REPLACEMENT OF ASSOCIATIONS OF MOLLUSC PLANKTON AND GIANT FILTER FEEDERS AFTER THE CRETACEOUS? Review round: 1 Reviewer: 3

Basic reporting: Writing is generally clear but sometimes fuzzy in few sentences. Some typos also pervade the MS. This is minor but I recommend that the authors review the paper for wording/style. L.23: fishES? Introduction: maybe too short. Authors can add for instance some known examples of potential similar replacements in the fossil record. l.32: mass extinction intervals: what does it means exactly? They are more short events than intervals. Maybe reword this sentence. l.36-38: This needs a few references. Also, it may depend on if you are talking about taxonomic richness and/or functional diversity. See e.g. recent works of Foster et al. on the PT and TJ extinctions. l.43: flood-basalt-eruptions => flood basalt eruptions l.49: Ammonoids “evolved a great disparity”: I suggest to rephrase this sentence. l. 50: “bizarre forms”: maybe refer directly to heteromorphs. l.51: please rephrase the beginning of this sentence. l. 53: “they also occurred worldwide and in great numbers”. I suggest to modify this sentence, e.g.: “they also abundantly occurred worldwide” l.63: maybe add in the sentence if this is true for all ammonoids and periods (?). l.66: a space is missing after “(iii)”. l.85-89: if possible, could be nice to highlight abbreviations for measurements in italics. l.90: “as demonstrated by”: I’m not very prone to use such terms in articles. Maybe “as shown by” (or equivalent) is more appropriate. Same apply l. 212 “undoubtedly” is likely too strong. l.90: “derived ammonoids”: unclear, please add a definition and details. l.113-117: Important point: I expect this is a mistake but please do not use L² to refer to a volume!! => L3. l.118-120: iteroparity vs semelparity: what is the most realistic based on the known fossil and modern data (if any)? l.121: “fecundity would further increase”: unclear for me, especially the link with growth of embryos after they were laid. Please add an explanation and potential references (even if these are for modern cephalopods). l.124: => Cretaceous l.134: “high fecundity corresponded with high mortality”: this may appear rather authoritative. You should explain why, is this always the case and add references on that topic. l.137: are we sure that belemnites were in direct competition with other coleoids? With all coleoids? l.136-140: In my opinion, such general sentence on belemnites should arrive earlier in the ms, e.g. in the same part authors present ammonoids and their first ontogenetic stages. It could also be nice to see a little bit more space devoted to belemnites in the introduction. l.178-185: this sentence is really too long to be well understood. l. 182 CTB: add definition of this acronym. l.178-185, l.189-191 and at some other places in the ms: authors often link potential trophic relationships among organisms with associated extinctions among the same organisms. I don’t say this is not the case but this should be nuanced as this link is far to be evident and may result from environmental changes or other biotic interactions. l.206-207: “compare the UNCOILING of the conch of Thecosomata with the COILING of ammonites”: in my opinion, this sentence sounds very strange. What are the potential “macroecological implications”? l.212-213: may add references on that point. l.213-215: a “few pteropods” “implies that they were abundant and widely distributed”… strange wording I think. Please clarify the sentence by making a better link with previous sentences. l.219: “instalment”: what does it means? l.233: late => Late? Fig. 2: Maybe shift the gastropod data on the other side, near the ammonite and belemnite data, for clarity. References: they are sometimes in chronological order, sometimes in alphabetical order. Maybe homogenize them if you have instructions from PeerJ. Experimental design: No comments. Validity of the findings: This is my first review for PeerJ and I am not sure how the following comments fit well here or should be placed in Comments for author. This work mainly resembles a short review on some “ecological replacements” that are observed during the KT transition. The working hypothesis is interesting because results support that ammonites and belemnites eggs and embryos may have served as food resources during the Cretaceous and that their disappearance at the KT boundary led to extinction of their potential predators. Such an idea is not totally new. The hypothesis of their replacement by holoplanktonic gastropods in food webs is also interesting as well as the discussion about potential new predators feeding on these small-sized organisms. We have nevertheless only rare direct fossil evidences for such feeding; the final discussion/conclusion is therefore partly speculative, but presented as such. To slightly enlarge the discussion and maybe yield slightly more balanced scenario, authors may preliminary answer to some associated questions: for instance, can other potential candidates than holoplanktonic gastropods have also served as potential food resources for filter feeding predators. What about crustaceans at that time? Are they too small? Too rare? Also to complement the discussion: are similar potential replacements (not necessarily complete) between an ammonite subgroup and another marine clade that has been documented (or speculated) in the Paleozoic, Triassic or Jurassic? This might be a point to address. Just a (probably naive) thought: can we imagine that largest ammonites also fed on smallest ones, thus reducing the number of available preys for other groups leading to an “equilibrium” inside the ammonoid clade? Additional comments: This short MS presents an interesting working hypothesis on ecological/functional successions during the KT transition. This is relevant for PeerJ. I suggest to slightly enlarge some discussion points and I have highlighted a few areas that may benefit from some clarifications or rewording. I recommend publication after minor revision.

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.

Title: PREVENTIVE EFFECTS OF SALVIA OFFICINALIS LEAF EXTRACT ON INSULIN RESISTANCE AND INFLAMMATION IN A MODEL OF HIGH FAT DIET-INDUCED OBESITY IN MICE THAT RESPONDS TO ROSIGLITAZONE Review round: 1 Reviewer: 1

Basic reporting: MR. Ben Khedher and colleagues investigates whether Salvia officinalis (Sage) leaf extract prevent insulin resistance and inflammation in high fat diet-induced-obesity mice model. It should be emphasized that 598 papers (see Pub Med) were written about the sage effects. Among these references, it is described that sage exerts hypoglycaemic and anti-dyslipidemic effects in diabetic subjects (https://www.ncbi.nlm.nih.gov/pubmed/27942500; https://www.ncbi.nlm.nih.gov/pubmed/25340127; https://www.ncbi.nlm.nih.gov/pubmed/21506190). This study may be considered original (inflammatory state related adipose tissue) and may follow a publication. In spite of that, some ambiguities need to be clarified. Problematic question: Why the authors chose to compare the sage effects to rosiglitazone properties, whereas this oral anti-diabetic molecule (Avandia©) has been severely criticized due to cardiac complications leading many deaths, such as myocardial infarction and then withdrawn from the market since 2002. The prestigious British journal NEJM evaluated the deleterious effects of rosiglitazone (https://www.ncbi.nlm.nih.gov/pubmed/17517853). Thanks to argue this aspect. Relevant question: Sage can also leads to cardiovascular complications effects like rosiglitazone? The authors did not investigate this point in their study (effect on blood pressure?) and no discuss in discussion section. Indeed, in conclusion, the authors confirm that: at low dose, Sage exhibits similar effects to rosiglitazone? Thanks to argue this aspect. In addition, it is recommended that Rosiglitazone appear in the article-title. For example: “Preventive effects of salvia officinalis leaf extract versus Rosiglitazone on insulin resistance and inflammation, in high fat diet-induced-obesity mice model” Experimental design: Diet: 60% fat by energy value is no sufficient. What is the ratio of diet lipid composition to saturated, monounsaturated and polyunsaturated fatty acids, respectively? Also, the composition to others nutriments: proteins, carbohydrates, vitamins and minerals? Fasting time: 5 hours is insufficient to study metabolism. 16 hours minimum. What protocol and reference? Insulin resistance (IR): The protocol used is not clear. Why to use Oral Glucose Tolerance Test and the Insulin Tolerance Test separately? In practice (experimental animal but not in human), IR is evaluated by the hyperinsulinemic euglycemic clamp. HOMA model is no sufficient. Please clarify this point. Obesity mice model or diabetic mice model? As the authors studied rosiglitazone (oral antidiabetic), It is important to confirm the mice C57Bl6 (transgenic model) is also a model of type 2 diabetes, and to be associated with insulin resistance. The authors have not studied. Please clarify this point. Statistical analysis: The correlation test (Pearson test) between the comparative effects between sage and rosiglitazone is missing. Student t-test is no sufficient. Validity of the findings: In vitro versus in vivo: The results are obtained from both the pre-adipocytes cells (3T3-L1) and mice (in vivo study). What relationship between these two studies, since the cells were not isolated from the obesity model mice (C57Bl6)? Anthropometric parameters: This study concerns obesity related to glucose tolerance. Although in Table 1 the authors present the weight variations, Results not show whether sage extract effect on Body mass index variation. In addition, the imaging data (Lean and fat mass were measured by Nuclear Magnetic Resonance) for the assessment of fat qualitative mass composition (subcutaneous or visceral) have not been shown. Lipogenesis versus lipolysis: There is confusion in the data to explain the lipids metabolism effects of sage. The authors confirm that sage exerts both lipogenic and anti-lipolytic effects at the same time (Line 267:... however sage-treated animals had a decrease in plasma NEFA and triglycerides levels, suggesting an inhibition of lipolysis…. Line 271:... the plant extract reduced lipogenesis…). Please clarify this point. Sage inefficacity or sage toxicity: Line 317:... This might explain the beneficial effect on food intake of sage low dose when compared to high dose… It’s not clear the dose effect of sage. If sage has no beneficial effect at high doses, can to become toxic? In this case, how will sage be correlated with rosiglitazone? Please clarify this point. Additional comments: Thank you to see critical above. In Figure 5, authors should correct that panel A: is anti-inflammatory cytokines and panel B: is pro-inflammatory cytokines.

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.

