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

Input:
Title: USE OF AERIAL THERMOGRAPHY TO REDUCE MORTALITY OF ROE DEER FAWNS BEFORE HARVEST
Review round: 1
Reviewer: 1

Output:
Basic reporting: The whole text is clear, generally well written and interesting.
Figures and photos are useful, well chosen.
The reference paragraph needs some small editing.
Experimental design: It is well described and documented. I would suggest adding the costs of the equipment
Validity of the findings: The whole device seems really effective and can reduce the fawn mortality in the cultivated fields
Additional comments: The paper is generally well written and interesting. The terms of the problem and the experimental design are well described. The devices can actually reduce fawn mortality. I would suggest adding an estimate of costs of the equipment (drone plus thermal imagine camera).  The reference paragraph should be better edited. I have here included some suggestions to improve the text in specific points.

Suggested corrections/notes for specific points:

row 50: fawns of roe deer
row 77: (Blodgett et al., 1995; Gardiner, 2006)
84: grey partridge
86-87: juveniles in danger press against the ground
87: Roe deer fawns are “hiders” and during the first 4-6 weeks mostly stay hidden alone in the vegetation.
90: as they often prefer
92-93 please cancel from “62%” onward. This is the birth timing for a specific population studied in Sweden. Please add a more generic sentence with a more general birth peak (for ex. 20th May-10th June). On the argument see (and quote) Linnell, Wahlström, Gaillard 1998 “From birth to independence: birth, growth, neonatal mortality, hiding behaviour and dispersal”, in Andersen, Duncan, Linnell, eds. “The European roe deer: the biology of success”. Scandinavian University Press (this is currently the best review).
94 (but also 134, 331, 427): game animals/species
98: in (West) Germany
101: and roe deer population size was quite lower than now
102: Please add something like “Since roe deer increase is mainly driven by the recruitment of juveniles, lowering fawn mortality is essential to maintain stable or increase the population”.
147: alfalfa or lucern
412: This paper documents
416: was published more recently (Jarnemo et al., 2002)
453: personnel (hunters, gamekeepers, game managers)
475: Meloidae
477: please put the year of publication in the correct place
479 (also 479, 513, 542): please use small letters in the title
481-482 (also 494): scientific name in italics
482: Viltrevy, Swedish Wildlife 1969; 6
489: breeding bird


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

Input:
Title: USE OF AERIAL THERMOGRAPHY TO REDUCE MORTALITY OF ROE DEER FAWNS BEFORE HARVEST
Review round: 1
Reviewer: 2

Output:
Basic reporting: Style and language are carefully chosen and used terminology is appropriate at most places. However, the manuscript does not always read easy. Most importantly, many sentences seem too long and somewhat complicated. Wording is confusing in some places and there are recurrent repetitions of the text throughout different sections of the manuscript (e.g. line 107 and 417-421). Second, the paper structuring, though formally mostly correct, is somewhat cumbersome. The presented work describes deals largely with two things. It aims at validating the use of thermal imaging cameras mounted on unmanned aerial vehicles (UAV) to detect middle-size mammalian species such as the roe deer fawns in the agricultural land. It also aims at demonstrating in field the applicability of the method to detect roe deer fawns in the conditions of central European agriculture with the goal of aiding prevention of machinery related fawn mortality. To conclude, the manuscript would benefit greatly from having a professional English speaker reading and editing the text in a way that would respect the natural logic of the paper that goes from 1) scientific validation of the method, 2) technical guidance for the reader with some account of the affordability for the hunting clubs, 3) example of applying the method in the wild (i.e. practical example of roe deer fawn detection on a few fields in the Czech Republic).

The account of the context for the presented study in the Introduction is adequate as is the use of literature. Some inconsistency in the format of citations both in the text of all sections and in the References shall be corrected. The last part of the Introduction shall give a clearer idea of the research question. Currently, the only lead is the sentence on the line 125-128 which is too vague. Is the aim of the study to validate the use of thermal imaging mounted on UAV for detecting middle size mammals? How was the validation performed? Given the method is proved valid for the purpose, i.e. the detection rates are reasonably high, is the next aim to show the applicability of the method in the natural settings including some technical guidance on using the tool for the reader?

Figure and legends. Visual quality of figures is excellent and their relevance adequate. Labelling is generally insufficient. Figure legends shall be fully explanatory for all variables displayed. Some of my concerns in detail below:
Figure 1. Consider displaying Lány premises by one type of symbol and the rest of study places by a different symbol.
Figure 2. Complement the figure legend with the place and date of the picture. E.g. “Typical alfalfa field used for searching roe deer fawns. The site is from this and this place, from this and this time.”
Figure 3. “Example of a plan…”, and put nd in superscript (2nd)
Figure 5. What is the graph on the bottom? It is not explained in the figure legend.
Figure 6. This looks more like a software interface when setting the parameters of the camera. Strictly speaking, it is not a “calculation” which would be represented by sort of formula/equation. Rather it is an  “example” of how you may set up the parameters in a study like yours. To me it feels like the primary aim of this figure is to illustrate that the software interface is user friendly and easy to deal with (even for hunters who may not be proficient in using such a type of device). Only after you convince a reader (hunter/hunter manager) that the method is easy to utilize and affordable, you can get it in practice.