Title: PREVENTIVE EFFECTS OF SALVIA OFFICINALIS LEAF EXTRACT ON INSULIN RESISTANCE AND INFLAMMATION IN A MODEL OF HIGH FAT DIET-INDUCED OBESITY IN MICE THAT RESPONDS TO ROSIGLITAZONE Review round: 1 Reviewer: 2

Basic reporting: The authors report positive effects of methanol extracts of Sage (salvia officinalis) on inflammation, obesity and diabetes related parameters both in vitro in 3T3L1 adipocyte differentiation model and in vivo in a high fat nutritional obesity mouse model. The authors appropriately conducted tests related to obesity and diabetic related studies that include glucose and insulin tolerance test and further evaluate the effects of Sage extract on adipokine leptin and inflammatory proteins. Although the in vitro data from differentiated 3T3L1 does not completely correlate with the in vivo data regarding the lipolytic effects of Sage, the authors successfully demonstrate positive effects in both models. In general, the manuscript is sound in logic and writing. The conclusions support the results. Appropriate statistical tests have been performed. Please refer to general comments for the reviewer’s critique. Experimental design: No comments Validity of the findings: No comments Additional comments: 1) Since the Sage extract contain several active compounds including toxic compound like alpha and beta-thujones, have the authors conducted a toxicity profile of the methanol extract of Sage. I would recommend a annexin-PI staining of the 3T3L1 cells and a TUNEL staining of frozen section of liver tissue after treatment with SAGE. 2) The authors claim that the presence of several ppar gamma and alpha agonists in the SAGE extracts may be responsible for the observed effects. I would recommend the authors to do a selective knockdown of PPAR gamma or alpha in 3T3L1 adipocytes and/or use the adipose specific knockout mouse model for the PPAR receptor gamma or alpha to prove the validity of results. Alternatively, in addition to the above suggested experiments, can the authors selectively treat cells with ppar gamma or alpha agonists observed in the Sage extract with mass spectrometry data alone or in combinations in vitro to demonstrate the validity of their results. 3) The reviewer would recommend the authors to represent all the in vivo bar graph data for ELISA as scattered or aligned dot plots.

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.

Title: PREVENTIVE EFFECTS OF SALVIA OFFICINALIS LEAF EXTRACT ON INSULIN RESISTANCE AND INFLAMMATION IN A MODEL OF HIGH FAT DIET-INDUCED OBESITY IN MICE THAT RESPONDS TO ROSIGLITAZONE Review round: 1 Reviewer: 3

Basic reporting: This is a well written manuscript that addresses the effect of sage extract on insulin sensitivity. Sufficient literature references and background context has been provided. Experimental design: How was the differentiation of adipocytes confirmed? Were adipocytes markers evaluated in the differentiated cells to validate the differentiation? Also, the markers should be tested when differentiation was carried in presence or absence of the extract. The authors have used two dosing concentrations of sage extract? How were these doses finalized? Also are these doses the weight of the total extract? What kind of validation was performed on the extract to check for the reproducibility of the contents and concentrations of the component of the extract? More details about the change in body weight from baseline before treatment to end of the treatment should be presented as a validation of the HFD. The authors have performed a post-hoc dunnett’s multiple comparison tests that compares the means of the treatment group to the control group. Maybe the authors should consider doing something like Tukey’s to compare all the groups so that they can also judge the difference in effects of the two treatment concentrations. Validity of the findings: The authors need to touch upon various aspects of HFD model in their discussion to tackle the drawbacks. Male mice are more susceptible to hyperglycemia and inflammation compared to female mice. Further more the authors purport that their study has an advantage over the previous studies since they have used a HFD model vs drug induced diabetes. However they also need to address issues like T2D is a more age associated disease and is more common in the older population. Studies hence caution the use of younger mice. Additional comments: No additional comments

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.

Title: PREVENTIVE EFFECTS OF SALVIA OFFICINALIS LEAF EXTRACT ON INSULIN RESISTANCE AND INFLAMMATION IN A MODEL OF HIGH FAT DIET-INDUCED OBESITY IN MICE THAT RESPONDS TO ROSIGLITAZONE Review round: 2 Reviewer: 1

Basic reporting: 1. BASIC REPORTING MR. Ben Khedher and colleagues investigates whether Salvia officinalis (Sage) leaf extract prevent insulin resistance and inflammation in high fat diet-induced-obesity mice model. It should be emphasized that 598 papers (see Pub Med) were written about the sage effects. Among these references, it is described that sage exerts hypoglycaemic and anti-dyslipidemic effects in diabetic subjects (https://www.ncbi.nlm.nih.gov/pubmed/27942500; https://www.ncbi.nlm.nih.gov/pubmed/25340127; https://www.ncbi.nlm.nih.gov/pubmed/21506190). This study may be considered original (inflammatory state related adipose tissue) and may follow a publication. In spite of that, some ambiguities need to be clarified. Problematic question: Why the authors chose to compare the sage effects to rosiglitazone properties, whereas this oral anti-diabetic molecule (Avandia©) has been severely criticized due to cardiac complications leading many deaths, such as myocardial infarction and then withdrawn from the market since 2002. The prestigious British journal NEJM evaluated the deleterious effects of rosiglitazone (https://www.ncbi.nlm.nih.gov/pubmed/17517853). Thanks to argue this aspect. Relevant question: Sage can also leads to cardiovascular complications effects like rosiglitazone? The authors did not investigate this point in their study (effect on blood pressure?) and no discuss in discussion section. Indeed, in conclusion, the authors confirm that: at low dose, Sage exhibits similar effects to rosiglitazone? Thanks to argue this aspect. In addition, it is recommended that Rosiglitazone appear in the article-title. For example: “Preventive effects of salvia officinalis leaf extract versus Rosiglitazone on insulin resistance and inflammation, in high fat diet-induced-obesity mice model” Experimental design: Diet: 60% fat by energy value is no sufficient. What is the ratio of diet lipid composition to saturated, monounsaturated and polyunsaturated fatty acids, respectively? Also, the composition to others nutriments: proteins, carbohydrates, vitamins and minerals? Fasting time: 5 hours is insufficient to study metabolism. 16 hours minimum. What protocol and reference? Insulin resistance (IR): The protocol used is not clear. Why to use Oral Glucose Tolerance Test and the Insulin Tolerance Test separately? In practice (experimental animal but not in human), IR is evaluated by the hyperinsulinemic euglycemic clamp. HOMA model is no sufficient. Please clarify this point. Obesity mice model or diabetic mice model? As the authors studied rosiglitazone (oral antidiabetic), It is important to confirm the mice C57Bl6 (transgenic model) is also a model of type 2 diabetes, and to be associated with insulin resistance. The authors have not studied. Please clarify this point. Statistical analysis: The correlation test (Pearson test) between the comparative effects between sage and rosiglitazone is missing. Student t-test is no sufficient. Validity of the findings: In vitro versus in vivo: The results are obtained from both the pre-adipocytes cells (3T3-L1) and mice (in vivo study). What relationship between these two studies, since the cells were not isolated from the obesity model mice (C57Bl6)? Anthropometric parameters: This study concerns obesity related to glucose tolerance. Although in Table 1 the authors present the weight variations, Results not show whether sage extract effect on Body mass index variation. In addition, the imaging data (Lean and fat mass were measured by Nuclear Magnetic Resonance) for the assessment of fat qualitative mass composition (subcutaneous or visceral) have not been shown. Lipogenesis versus lipolysis: There is confusion in the data to explain the lipids metabolism effects of sage. The authors confirm that sage exerts both lipogenic and anti-lipolytic effects at the same time (Line 267:... however sage-treated animals had a decrease in plasma NEFA and triglycerides levels, suggesting an inhibition of lipolysis…. Line 271:... the plant extract reduced lipogenesis…). Please clarify this point. Sage inefficacity or sage toxicity: Line 317:... This might explain the beneficial effect on food intake of sage low dose when compared to high dose… It’s not clear the dose effect of sage. If sage has no beneficial effect at high doses, can to become toxic? In this case, how will sage be correlated with rosiglitazone? Please clarify this point. Additional comments: Thank you to see critical above

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.

Title: PREVENTIVE EFFECTS OF SALVIA OFFICINALIS LEAF EXTRACT ON INSULIN RESISTANCE AND INFLAMMATION IN A MODEL OF HIGH FAT DIET-INDUCED OBESITY IN MICE THAT RESPONDS TO ROSIGLITAZONE Review round: 2 Reviewer: 2

Basic reporting: This reviewers comments have been adequately addressed. However the reviewer would appreciate that the authors show a toxicity profile of SAGE extract with a more robust assay like annexin v-PI staning. Experimental design: No comments. Validity of the findings: No comments. Additional comments: Acceptable revision. No further comments.

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.

Title: PREVENTIVE EFFECTS OF SALVIA OFFICINALIS LEAF EXTRACT ON INSULIN RESISTANCE AND INFLAMMATION IN A MODEL OF HIGH FAT DIET-INDUCED OBESITY IN MICE THAT RESPONDS TO ROSIGLITAZONE Review round: 2 Reviewer: 3

Basic reporting: None Experimental design: None Validity of the findings: None Additional comments: No additional comments

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.

Title: BEYOND HARM’S REACH? SUBMERSION OF RIVER TURTLE NESTING AREAS AND IMPLICATIONS FOR RESTORATION ACTIONS AFTER AMAZON HYDROPOWER DEVELOPMENT Review round: 1 Reviewer: 1

Basic reporting: Overall, the writing and style of the manuscript is high quality, concise, and easy to follow. The paper utilizes pre- and post-dam data to analyze the impact of a dam’s reservoir on one species, which is an important, but many times neglected, element of understanding the realized impact of hydropower projects. A minimal amount of text (specific suggestions given) added to the methods and results will aid in reader understanding. Given the high predation of turtle nests by humans, the authors should acknowledge the complex impact of dams on local humans, specifically that human-ecosystem relationships change in ecosystems altered by dams. Human predation on the turtles is discussed in one paragraph of the discussion, and there is just one sentence stating that humans in the submergence zone are compensated but that these turtles have not been considered. No acknowledgment is made by the authors that this compensation to humans is rarely, if ever, equivalent to the economic loss suffered by the displaced peoples (McCully 2001). It would be appropriate to briefly discuss the situation of these displaced peoples, if known from personal communication (potentially 244 families according to the EDP annual report 2013), and how this could potentially relate to the increased pressure on turtle nests, especially given the higher density of (and ease of finding) nests. Given the impact of dams on turtle nest-areas, it seems like decreasing human predation on turtle nests would be a critical component of turtle conservation in this area and a necessary component of mitigation efforts (in addition to those mitigation efforts suggested by the authors: BACI studies, restoring bank and island habitat, and protective legislation). Experimental design: The experimental design is well suited to answer the proposed research question, how far upstream the reservoir impacted one species of turtle and whether this result was accurately reflected in the environmental impact assessment. Line 123: Remove “potential and effective” before turtle nest-areas, as the description comes in the following paragraph. Line 130: Please add one sentence clarifying how you determined potential nest-areas. Was this done by walking the banks and mapping all areas in the 66 km stretches of riverbank not covered by rock/boulders/root/clay substrates? Please move the other sentence describing potential nest-areas (Lines 146-147) to this earlier description. Please also state in this description that potential does not include effective (this was made clear by Table 1). What about the nest-area designations being potential and actual (instead of effective) nest-areas? Effective may suggest to some readers that the nest was successful in producing young. Validity of the findings: I found the majority of the results presented in a clear manner. Readers would be interested in the authors’ speculation in the discussion on how changing water levels in the reservoir would affect turtle nesting habitat and how the dam and altered flows could affect migration patterns. Line 171: Why do you state the percentage of area that is reduced along 60.1 km and not the full study area of 66 km? Figure 3: Panels A and B are redundant. Please remove. “C” should be capitalized. Please report an equation and R^2 values on the plots for the lines of fit. Additional comments: Text to add to the “Study area” section: What type of dam is Cachoeira Caldeirão? Who paid for the dam and completed and paid for the EIA? Be consistent in using pre and post vs pre- and post- Include the product information for Rapideye Clayey should be clay Consistent spacing between numbers and units (Line 152) Line 189: Remove the statement of novelty Line 233: Mention that the Congdon paper is referring to the Blanding’s turtle and the location of the study area. Line 289: Add “are” to reservoirs are built

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.

Title: BEYOND HARM’S REACH? SUBMERSION OF RIVER TURTLE NESTING AREAS AND IMPLICATIONS FOR RESTORATION ACTIONS AFTER AMAZON HYDROPOWER DEVELOPMENT Review round: 1 Reviewer: 2

Basic reporting: Summary Norris et al. investigated the effect of the construction of a dam on the reproductive success of an aquatic turtle in the Amazon region. The results indicated that the construction of dams had negative effects on the reproductive success of this species. Additionally, the authors proposed some strategies to mitigate these effects. Overview The article is well written and easy to follow. The analyses are adjusted to theobjectives of the study and the results are adequately discussed. The study has a strong point, which is that it compares the success of nesting before and after the river regulation; however some sections of the manuscript would benefit from further literature support. The study has also important implications for the management of turtle populations in basins impacted by dams. Experimental design: Overview The article is well written and easy to follow. The analyses are adjusted to the objectives of the study and the results are adequately discussed. The study has a strong point, which is that it compares the success of nesting before and after the river regulation; however some sections of the manuscript would benefit from further literature support. The study has also important implications for the management of turtle populations in basins impacted by dams. Validity of the findings: The analyses are adjusted to the objectives of the study and the results are adequately discussed. Additional comments: Specific points Abstract The introduction of the abstract is excessively long (5/13 lines). I suggest to the authors to reduce it. The authors could use this space to explain (in the last sentence) some of the mitigation proposals. Introduction In general, it is well structured, although the authors could consider a more general approach at the beginning of the introduction (e.g. effect of dams on riverine biodiversity). Ln 63-Ln 67. These effects have no interest in the current study, although they could be cited in an introduction to the ecological impact of dams/river regulation. Perhaps a general revision about these impacts could be placed at the beginning of the introduction (including population isolation, reservoirs for invasive species, …). Ln 64-67. These lines could be moved to the end of the introduction and used to formulate and support a hypothesis. Methods The authors could briefly describe the climate of the study area. This would help the reader to understand the nest-area survey timings. They could also (briefly) discuss some aspects of the target species (eg distributional range, natural history -focusing in breeding aspects-, IUCN status) that authors believe could provide more background and strength to their discussion. Non-Brazilian readers may find the article interesting but largely ignore the geographical and ecological frame of this study system. Ln 118. Are there no references in the literature describing the nesting season of P. unifilis? Ln 121. I suppose that the difference in the length transects (2011 vs 2015/2016) is explained by the inclusion of a control transect. It should be detailed. Ln 122. Authors have to specify which are these conditions and include appropriate references. Ln 134. Authors could consider include references to this method of survey. What are the ‘systematic substrate searches’?. Ln 158. The software used to implement the GLM should be specified. Also the distribution family assumed for the dependent variable (nesting beaches / km2). Discussion Ln 248. Authors should consider include these references. Ln 268-279. I find this part of the discussion interesting and ‘ethically’ justified, but completely out of context. In my opinion, the discussion should focus on the subject of study. Here the authors could provide some examples (references) explaining that restoring nesting habitats in aquatic turtles is a successful strategy. This would support the mitigation measures proposed below. Ln 281-294. Apart from the negative impact on nesting, the dam and river regulation may involve some other pernicious effects (some indirect, mediated by predators or competitors) on turtle populations??. Authors should comment this here or just in the previous paragraph.

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.

Title: BOUNDARIES IN GROUND BEETLE (COLEOPTERA: CARABIDAE) AND ENVIRONMENTAL VARIABLES AT THE EDGES OF FOREST PATCHES WITH RESIDENTIAL DEVELOPMENTS Review round: 1 Reviewer: 1

Basic reporting: No comment. Experimental design: No comment. Validity of the findings: No comment. Additional comments: This interesting manuscript is dealing with the identification of boundaries in environmental variation and ground beetle community structure using triangulation wombling. Moreover, the authors tried to determine whether these boundaries are related. The topic of the manuscript is very interesting and relevant, since ecological responses to the presence of habitat edges are one of the most extensively researched topics in ecology. The main strength of this manuscript is the use of a novel and powerful spatial partitioning technique, the triangulation wombling, to identify boundaries. Objectively identifying boundaries is a key topic in edge studies, as different groups of organisms may operate at different spatial scales, and what appears as a homogenous patch to one species may comprise a very heterogeneous patchy environment to another. Earlier this manuscript was submitted to the Ecosphere and the Ecology and Evolution and was thoroughly reviewed. Based on the reviewers’ comments, the authors completely re-wrote and substantially improved the manuscript. The present form of the manuscript is generally well written. The analyses are well explained and straightforward, and the discussion is sound. The most recent edge studies on ground beetles (e.g. Urban Ecosystems, 20: 971-981, 2017; Forest Ecology and Management, 384: 371-377, 2017; Journal of Insect Conservation, 20: 49-57, 2016; Ecological Research, 31: 799-810, 2016), however, are not referred in the manuscript. Please cite and discuss these papers. Based on the above, this manuscript is valuable and should be published.

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.

Title: BOUNDARIES IN GROUND BEETLE (COLEOPTERA: CARABIDAE) AND ENVIRONMENTAL VARIABLES AT THE EDGES OF FOREST PATCHES WITH RESIDENTIAL DEVELOPMENTS Review round: 1 Reviewer: 2

Basic reporting: I am not an expert on beetles, pit traps, nor triangulation-based methods, so I can only comment from a general science and statistics perspective. I don't know the "triangulation wombling" was explained well for a journal of general readership. E.g.: "Second, we calculated a rate of change and its orientation for each triangle using the values of a variable of interest at the triangle’s vertices." Rate of change of what with respect to what? How do you define the "orientation" of a triangle? I had to look up the referenced book to determine that "traingulation wombling" involves a finite-difference method for approximating spatial derivatives in multiple dimensions and orientation is that of the approximated gradient. This method naturally would involve some assumptions about continuity that I doubt are checked in practice, but for detecting "boundaries" that run perpendicular to large gradients perhaps that doesn't matter. From a statistical perspective, the layers of estimation atop the data were a bit uncomfortable. First, finite-difference derivatives are calculated. From that boundaries are detected at p=10% significance and with various thresholds. Then the boundary overlaps were calculated, also at p=10% significance. This looks like a typical use of this method, but I do not find it ideal. Otherwise, the paper looks fine to me. Experimental design: no comment Validity of the findings: no comment Additional comments: no comment

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.

Title: RESOUNDING FAILURE TO REPLICATE LINKS BETWEEN DEVELOPMENTAL LANGUAGE DISORDER AND CEREBRAL LATERALISATION Review round: 1 Reviewer: 1

Basic reporting: The article is generally clear and well written, and the coverage of literature is appropriate. The tables and figures are clear. If anything, the data are over-analyzed, but this is understandable given that the results are largely negative with respect to the hypotheses under test—and with respect to previous claims. Some of the statistical techniques will be unfamiliar to many readers, but as far as I can tell they are appropriate. I think the formula for computing LI should be given, along with more detail as to how their significance was tested for the assignment into laterality groups (binomial? T-test?). The data have been made publically available. Experimental design: One concern with the design is that the study was based on twins, and there might be some question as to whether the results can be generalized to nontwins. This is perhaps a relatively minor issue, although there is some evidence, again inconsistent, that twins may be slightly more likely to be left-handed than nontwins (as claimed, for example, by Annett). It might be worth citing the very large-scale study by Medland et al.. (2009, Neuropsychologia, 47, 330-337) showing effectively no difference in handedness between twins and nontwins, or between MZ and DZ twins. The idea that MZ twins of opposite handedness derive from mirror-splitting of the ovum can probably be dismissed, as the authors suggest, and it might be worth noting that the left-handed member of mirror pairs is typically left-dominant for language—just as left-handers in general are (e.g., Badzakova-Trajkov, et al., 2010. Neuropsychologia, 48, 3086-3093). The authors cite McManus (1981) as evidence that handedness is unaffected by birth stress, although there are (or were) claims to the contrary, and there may be more than just birth stress that differentiates twins from nontwins. It seems that the groups were not completely matched on IQ, and given the standard deviations I suspect the difference may be significant. Of course this partly reflects the language impairment in the DLD group, but I wondered whether they might be matched on nonverbal IQ. It would also have been useful to know whether there were differences between the MZ and DZ twins. I realize that this will be the topic of another report, but I did wonder whether it might qualify the results reported here. The sex difference might also be a (minor) issue, and I think it is generally better to include possible confounding effects in the model rather than dismiss them if their effects are not significant. These are actually fairly minor points, and the results are compellingly negative even if they are considered. Any minor deficiencies in design can be weighed against the fact that the samples were impressively large, and the analysis comprehensive. I might add though that previous reports of significant effects should not be attributed to lack of power, which has to do with Type II error, not Type I error. Validity of the findings: In spite of some minor issues of design, the results are impressively negative with respect to the hypotheses being assessed. Acceptance of a null hypothesis (or rejection of the experimental hypothesis) is always an issue, given the logic of hypothesis testing, but here the p-values are impressively large. I wondered if the finding of a slight right-hemisphere bias for the TD group might reflect different strategies during the fTCD sessions. Perhaps some of the participants were more likely than others to incorporate mental imagery while describing the cartoon action. I wonder if the results would have been different had a task without a visual component, such as word generation, been used. The authors do acknowledge that use of different measures of cerebral asymmetry might yield different results. Additional comments: This is a bold and commendable report, especially given that it seems to contradict some of your own previous findings.