Raw data were not supplied.
Experimental design: The primary research seems to fit the Scope of the Peer J journal.
The research question is at present not developed sufficiently and shall be elaborated on in more detail, particularly at the end of the Introduction (see my comment above).
The investigation was performed to a high technical and ethical standard.
Methods behind using and setting the cameras and UAVs are described with sufficient detail. However, the field procedures lack enough information to allow replication of this study. Specifically, the following shall be explained: how many plastic containers were placed in the grass at every time (line 383)? How did you choose the location of the containers in the field? Randomly? Did the pilot of the UAV know the location of the containers he/she was searching for? How many replications were performed? From table 1 it looks like the number of replications may have been 2-10 which is not too many. When did you perform the flights? Some information on the time of the day is provide on line 332 but I can not recall seeing any information on the part of the year. How exactly did you determine the optimal speed being 4-6 m/s (line 358)? Did you evaluate detection rates of fawns at different speeds and found this to have highest detection rate? Why did you use different sizes of scanned areas (Table 1)? If the aim was to determine optimal flight height/altitude in an experimental approach, one has to keep everything same in the replication except manipulating the factor of interest (here flight height, or perhaps flight speed). If one does manipulate the two factors (here flight height and area) how can you be sure the optimal height is not in some way confounded by changes in the scanned area?  During the validation, you can derive detection rate of containers as you know their location but how did you conclude the detection rate is 100% in searching for live fawns? How can you be sure that you did not miss any fawn by the camera? I assume there is still a chance that you miss a fawn, it just happens to survive the mowing and so you do not find its corpse.
Validity of the findings: The results seem pretty strong. However, there are possible flaws in the experimental design that need some addressing from the authors (please see my comments above). Also, I am not sure you can really state you have 100% detection rate. OK, perhaps you can get close to 100% but not exactly 100%. How can you assure the camera/software or whatever of the technicalities never get in a problem? Given there is always a human element, how can you be sure you do not overlook a fawn during the flight? These may be too anxious concerns, but authors shall provide rigorous arguments for a reader not to have any of such.
Additional comments: Below please find some minor comments concerning mostly style, language and typing errors.

Majority, if not all, of citations refer to agricultural practice in Europe. Agricultural policies in Europe are different from other areas, e.g. USA (http://www.momagri.org/UK/points-of-view/A-comparative-approach-to-European-and-American-agricultural-policies_798.html
  or
http://www.europarl.europa.eu/RegData/etudes/ATAG/2016/586615/EPRS_ATA(2016)586615_EN.pdf ). For this reason, I encourage the authors to make clear early in the text that the paper is dealing with a problem mostly relevant for the European agricultural practice.

Citations in the text: inconsistent use of commas and semicolons in the text citations. Comma is missing, f. ex., on line 77 in „Blodgett et al. 1995“, when it shall be „Blodgett et al., 1995“. The citation on line 77 is „Blodgett et al. 1995, Gardiner; 2006“, while it shall be „Blodgett et al., 1995; Gardiner, 2006“. Similarly line 79, 81, 82, 91, and elsewhere in the text)

Abstract
Line 55: UAV not AUV

Introduction
Line 69-71 and 74-75: The two sentences are very similar and so repeating them is redundant. Please consider merging the two sentences.
Line 72: Could you please provide some numbers that would illustrate how dramatic the increase in mortality related to agricultural operations may be during the years?
Line 76: Maybe better to directly state which taxonomic groups (beetles, Orthoptera,..?) are concerned.
Line 77: “in birds” better than “class Aves”
Line 82: According to BirdLife Int (http://datazone.birdlife.org/home) the English name of Crex crex is Corncrake, Perdix perdix is Grey Partridge.
Line 84: Can you please provide any reference here?
Line 93: Is the 25th of May and 7th of June a typical fodder harvest season? If so, where? CZ? How about annual variation?
Line 94-101: please use litter and fawns. Please be consistent in using terms litter and fawn throughout the manuscript
Line 96: please delete “Altogether with”
Line 96: Better provide the exact years of study and add citation. E.g. “…to be within 25-44% during the years XX-YY (citation)”
Line 98-99: Consider rephrasing to make the text shorter. E.g. “In Germany, the number of killed roe deer fawns was estimated at 84 000 individuals in a single year 1997 (Kittler, 1979)”
Line 106: consider moving the sentence “In the past,… (Jarnemo et al., 2002)” to the line 102, after the sentence “…crops harvest.” In that way you would start with what was done in the past and follow with more recent approaches including the thermal imaging cameras as the most state of the art approach.
Line 110: Please consider deleting the part of the sentence “using such…process. In this case,” in order to omit the redundant text. The proposed shorter version may sounds, e. g., as following: “However, the protection of detected game…operations.”
Line 113: is the increased labor demanded by the farmers the only reason? What are the financial costs/benefits of using the cameras/UAVs for the farmers/hunters?