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.

Title: RESOUNDING FAILURE TO REPLICATE LINKS BETWEEN DEVELOPMENTAL LANGUAGE DISORDER AND CEREBRAL LATERALISATION Review round: 1 Reviewer: 2

Basic reporting: The article meets the necessary standards - it is clear, with professional English used throughout. The article does cite sufficient and relevant references although I note that one review that seems very relevant and complementary to this paper has not been cited - Mayes et al Dev Med Child Neurol. I think this was a 2014 or 2015 publication and am sure it focused on MRI evidence for or against the lateralisation hypothesis. It concluded that the field is a mess due to small numbers of participants, inconsistent MRI methodologies in terms of data acquisition and analysis methods etc. Whilst an MRI method was not applied in this paper, I would imagine that the findings of this review would serve to further strengthen the rationale for yet another study in this area (ie you address a number of the limitations of the field reviewed in their paper)? The paper has a professional article structure and is self-contained with relevant results to the hypothesis posed. Experimental design: Question is well defined. Strong participant numbers for this field. Rigorous investigation performed to a high standard. Validity of the findings: No comment Additional comments: This is a careful body of work that builds on past related work by this leader of the field and team. I wonder if there is any room for inclusion of genetics discussion in this paper? Is Clyde Franks and team at Max Planck Institute in Nijmegen working in this space re genetics. Would be worth adding in a few lines on what has been found around hereditary links for language, lateralisation and handedness. Could this be an important missing variable in the debate in this field - ie we keep focusing on brain and behaviour but are genes missing here to make this a more sophisticated argument and get us into more specific genotyping of cases ...presumably this is a highly complex genetic relationship that will not be unravelled for many decades - but perhaps worth a mention as regards future direction...

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.

Title: IS THERE A LINK BETWEEN AGING AND MICROBIOME DIVERSITY IN EXCEPTIONAL MAMMALIAN LONGEVITY? Review round: 1 Reviewer: 1

Basic reporting: citations are missing Experimental design: The issues with experimental design are closely linked to the validity of findings, so these are commented on below. Validity of the findings: This study investigates the anal microbiome of Myotis myotis collected at different localities in France. The authors frame the analysis of microbiome composition in terms of aging, as these bats can be long lived. The authors find no significant differences between sexes or ages, and conclude that this species has a static anal microbiome and that this is relevant to aging. They also comment on the abundance of Proteobacteria and certain functional categories that were inferred and how they think these observations are relevant to aging. There were multiple aspects of the interpretation and data presentation of this article that are problematic. One of the most obvious flaws is that the microbiomes are said to be 'stable' over time. In the discussion it is stated that microbiomes are "unchanging" between ages. Although not explicitly presented, it appears, for example from consideration of the range in number of OTUs observed from sample to sample, that there is considerable variation in this dataset (interindividual). How does one reconcile the wide latitude in differences among individuals with the notion that there is an aspect of stability that helps protect bats from aging? How does the fact that the dataset appears to only include bats that are not at the upper end of the life span influence the ability to design a robust study to look at microbiomes in bat aging? It seems that it would preclude the ability to address the question, and that the comparison of results in humans and mice is not valid. Given that these bats appear to roost in groups, how could microbiome dispersal effects arising from social interactions (Elife. 2015 Mar 16;4. doi: 10.7554/eLife.05224.) erase any age-related patterns? Bats were collected at different localities, but the effect of locality, was not assessed. If only some bats are considered to be long lived (if this is the case), how is the observation that Proteobacteria is apparently a bat-wide association relevant to aging? The discussion of functions inferred from the metagenomes appears to only include a list of general functions that are the most abundant in bacterial genomes in many datasets. I don't see any function frequencies that stand out from the norm for a digestive microbiome, although the authors conclude that the presence of metabolic pathways, among others, is somehow signal for the specific bat-bacteria energetic relationship. It doesn't seem likely that tabulating the frequency of KEGG terms summarized at some ontological level, and summarized across all bacteria in a microbiome, then doing a test for frequency differences between groups would be empowered to understand any age-related functional differences. It may be worthwile for the authors to consider methods presented in this recent work (Integr Comp Biol. 2017 Jun 28. doi: 10.1093/icb/icx011. ). The authors rarefy their dataset. There has been a sound statistical justification for not doing so. Please see (PLoS Comput Biol. 2014 Apr 3;10(4):e1003531. doi: 10.1371/journal.pcbi.1003531.). The authors state that there was "apparent separation of bat to other mammals in PCoA". Visual inspection of the plot does not support this statement (Sup. Fig. 5), but more importantly, such statements must be based on statistical testing, and this is not the case. Although studying the occurrence of competitive exclusion in microbiomes is interesting, it doesn't seem that the putative identification of bacterial genera that belong to families that include pathogens warrents any mention of this ecological phenomenon. There is no functional analysis presented to support that observed lineages compete for niche space with known pathogens. The conclusion of this manuscript is very speculative, and not supported by the data. For example, there appears to be no data connecting Proteobacteria, any of the observed functions, and biology that has effects on longevity. Additional comments: No additional comments

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.

Title: IS THERE A LINK BETWEEN AGING AND MICROBIOME DIVERSITY IN EXCEPTIONAL MAMMALIAN LONGEVITY? Review round: 1 Reviewer: 2

Basic reporting: Graham Hughes and colleagues present an interesting article about the gut (anal) microbiota variation across age in bats. The manuscript is well written and well supported using many references, and the structure of the article is correct. Besides, I believe this is the first paper dealing with this topic in Chiropterans, thus it is a timely and original work. They found no effect of age, which contrasts with the general trend observed in other organisms where microbiota composition varies and becomes more diverse through development. I think this finding is relevant. However, the employed methodologies (or at least the way to present them) have major limitations, and I think they are not appropriate to in depth in the interpretation of the results. I feel that many interpretations of the data and conclusions are too courageous considering the employed approach, namely DNA metabarcoding + Picrust, and I also believe the methodological aspects of the study, or at least the way of presenting them, can be considerably improved. Experimental design: Regarding the methodology, I think there are two major issues that need modification or further explanation. First, I miss information about negative controls. I have personally used the Illumina Nextera kit and I know how easily cross-contamination and amplification of environmental bacteria can occur. Thus, it is necessary to mention how the effect of these putative problems was controlled, because cross-contamination could make samples more similar (resulting in no differences between groups) and diverse (resulting in larger phylogenetic diversity than previously reported) that what they actually are. I don’t say the results of this study are artifactual, but this experimental design definitely requires negative controls to avoid such problems. Second, I am also concerned about rarefaction curves and sequencing depths. I can think about two intepretations of the sentence “Samples were rarefied…“ in the methods section, but both are incorrect from my point of view. FIRST INTERPRETATION (most likely)- First the authors say that the data was rarefied to 8000 OTUs, which I think makes no sense. I think they mean they randomly picked 8000 sequences per sample to level sequencing depth to 8000 sequences. I wouldn’t say that’s rarefying but leveling. Besides, leveling to 8000 OTUs does not make sense, since the number of OTUs of each sample will show the actual diversity. It should be 8000 reads or sequences, depending on when the leveling step was carried out. I guess it was done after quality filtering, thus I would use 8000 sequences. In addition to terminology though, it is not clear how the authors assessed the sequencing depth, and if the employed sequencing depth of each sample was enough for correctly characterizing each amplicon product. They present average values of phylogenetic diversity in Figure 1, but I believe individual information should be provided. In this kind of metabarcoding studies interested in compositional and diversity differences I believe it is necessary to generate OTUs x sequences rarefaction curves for EACH sample, to estimate whether sequencing depth was enough to cover the entire diversity. This can be easily done using the R packages vegan, iNext or DivE. Finally, there are 11 samples with less than 8000 reads according to Table 1, and it is not clear what the authors did with these samples. I think they should be discarded. SECOND INTERPRETATION (less likely) - If what the authors did was to use a rarefaction and extrapolation method (iNEXT r package, for instance) to estimate the diversity at 8000 sequences (not OTUs) regardless the samples had more or less sequences than 8000 this approach is extremely risky, since diversity estimates will have very uncertain when the number of sequences is very low. Either case, these methodological issues should be addressed before publishing. Validity of the findings: The validity of the findings is related to the comments mentioned above. Additional comments: I also have some minor edition suggestions: Abstract “The anal microbiome was sequenced” > make clear you did metabarcoding and not shotgun sequencing, since as it is written now, it seems you have sequenced the whole microbiome, more so when you talk about function in the next sentence. “Functional gene categories expressed in the M. myotis microbiome” suggest you did transcriptomics, which is not true. Thus, I would use “functional gene categories inferred from metabarcoding data” instead. Introduction Line 64: species > organisms Line 67: make the first sentence more explicit. I suggest: “we have used DNA metabarcoding of the bacterial 16S rRNA gene” (which implies the use of HTS) Lines 99-112: it should be written somewhere that the weaning/nursing status of juvenile bats was not possible to determine (as it is said in the discussion). Besides, in the discussion, it would be appropriate to add a sentence about the most probable feeding state of the sampled bats based on capture date and average weaning time of Myotis myotis bats. Lines 120-122: split the library building step and the actual sequencing. It would be more appropriate to write something like: “all 52 samples were built into Illumina sequencing libraries using the Nextera XT (…) and sequenced in a MiSeq platform using 250PE chemistry” Line 123: mention which scripts from Fastx toolkit were used for the quality filtering Line 132: I know Picrust requires Greengenes, but according to my personal experience the taxonomy assignment success is larger using Silva, and ideally different databases should be combined (Silva, RDP, Greengenes, NCBI…) (just a comment for the authors). Line 137: mention the software/script used for rarefaction/leveling/extrapolation… I hope the authors will address all the issues raised in this report and the improved manuscript will be published in PeerJ, as I think it will be a nice contribution to the literature. All the best and good luck Antton Alberdi University of Copenhagen

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.

Title: IS THERE A LINK BETWEEN AGING AND MICROBIOME DIVERSITY IN EXCEPTIONAL MAMMALIAN LONGEVITY? Review round: 2 Reviewer: 1

Basic reporting: No problems. Experimental design: No problems. Validity of the findings: No problems. Additional comments: I believe that you provided a very diligent and well-rounded respond to my previous comments. Good work.

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.

Title: IS THERE A LINK BETWEEN AGING AND MICROBIOME DIVERSITY IN EXCEPTIONAL MAMMALIAN LONGEVITY? Review round: 2 Reviewer: 2

Basic reporting: The authors have addressed all the issues I raised in the previous review round. Experimental design: The authors have addressed all the issues I raised in the previous review round. Validity of the findings: The authors have addressed all the issues I raised in the previous review round. Additional comments: The authors have addressed all the issues I raised in the previous review round.

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.

Title: ACTIVITIES CONCENTRATION OF RADIOCESIUM IN WILD MUSHROOM COLLECTED IN UKRAINE 30 YEARS AFTER THE CHERNOBYL POWER PLANT ACCIDENT Review round: 1 Reviewer: 1

Basic reporting: Comment to manuscript : #20396 Activities concentration of radiocesium in wild mushroom collected in Ukraine after the 30 years from the Chernobyl Power Plant Accident (#20396) General comment: Useful data; can be accepted after some upgrade (also with a recent literature) Suggested improvements: Is: This accident released huge amounts of radionuclides, including radioiodine and radiocesium, into the environment, contaminating the lands of Ukraine, the Republic of Belarus, and the Russian Federation (Taira Y et al., 2011). Suggested correction: This accident released huge amounts of radionuclides, including radioiodine and radiocesium, into the environment, contaminating the lands of Ukraine, the Republic of Belarus, and the Russian Federation (Taira Y et al., 2011). A cases of a hot spot contamination with radiocaesium and high levels in mushrooms has been recorded also in the nearby countries such as Poland, Sweden or Norway while less elsewhere in Europe (Bakken and Olsen 1990; Cocchi et al. 2017; Falandysz and Borovička 2013; Falandysz et al. 2015, 2016; Strandberg, 2004; Zalewska et al. 2016) Examplum gratia: [ Bakken and Olsen, 1990, Accumulation of radiocaesium in fungi. Can J Microbiol 36:704-710 Cocchi et al. , 2017, Radioactive caesium (134Cs and 137Cs) in mushrooms of the genus Boletus from the Reggio Emilia in Italy and Pomerania in Poland. Isotopes Environ Health Stud 53:doi.org/10.1080/10256016.2017.1337761 Falandysz and Borovička. 2013, Macro and trace mineral constituents and radionuclides in mushrooms – health benefits and risks. Appl. Microbiol. Biotechnol. 97, 477-501 (2013). Falandysz et al. 2015, Evaluation of the radioactive contamination in Fungi genus Boletus in the region of Europe and Yunnan Province in China.. Appl. Microbiol. Biotechnol. 99, 8217-8224. Falandysz et al. 2016, Determination of activity concentration of 137Cs and 40K in some Chanterelle mushrooms in Poland and China J Environ. Sci. Poll. Res. 23, 20039-20048. Strandberg, 2004, Long-term trends in the uptake of radiocesium in Rozites caperatus. Sci. Total Environ. 327, 315-321. Zalewska et al. , 2016, Radiocaesium in Cortinarius spp. mushrooms in the regions of the Reggio Emilia in Italy and Pomerania in Poland. Environ. Sci. Poll. Res. 23, 23169–23174.] Is: Radiocesium is known to concentrate in wild mushrooms (Hoshi et al., 2000; Travnikova et al., 2001; Hoshi et al., 1994; Kakuda et al., 2006; Smith et al., 1993; Bulko et al., 2014). Suggested: Radiocesium is known to concentrate in wild mushrooms (Hoshi et al., 2000; Travnikova et al., 2001; Hoshi et al., 1994; Kakuda et al., 2006; Smith et al., 1993) and, as mentioned, the most contaminated originated from the regions of Ukraine, Gomel in Belarus or a countries north and west of Chernobyl (Bakken and Olsen 1990; Bulko et al., 2014; Falandysz et al. 2015; Grodzinskaya et al. 2003 and 2013). A.A. Grodzinskaya, M. Berreck, K. Haselwandter, S.P. Wasser. Radiocesium contamination of wild-growing medicinal mushrooms in Ukraine. Int. J. Med. Mush. 5, 61-86 (2003). A.A. Grodzinskaya, S. A. Syrchin, N.D. Kuchma, S.P. Wasser. Macromycetes accumulative activity  in radionuclide contamination conditions of the Ukraine territory. Part 6. - P.217-260, 368-373. In: Mycobiota of Ukrainian Polesie: Consequences of the Chernobyl disaster. Kiev: Naukova dumka, 2013. (In Russian). Is: ……. locally produced foods, especially edible wild plants and wild mushrooms, which are traditional parts of the regular diet (Orita et al., 2016). Suggested: ……. locally produced foods, especially edible wild plants and wild mushrooms, which are traditional parts of the regular diet (Orita et al., 2016). Is: …. radiocesium in each type of mushroom .. Suggestion: … type? What do you mean? (should be “species of mushroom”)? Is: … (p<0.01) and L. scabrum (p<0.01) …… Proper record is: (p < 0.01) and L. scabrum (p < 0.01) ….. Is: Our study has several limitations. First, we could not evaluate the relationship between radiocesium concentrations in mushrooms and the concentrations in in the soil. Second, additional analytical uncertainties arose because the committed effective doses from dietary intake of mushrooms cannot measure the day-to-day variations in individuals in Ukraine. Suggested: Our study has several limitations. First, we could not evaluate the relationship between radiocesium concentrations in mushrooms and the concentrations in in the soil. Second, additional analytical uncertainties arose because the committed effective doses from dietary intake of mushrooms cannot measure the day-to-day variations in individuals in Ukraine. A household treatment can have a high impact on content of metallic elements and radionuclides in cooked mushroom and usually a decrease of activity concentration can be expected (Drewnowska et al. 2017; Steinhauser and Steinhauser, 2016)) but this aspect was not considered in our assessment. Drewnowska et al., 2017. Leaching of arsenic and sixteen metallic elements from Amanita fulva mushroom. LWT - Food Sci technol 84:861–866 Steinhauser and Steinhauser, 2016, A simple and rapid method for reducing radiocesium concentrations in wild mushrooms (Cantharellus and Boletus) in the course of cooking. J Food Prot 79:1995–1999 Other remarks: All results (data) have to be rounded and only two significant figures have to be shown!!! Not: 583 (27–1,838) But: 580 (27–1,800) It also matters as relates to assessed exposure (mSv). Experimental design: Is Ok. Validity of the findings: Good Additional comments: Useful data; can be accepted after some upgrade (also with a recent literature); look into a detail comment

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.

Title: ACTIVITIES CONCENTRATION OF RADIOCESIUM IN WILD MUSHROOM COLLECTED IN UKRAINE 30 YEARS AFTER THE CHERNOBYL POWER PLANT ACCIDENT Review round: 1 Reviewer: 2

Basic reporting: There are many important literature references missing in the text regarding radiocaesium concentration in mushrooms in areas affected by Chernobyl accident. For example, there is no mention to the ban of mushroom collection, and how it affected the internal dose due to their consumption. Raw data are shared. A map showing sampling points is missing. Experimental design: The analysis of the radiocaesium concentration in mushrooms in Ukraine and how it may be related to those collected after Fukushima accident is an interesting topic. However, there are only data for three species of mushrooms in Ukraine and none for Japan. The authors use raw and dried mushrooms, this is quite confusing, since a dried mushrooms is not cooked. It would be better to express this in terms of fresh and dry mass. It is not clear if the samples were measured dried or not. Validity of the findings: No comment Additional comments: The novelty and the importance of the manuscript is very low, as it only gives the mean value of three species of mushrooms from Ukraine. There is no comparison with data from mushrooms collected just after Chernobyl accident. There are many significant references missing. The authors only focused on previous work done mainly by Japanese scientist in that area. In my opinion, this manuscript show only that the authors collected a lot of mushrooms samples and measured them. Therefore my recommendation is rejection.

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.

Title: ACTIVITIES CONCENTRATION OF RADIOCESIUM IN WILD MUSHROOM COLLECTED IN UKRAINE 30 YEARS AFTER THE CHERNOBYL POWER PLANT ACCIDENT Review round: 1 Reviewer: 3

Basic reporting: The article is nicely written, clear and lucid English. Sufficient literature citation. I just mention one more article in line 52, 53 of introduction part. Six references have been cited. The following reference, which is newer than some of the reference cited also strongly advocates your comment in the relevant section. Therefore may be included: B Mukhopadhyay et al., J. Radioanal. Nucl. Chem. 273 (2007) 415-418. Experimental design: Research question is well defined, relevant and meaningful. Methods described with sufficient detailing. According to APS, now year is abbreviated as 'a' (annum), when half life of a radionuclide is described. Please change '30 y' to '30 a' while mentioning half-life of Cs-137. In line 94 you described the detection limit of 137-Cs 28.5 Bq/kg (as you have calculated from median value). But it must be much less for HPGe detector for 137 Cs. I think you should calculate detection limit of 137-Cs from the background. You have not mentioned how much dry mushroom was taken for measurement of 137Cs and in what geometry. This is important information for gamma spectrometric measurement. Validity of the findings: Data is robust, statistically sound and controlled. I would be nice if you (i) describe any significant physical variation of the mushrooms from contaminated regions and non-contaminate regions taken from anywhere of the world. (ii) In figure 3 and 4, first data has very high error as calculated by you. Can you opine for this in discussion section. Additional comments: As mentioned above.

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.

Title: ON REVEALING THE GENE TARGETS OF EBOLA VIRUS MICRORNAS INVOLVED IN THE HUMAN SKIN MICROBIOME Review round: 1 Reviewer: 1

Basic reporting: The article has to be rewritten mainly for the language. The approach is novel yet there are a few important lacunae, the most obvious being how does the work relate to African population where Ebola is prevalent. Are there any studies on the skin biome from African regions where Ebola is Endemic? How often is Ebola transmitted through skin contact? Experimental design: Use of different strains of the bacteria may further help to expand the study and verify if microRNAs are indeed important. Are any other proteins closely associated with the pathological process that could be identified through this approach? For example any other cytokine? Validity of the findings: The findings look interesting and a little more detail study explaining why macrophages and not DCs maybe relevant in this case needs to be supported.The skin is home to the Langerhans cells which are mostly associated with the immune response which the authors have not described in detail. Additional comments: Please check for grammar and spelling

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.

Title: ON REVEALING THE GENE TARGETS OF EBOLA VIRUS MICRORNAS INVOLVED IN THE HUMAN SKIN MICROBIOME Review round: 1 Reviewer: 2

Basic reporting: Overall, the writing is clear. The structure of the paper is complete except that a conclusion or discussion is missing. Experimental design: The scientific question is novel. More evidence and discussions need to be provided to make a valid conclusion. See below. Validity of the findings: The experimental data is controlled. However, in Figure 3, the p-value has not enough power for it was calculated using only 3 data points. Additional comments: This paper discovers short RNA sequences in P. acnes that might act as microRNA to regulate THSD4. It also found that the short RNA sequence PA-mir-2-3p-5 from P. acnes and micro EBV-miR-2-3p from Ebola can synergistically regulate this gene, through which they claim that these short RNA sequences may protect human from evasion of Ebola. The idea of this paper is novel and very interesting. However, I have several major concerns: 1. Although the short RNA sequence PA-mir-2-3p-5 from P. acnes and microRNA EBV-miR-2-3p from EBOV can synergistically regulate THSD4, the roles of those short RNA sequences in protecting human from evasion of EBOV is not clear. More evidence should be provided to show how P. acnes may prevent EBOV from evasion of the human. 2. It is not clear that increasing of the expression of the gene THSD4 play an important role in the human immune system. In Figure 3, both PA-mir-2-3p-5 and EBV-miR-2-3p can upregulate the expression of THSD4 mRNA. However, the functionality of P. acnes and EBOV are completely different. Please provide some discussion on the possible functionality of THSD4. 3. It would be helpful to explain the reason of using P. acnes in this study. Is it possible for other bacteria, rather than P. acnes, to play an important role in preventing EBOV from evasion of human? Minor comments: 1. Several grammar errors: line 83-84, the sentence is composed by two sentences. Thus there should be a dot in the middle, not a comma line 123, should be a dot before TargetScan line 145, should be a comma after 48 hr line 155, missing subject before “provide" line 181, that are complementary line 190, should be “is” not “has” line 221, should be a comma after "control" 2. Typos line 130, nucleotides 2-7 line 109, the font of the capital T is different. Table 1, last row, Human[40], where is [40] from? 3. line 91-92. Thus, miRNAs constitute one of the most abundant classes of gene regulatory molecules in animals. The abundance of miRNA in human is not necessarily applied to in other animals. Please provide other evidence to support this statement.

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.

Title: ON REVEALING THE GENE TARGETS OF EBOLA VIRUS MICRORNAS INVOLVED IN THE HUMAN SKIN MICROBIOME Review round: 2 Reviewer: 1

Basic reporting: No comment Experimental design: No comment Validity of the findings: Transmission is through body fluids or broken skin indicating contact with the blood/lymphatic system.So again the question remains how biome on intact skin are related to Ebola transmission. Additional comments: The role of Langerhans Cells seems to be vital for transmission of the virus through skin. So how do micrbiomes affect the Mobility of LCs?

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.

Title: ON REVEALING THE GENE TARGETS OF EBOLA VIRUS MICRORNAS INVOLVED IN THE HUMAN SKIN MICROBIOME Review round: 2 Reviewer: 2

Basic reporting: The conclusion was added at the end of the result section. Experimental design: no comment Validity of the findings: no comment Additional comments: First of all, thank the authors for the revision. The paper is much easier to read now. The conclusion is still a little confusing that "our findings demonstrated that these P. acnes sequences may act as miRNAs and protect humans from EBOV infection by regulation of the expression of their common target gene THSD4.". The first half of the conclusion is derived based on the results in Figure 2. But the second half of the conclusion is still not supported. By what kind of mechanism that P. acnes prevent EBOV from evasion of the human with the help of THSD4? Since this is the key finding in the paper, please provide rigorous investigation. Otherwise, please limit the statement to supporting results alone.

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.

Title: GEO-REFERENCING BIRD-WINDOW COLLISIONS FOR TARGETED MITIGATION Review round: 1 Reviewer: 1

Basic reporting: Clear, concise, an accurate professional English used throughout. Except for Nelson (2015) which I may have missed in the text, all other citations are appropriately in text and the Reference section. The figures and supplemental materials are useful, appropriate, and complement the text. Meets all your standards in my view. Experimental design: Although a rigorous experimental design is not appropriate given the objectives of this study, the authors adequately explain this in detail and provide extensive justification for what was done, how, and the marked value for future studies; fundamentally, providing a means of gathering more efficient and comprehensive quantitative data on incidents of these important conservation events. Ample information and description is provided for replicating and enacting the proposed advancements. Meets all your standards in my view. Validity of the findings: Introduces and encourages an enhancing data gathering technique to aid current and future work on this subject. Conclusions are generally meaningful, useful, and supported by case study results presented and interpreted. A valuable contribution is the comparison to other online sources (programs - eBird, FLAP Mapper, but why not dBird (?) from New York City Audubon) with similar intents. The authors offer detailed and convincing evidence that their recommended iNaturalist use (methods of data entry) offers the most utility. Meets all your standards in my view. Additional comments: Compliments for recommending a useful and valuable tool to further study this important conservation issue for birds and people.

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.

Title: GEO-REFERENCING BIRD-WINDOW COLLISIONS FOR TARGETED MITIGATION Review round: 1 Reviewer: 2

Basic reporting: I feel the manuscript could be tightened up in places, though my criticisms are all quite minor. They are detailed here. Presentation of information: Introduction - Line 20: I would mention the leading cause of anthropogenic bird mortality and how bird-window collisions compare in terms of number of casualties. Lines 27-34: I suggest combining the first three paragraphs into two, with the break after “(i.e. ‘greening’)”. The current paragraph breaks interrupt the narrative. Lines 34-50: This is useful as a case study but the paragraph overall is difficult to read due to the many internal citations. How stable are the urls cited here? Are they all necessary? Consider paring down this paragraph. Lines 61-64: This sentence (“The goal of….”) is awkward in context; needs smoothing to better align with previous and following sentences Methods - Lines 99-101: You should mention explicitly what led to Duke's decision to retrofit the building. One glaring omission from the Methods is the survey methodology itself. When were surveys done? What time of day? What was done with the carcasses - were they salvaged and deposited in a museum? Was any information noted on weather conditions, wind direction, amount/height of vegetation surrounding windows, etc? How exactly was iNaturalist used on the ground during your surveys? Results - Lines 155-161: I think this background - specifically, why birds collide with windows, including references dating back to 1946 - should be raised earlier in the paper. By doing this, the summary of the effectiveness of the retrofit would be more succinct. Lines 163-165: Reference these percentages to survey years - 2014-2016. Line 168 - this is important to highlight, that retrofitting can successfully eliminate collisions! I would add emphasis and broader context to this point. Minor copy edits: Line 13 - throughout the paper, you use data as a singular noun. While technically correct either way, I think your sentences read better when "data" is treated as plural. Line 39 - the franchise has been, not have been Line 44 - insert comma after "collisions"; insert "the" after "and" Line 51 - insert comma after "limited" Line 57 - see comment on "data" above Line 66 - smartphone should be one word Line 110 - "that have been" Line 112 - cities, not areas Line 115 - "four-conjoined" should not be hyphenated Line 124 - delete "building" Line 132 - delete "is" Line 163 - insert comma after "Center" Line 172 - insert comma after "collisions" Line 177 - delete "a" Line 184 - see comment on "data" above Experimental design: No comment (see "Validity of the findings" below) Validity of the findings: I think this is a great paper that furthers the cause of citizen science initiatives by introducing iNaturalist as a tool to provide useful and replicable data in a specific context. Although the design of the present study is beset by problems, including small sample size and lack of controls, the authors are well aware of this and state explicitly that their conclusions should be interpreted with this in mind. To me, the chief contribution of this paper is to highlight the rigor that iNaturalist provides over subjective descriptions of survey locations provided by study participants. This is something that is badly needed in many citizen science endeavors, and widespread adoption of the methodology presented here will improve the quality and replicability of all studies for which georeferenced localities are an essential component of the dataset. The study therefore has applicability far beyond documentation of bird-window collisions, and this broader benefit to the scientific community far outweighs any shortcomings in their survey design. I would add that, based on my own experiences monitoring bird-window collisions, I am quite confident that greater sample sizes would only bolster their conclusions, and that appropriate controls would be difficult or impossible to incorporate into any study on bird-window collisions, considering the practical and legal issues involved with surveying buildings. The authors have done a commendable job within these constraints. Additional comments: No additional comments

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.

Title: GEO-REFERENCING BIRD-WINDOW COLLISIONS FOR TARGETED MITIGATION Review round: 1 Reviewer: 3

Basic reporting: The writing is clear and understandable with only minor edits needed, see below. The literature cited is satisfactory but does include several incomplete references. Experimental design: The authors point out that there is insufficient experimental setup to warrant statistical analyses. The reporting is mostly qualitative in nature but is sufficient given the nature of the approach. Validity of the findings: The authors have done a good job of summarizing the data. Additional comments: Manuscript 21227 PeerJ Overall Assessment The authors report on a new method to better track the location of birds killed by window strikes. Most studies simply record the wall of the building, or perhaps the direction of the wall, at a very coarse scale. Without more specific information, attempts at mitigation may be more difficult if there are particular areas of the wall that are more likely to be prone to bird strike. The authors argue that GPS locations are better than simply compass directions. The authors apply these findings to two other locations where data was input using iNaturalist, a citizen science program for reporting natural history observations. The authors recommend using iNaturalist in an effort to thwart large-scale bird-window collisions. The authors point out that a recent approach used by Loss et al (2015) involves a google form datasheet that lacks a space for GPS coordinates. In addition, the data platforms recommended by Loss et al do not contain any public datasets so the present authors warn that these databases are not being used by scientists or members of the public interested in bird-window kill data. As a result, the authors recommend yet another platform, iNaturalist, because it allows for GPS coordinates, crowd-sourced identification, and citizen science data collection. Because the dataset is small and without proper control design, the authors do not use statistics but, instead, report the results as qualitative statements. I think figure 1 illustrates quite well what the issues are relative to using simple cardinal direction vs GPS coordinates. The kernel function in figure 1C and the point density function in 1D show clearly where the problem areas on the buildings are. Overall I think this approach is important and will make a nice contribution to the burgeoning field of bird-window mortalities. Line Edits 20 If you are going to tease the reader with “second-greatest”, you should probably let them know what the highest source is. 25 “bird-deterrent films”, compound modifier should be hyphenated 29 Inconsistent single vs double quotes for quotations. See, for example, line 227 where the authors write “zoom in as you see fit” 39 franchise is singular so it should be “has been publicly” 40 The authors imply that it was up to the Minnesota Vikings to invest in a bird-friendly facility. But, the stadium is not owned by them. “U.S. Bank Stadium is owned by the state of Minnesota, represented by the Minnesota Sports Facilities Authority. The Minnesota Sports Facilities Authority (MSFA) was established by the legislature in 2012 and charged with the design, construction and operation of U.S. Bank Stadium. The MSFA consists of five members and is chaired by Michele Kelm-Helgen. The other members are John D. Griffith, Tony Sertich, Bill McCarthy and Barbara Butts Williams. Ted Mondale serves at the CEO/Executive Director.” Source: https://www.usbankstadium.com/stadium-info/faq. As such, it seems the criticism should be redirected to the state of Minnesota, not to the Vikings. 42 The authors should reference the statement “according to the popular narrative…” 57 Data are plural so it should read “so data are not always” 65 coordinates are plural so it should be “suggested by Loss et al. (2015) are GPS coordinates” 94 Better to use “autumn” instead of “fall”. The latter is generally only used by North Americans. 96 Statement about methods being adopted by dozens of campuses should be referenced. 110 Suggest inserting “that” so it reads “Two urban areas in the United States that have been” to match the syntax “are downtown….”. 129 The abbreviation “Penn” has not been formally introduced. 136 Better to write “collisions reported” 233 (and 45) Better to use the full name National Audubon Society. “Audubon” is short form. Reference Inconsistent capitalization for titles. Bayne et al uses Sentence style. But, Borden and Lockhart capitalize each word. Audubon reference is incomplete. Forbes reference is incomplete. Klem 1990 does not list the journal name. Nelson reference is incomplete. Ocampo-Peñuela et al. 2016 does not list journal name.

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.

Title: ON THE EXPONENT IN THE VON BERTALANFFY GROWTH MODEL Review round: 1 Reviewer: 1

Basic reporting: the English is clear and references and background context are sufficient. No issues with the structure of the papers , figures or the tables. article met these criteria. Experimental design: the experimental design area meets the criteria. The research question is defined and relevant. The investigation was reported and carried out to technical standards. The authors appear transparent in addressing issues with the data sets and analyses. Methods reported in sufficient detail. Validity of the findings: the authors are clear on the validity of their findings. The authors used data from numerous sources for their analyzes and were transparent and the conclusions are well stated. There is no over speculation. Additional comments: Line 23 – size but also mass of the animal Line 55 – how many of these data sets included repeated observations (measurements ) on individual animals? Really better analyses If mixed model approaches used like that of Strathe et al. ? Strathe, A. B., A. Danfær, H. Sørensen, and E. Kebreab. 2010. A multilevel nonlinear mixed-effects approach to model growth in pigs1. J. Anim. Sci. 88:638-649. doi:10.2527/jas.2009-1822 Note line 81 – BW to the 0.75 is used widely in animal sciences as metabolic size. Line 194 -195 – did you also check enough points before and after the expected inflection point ? Data with 1 or 2 points above or below the IP – results in greater SE for the model parameters .. it is not clear why the transformation was done in figure 6 and how often this was done in the data sets note the variation in Bw increases as animals grow and ordinary least squares do not account for this and using the means at each age as mentioned in line 203 --- does not really take the equal variances into account as the SE of the BW means increase as BW increases – it should be discussed what was assumed and the better methods of doing the statistics when actual data is available included mixed models and accounting for heterogeneous variances

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.

Title: ON THE EXPONENT IN THE VON BERTALANFFY GROWTH MODEL Review round: 1 Reviewer: 2

Basic reporting: The manuscript is generally well-written. Yet, I recommend authors to address the following: 1) Authors present parts of their methods and results in other sections of the manuscript. Please move lines 57-58 (including Fig 1) and lines 387-401 to the result section. Secondly, please move line 33 “this paper assumes b=1”, Lines 100-101 “Figure 2 displays ....”, lines 115-118 and lines 356-359 to the method section. 2) Multiple times the line of reasoning is unclear to me. Please clarify: o Lines 26-30 and how this relates to the text before. Improved otolith analysis is not an application. Why do authors also write a full sentence about starfish populations? This is not related to their own work. Please broaden the first paragraph of the introduction. Potentially with text from lines 119-122. o Explain 41-43 in more detail. Explain why authors describe the inflection point? Is this detail needed in the introduction? o Again for lines 72-77, is this amount of detail needed in the introduction? If so, please rephrase. o Lines 117-118. Is this comment related to the literature or to the work described in this study (if the latter, please move to method section). o Lines 122-126 change to: In addition, a search in google scholar identified approximately 24800 papers related to the von Bertalanffy growth model in fish (using the search terms “...”). The use of VBGF in the literature was also specifically surveyed for elasmobranch species, showing that the VBGF model was studied twice as often as any other model (Smart et al. 2016). o Lines 146-148: what is the “weight” at the inflection point. Secondly, how can it vary 0% of mmax. Please clarify. o Lines 368-369 Why is this sentence included? For which type of animals do authors expect sex change? Has this been reported in the data they use? 3) Authors use Excel annotation to describe how they analyzed their data (e.g. line 225, 230, 232-235, 249). This makes the text unclear. Please rephrase (to standard text annotation) and include all Excel code in supplement. 4) Change lines 155-193 from text into a table with numbers, names, references and additional information on the growth data (number of time steps (days, months, years), whether the data is based on an individual growth trajectory or the mean of a group and whether the original data is in length or weight). Experimental design: The authors did a very good job to get an extensive dataset on growth to analyze their research question. The question is well-defined and highly relevant. No further comments. Validity of the findings: The work essentially shows that there is large variability in parameter a when other parameters (mmax, q and m0) are also optimized. This highlights that different combinations of these 4 parameters fit the data best, but that the data quality is not good enough / the growth model too flexible (with many free parameters) to quantify parameter a (and q, mmax and m0) in a consistent way. What is interesting is that in many species parameter a can vary over a very large range (between 0-0.99) without affecting the fit to the data. This is to me the main finding of the paper and it is also described by the authors in lines 410-414. Given that combinations of the other parameters affect parameter a so much, I have some suggestions/comments about the other parameters: o The mass at age 0 (m0) is largely similar for fishes (see Andersen et al 2008 Theoretical Population Biology), while m0 might be known for the other species in the dataset. It is perhaps better to derive m0 from the literature (instead of optimizing this parameter). This will reduce the flexibility of the optimization, since there are less “free” parameters, and hopefully lower the parameter space that can fit the data in an adequate way. o Authors have constrained Mmax and assumed that this parameter needs to be higher than the maximum observed mass. Authors state mmax can exceed the maximum observed mass up to 100 times (lines 275-276). I’m not convinced with this as any mmax that is ~2-100 times larger than the observed maximum basically shows that the data is not strong enough to do the analysis (no data point close to mmax). This is very important as uncertainty in mmax is directly affecting parameter a (as these parameters are dependent on each other in the model). Hence, I would suggest removing all the species that have a mmax that is much higher (e.g. 2 x as high) than the maximum mass observed in the data. o Finally, it is not clear to me why authors did not optimize parameter b. Is there evidence in the literature that b should be 1? How will variation in b affect the optimization of parameter a? Furthermore, I suggest to focus less on the fact that a=0.67 is weakly universal for fish, also because the percentage of non-rejection (fig 8) varies weakly when parameter a is larger than 0.65 (probably the effect of 1 or 2 fishes). If authors would have chosen a threshold of 80% or 85% (see line 106), all values higher than 0.65 of parameter a would be defined as weakly universal. It now seems as if VBGF is supported by the data, while the 0.75 exponent not. However, the differences are very small and as such I find the choice of threshold (90%) a bit misleading. Lastly, the difference between fish and non-fish is also a difference between highly controlled experiments (where food is not limiting) (non-fish) versus natural conditions (most fish). As such, I would not do a comparison as in Table 3 and only show that both fish and non-fish species have a large range of values for parameter a that fit the data adequately. The difference between fish and non-fish seems to be an artefact and authors provide no biological explanation why fish potentially differ from other taxonomic groups. As such, I also recommend removing lines 18-19 in the abstract. Additional comments: Line 47 Can authors provide more explanation what (mt/mmax)^1-a exactly is. And what are the units (mass per time)? Line 83 please explain “noisy data may hide” or remove. Lines 201-202 please clarify this sentence. Lines 212-215 please explain how this is standardized across the 60 datasets? Lines 217 Why has parameter b a value of 2 in Figure 5? Previously it was stated that the value of parameter b is assumed to be 1, please explain? Line 295 remove “that” Lines 294-298 maybe add “ FNR shows the parameter space that fits the data adequately. The lower the FNR the less uncertain the value of parameter a”. Lines 299-300 please clarify where this is justified (discussion section?). Lines 402-409 I’m very much agreeing with the conclusion, yet the conclusion is not supported by the last lines of the abstract. Please rephrase the ending of the abstract. Lines 413-414 do authors think this is only a data quality aspect or perhaps also the result of a growth model with too many “free/ unknown” parameters? Figure 1 which m0, mmax and q are used for the two lines? Do the lines differ in these parameters? Figure 5 what is the selection criterion from Knight 1968? Is this explained in the method?

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.

Title: ON THE EXPONENT IN THE VON BERTALANFFY GROWTH MODEL Review round: 2 Reviewer: 1

Basic reporting: English is clear -- and revisions improved the paper Experimental design: the question is well defined -- and revisions add clarity --- no issues Validity of the findings: findings are valid -- no issues -- conclusions stated well -- Additional comments: the revisions have added clarity to the paper -- less potential reader misunderstandings with the revisions. --ready to be published -

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.

Title: ON THE EXPONENT IN THE VON BERTALANFFY GROWTH MODEL Review round: 2 Reviewer: 2

Basic reporting: no comment Experimental design: no comment Validity of the findings: no comment Additional comments: The authors did a good job in revising the manuscript. Below some minor comments to improve clarity. Line 35 maybe rephrase to “and applications in ecology, e.g. understanding outbreak dynamics (Pratchett 2005). Lines 62-69 This is a bit confusing as the first sentence is about mass growth, while the rest relates to length growth. As authors state later, typically length growth with exponent a=0 is equal to mass growth with exponent a=2/3. Hence, “a=0” at the start of line 63 is not equal to the second “a=0” in the same line. Maybe call the length exponent aL. Line 64 Note that Koch et al 2015 is about shellfish. Please change reference or sentence. 198-208 not fully clear when these tests are used in the analysis, e.g. what are location parameters and where are they compared? Lines 218-243 could be moved to discussion Lines 253-254 maybe include, “resulting in 100 models for each dataset” as authors refer in line 304 to “the 100 models”. Line 442 change “here” to “there” Line 450 I suggest to rephrase “model (1)” to be understandable for someone who only reads the conclusion paragraphs Line 452 perhaps add: as the biological meaning of the model exponent, and the variation in the exponent, is not clear. Line 453 Authors could discuss the consequences of their findings a bit further (perhaps in relation to fisheries management). What do the findings imply in relation to the widely used estimated growth parameters in FishBase and the approximately 25,000 other papers that modeled fish growth? More a matter of style, but I would rephrase full sentences in round brackets, e.g. line 212-213,310-311, 425-426 Figure 2: maybe include mmax and minfl in fig2a Figure 9, the example does not show that the combination of male and female data leads to “higher residuals making refutations more difficult and increasing FNR” (line 417-418), right? Yet, it shows there are large differences between males and females. So maybe move reference of figure 9 to line 416-417 “In case that these groups had a different growth pattern (different optimal exponents) (Figure 9)” Figure 9 include y-axis label

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.

Title: SMALL-SCALE SCREENING OF ANTICANCER DRUGS ACTING SPECIFICALLY ON NEURAL STEM/PROGENITOR CELLS DERIVED FROM HUMAN-INDUCED PLURIPOTENT STEM CELLS USING A TIME-COURSE CYTOTOXICITY TEST Review round: 1 Reviewer: 1

Basic reporting: no comment Experimental design: Well-designed Validity of the findings: The finding that the slow-acting drug, methotrexate has cytotoxic effects on human iPS cell-derived NSPCs specifically but not on human tissue-derived NSPCs is new and interesting. Additional comments: This article provides very valuable information for the development of drug discovery toxicology, and could be published if the authors modify the text as directed. 1, Time-course assay was performed by image-based and noninvasive procedure. I think that it is better for the authors to emphasize this a little more. Because the acceptance of image-based morphological assays of cells and living matters has been increased with advances in information technology currently. 2, Authors should describe a relational expression to calculate the percentage of neurosphere size indicated in y-axis of Fig. 3. 3, Authors should indicate the number of samples used in the experiment.

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.

Title: SMALL-SCALE SCREENING OF ANTICANCER DRUGS ACTING SPECIFICALLY ON NEURAL STEM/PROGENITOR CELLS DERIVED FROM HUMAN-INDUCED PLURIPOTENT STEM CELLS USING A TIME-COURSE CYTOTOXICITY TEST Review round: 1 Reviewer: 2

Basic reporting: The paper is well organized. Experimental design: 1. In the time-course assay, the drug responses of cells may be varied by the neurosphere sizes. Why did the authors use the cell density of 200 cells/micowell? 2. Did the authors exchange the culture medium including drug in the time-course assay? Validity of the findings: This study describes the efficiency of time-course cytotoxicity assay using 3D (neurosphere) culture. The idea that this assay applies as a backup safety measure for stem-cell therapies is unique and original. Furthermore, this assay has potential of wide use for drug screening method, because the protocol is very simple. The findings are novel and consistent. However, the following point is unclear. It is difficult to understand the degree of cytotoxic effect in the time-course assay, compared to the endpoint assay. Please add the discussion about the criteria for judgment of cytotoxicity on the time-course assay. Additional comments: No additional comments

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.

Title: SMALL-SCALE SCREENING OF ANTICANCER DRUGS ACTING SPECIFICALLY ON NEURAL STEM/PROGENITOR CELLS DERIVED FROM HUMAN-INDUCED PLURIPOTENT STEM CELLS USING A TIME-COURSE CYTOTOXICITY TEST Review round: 1 Reviewer: 3

Basic reporting: No comments Experimental design: The authors wisely combined two methods, one using the ATP measurement for cell proliferation/viable cells and cytotoxic effects of drugs after 2 days and the second (MSA assay) to follow the real time effects of the drugs by measuring the neurosphere size during 7 days. This expermimental design was relevant and meaningful. Validity of the findings: The combination of these two methods allowed to refine the effects of drugs concentration and with time. These results allowed to classify cisplatin and etoposide as fast-acting drugs with early cytotoxicity, and mercaptopurine and methotrexate as slow-acting drugs with late cytotoxicity. These results highlighted the need to reassess cytostatic drugs from the viewpoint of their time-dependent action. These effects were obtained in vitro and should be confirmed in vivo. Additional comments: This manuscript approaches scientifically how to overcome the risks of teratoma formation and the overgrowth of transplanted cells. This manuscript describes concicely but very convingly and elegantly the specificity of drugs able to reduce the potential tumorigenicity of transplanted cells. The paper is well written with figures highly informative.

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.

Title: SMALL-SCALE SCREENING OF ANTICANCER DRUGS ACTING SPECIFICALLY ON NEURAL STEM/PROGENITOR CELLS DERIVED FROM HUMAN-INDUCED PLURIPOTENT STEM CELLS USING A TIME-COURSE CYTOTOXICITY TEST Review round: 1 Reviewer: 4

Basic reporting: The article is well written, clear description of findings. The topic is very important, Experimental design: The experimental design is good, although the work would benefit from inclusion more compounds into studies Validity of the findings: The findings are valid, but somewhat limited Additional comments: The recommendation would be to extend study to include more compounds. Also, the paper would benefit from more discussion of challenges of cell therapies and guidance for the characterization of stem cell tissues for transplantation. For example, recent works of S. Yamanaka were related to characterization of proliferation potential of stem cell derived tissues and also genotyping of cell lines used for transplantation. More discussion may be beneficial for the paper.

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.

Title: MURINE PLURIPOTENT STEM CELLS THAT ESCAPE DIFFERENTIATION INSIDE TERATOMAS MAINTAIN PLURIPOTENCY Review round: 1 Reviewer: 1

Basic reporting: The English language needs to be improved throughout all parts of the manuscript to ensure that the text becomes clearly understandable. The language should be reviewed by a native English speaking colleague or a professional service for language editing. I will try to exemplify this for parts of the abstract only: The first half-sentence of the background is understandable; afterwards the reader has to guess what the authors want to say. Methods: abbreviations such as MES, EGFP, iPSCs are not defined and at least MES is an ambiguous abbreviation. Moreover, iPSC are generated by reprogramming, which might involve the transduction of cells by a virus to introduce the reprogramming factors. However, it is wrong to write that iPSCs were generated by virus infection as the authors did. I am also not a native speaker but it is clear that the sentence “And the differentiate ability were study by the assays of in vivo teratoma formation” is understanable or interpretable but it is not a correct English sentence. Results: MES-FT and iPS-FT as MES-ST and iPS-ST are not explained. These abbreviation are even not explained when they are first used in the manuscript, i. e. Fig 2, making the understanding of the manuscript very difficult. Due the problems regarding the language, it is currently difficult to comment on the scientific background. However, the introductory statement that in vivo differentiation systems are superior to in vitro differentiation systems and even “more adapt to clinical application” is not based on sufficient evidence. The structure of the article appears to be conform with PeerJ standards. However, the quality of several figures needs to be improved. On all pictures showing cells or tissues scale bars are missing. All figures have very poor legends. Data from which MES and iPS cells are shown in Fig. 2 (is it OCT4-MES and TG iPS1-7)?. In Fig. 2 (TR-PCR) several bands for the housekeeping gene Hprt are not clearly visible making the results for the other genes analyzed questionable. In Figs. 3a, 5c, 5d no specific tissues within the tumors can be identified since the magnification is not sufficient. In Fig 3b, I can identify an endodermal and a mesodermal differentiation but no ectodermal differentiation. Therefore, these pictures do not convincingly demonstrate pluripotency of the cells that were injected to generate these tumors. In Fig. 3c and 5e, the OCT staining is also not very convincing, the brown color looks more like a background staining (and this is different from Figs 3d and 5f). Fig. 7 is, in view of the raw data provided for the qPCR experiment, highly suspicious for showing means plus SD of technical replicates only. Technical replicates would not justify the statistical analysis. Moreover, the use of a Student’s t-test is likely not justified since it requires normal distribution data and equal variations between the data sets. Moreover, which “mES” are shown here? In addition,, some genes are indicated correctly (first letter in capitals) but most others are not. Raw data are supplied for the qPCR data only. The study by Pei and colleagues is in principle interesting. While many studies have shown the presence of cells expressing pluripotency markers in teratomas (some of which could have been cited), this study suggests that pluripotent cells can be recovered from teratomas and can be sequentially transplanted to generate new teratomas. This has to my knowledge not been shown before (assumed that the tumors are teratomas). However, the tumors shown cannot be clearly identified as teratomas in this manuscript. Diagnosis of a tumor as a teratoma require the presence of tissues derived from all three germ layers and this is not clearly shown. Moreover, the PCR and qPCR data are currently also not convincing (see my comments above). Experimental design: The design of the study is in principle reasonable and the research question is relevant. The methods used are generally very poorly described (see e.g. “mouse strains” which contains hardly any meaningful information so that strains including the genetic background cannot been identified). Moreover, no antibodies used are identified unambiguously and no information on primers are given. The teratoma experiments are hardly described; it is, e.g., not mentioned to which tissue the cells have been injected or how long the tumors were grown. More similar examples could be given easily. Validity of the findings: The technical standard of the experiments (or the results presented) is in my view currently not sufficient to justify publication. The authors need to provide data, which unambiguous demonstrate that the tumors are teratomas and they need to provide sound gene expression data. Currently, the conclusions are rather broad and not very specific with respect to the data shown. However, with respect to the language it is difficult to assess these issues. Additional comments: The basic critique regarding the experiments (unambiguous demonstration that the tumors are teratomas; providing sound gene expression data) needs to be addressed and the methods needs to be described according to common standards. If this is possible, the authors should work on issues such as language and presentation in the context of the literature.