Materials and Methods
Consider moving text from the line 130-135 to the last paragraph of the Introduction
Line 133-134: This is repetition of line 85-90 and can be omitted.

Study area
Line 138: better “…in selected 9 locations in the Czech Republic, from which XX site are experimental sites of the Czech University of Life Science Farm Estate Lány (Fig. 1)” Please state the size of the study areas, e. g. by referring to table 2 (please note that you need to refer to it as table 1 if it’s the first table to be referred to). What did the study sites look like? All fields for fodder production? Please see the comment to line 391 for inconsistency in the number of study sites.
Line 139: it seems there is something wrong with the coordinates. E 13° 48’?
Line 139: abbreviation CULS shall be explained here. It only is explained later, on line 146.
Line 141-142. The last sentence “Selected…in Fig. 1” is redundant and can be omitted.
Line 146-149: better to merge the two sentences as stating both is redundant. E.g.: Leave the first sentence “…to hide in and feed on” and delete the sentence “Alfalfa is not only…landscape”.

Equipment
Line 157,167, 308, 312, 329 and elsewhere: “2nd” (nd in superscript)
Line 161-163. I am bit confused. Cheaper solution than what? So, you used two types of thermal cameras? You mention lower resolution in one type. Did you in any way test/analyze the effect of lower resolution on detection rates of the roe deer fawns?

Thermal imaging camera and its setting
Line 172: at instead of @
Line 183: I am sorry, I do not understand. Do you say that you monitor area of 20 ha in 15 min. by one flight? What is the average UAV flight time? How did you manage to fly for longer than is average flying time? Did you save batteries in some way?

Setting of thermal imaging camera parameters
Line 201-204: the sentence is long and reads difficult. Consider shortening, e.g. by adding brackets “…influence (i.e. atmospheric temperature, relative atmospheric humidity), and …” and by deleting “are required” at the end of the sentence.
Line 215:  any citation available here?
Line 234: “given” or “required” seems better than “this”
Line 241-243: I am confused. There is no account about the manual mode in the paragraph. Should the sentence on line 241 read as “In the manual mode, the upper and lower…”?
Line 246: “time periods” better than “moments”?
Line 246: problem for what? For detectability rates?
Line 252: “optimal” better than “ideal”?

Temperature homogeneity of thermogram
Line 261: NUC is not defined here, it is defined only later in the text, on line 268. Please define NUC here.
Line 280: “…significantly greater (Fig 5c?)”
Line 281: better “smaller” than “lesser”
Line 286: How much lower is the probability of non-detection? This and similar issues about detectability rates shall be elaborated on in the text as those seem critical aspects of the utility of the described method.

Maximum flight altitude setting
Line 294: the sentence seems awkward. Please reformulate. Also, it seems there shall be a citation instead of “[32]” at the end of the sentence.

Applied UAV
Line 313: there is a different hexacopter type stated in the legend of the figure 7. Please correct.
Line 316: what is the difference in detection rates between manual and computer control?

Data collection
Line 331: move the first sentence to line 335 after “.. . 108/97 Coll.).” and before “Morning…”
Line 332: for the sake of consistency (see e.g. line 367), please use “4 to 6 am” instead of “4 to 6 o clock in the morning”
Line 339: Do you mean “applicability” rather than “availabilitity”?
Line 352: There is no “section 3.1” anywhere in the manuscript
Line 358 and 373: better be consistent and either use m/s or km/h throughout the manuscript.

Fodder crops harvest technology
Line 371: Was JD7930 used at all sites?

Results
Line 388: Avoid vague statements of the type “this and that characteristics is given in table 1.” (see also line 400). Be specific. Describe the detection rates here, how these varied as a function of the flight height (and other characteristics).

Table 1.
Line 391: “Results” of the test flights performed at 13 sites in the Czech Republic during 2016-2018.

Discussion
Line 417: delete “which was also described in this publication”
The work by Jarnemo et al 2002 is already mentioned in the Introduction (line 106-108). Why to repeat it here?
Line 420: “as” better than “when “,
Line 420: “…of bag 18 were moved by their mother on the day when the sacks were set out and 3 fawns were moved by their mother on the next day”
Line 422: Remove the sentence “Plastic …spacing” and put it in the following sentence, e.g. “Distribution of one plastic bag per hectare (as in Jarnemo et al. 2002), requires….., which are common in present agricultural landscape in the Czech Republic (Figala et al. 2001).”
Line 425: “advantage of the plastic bag method…” rather than easy-to-confuse “described method”.
Line 427: what game species were detected?
Line 428: remove “In this case,”
Line 431: “safer” better than “safe”;
Line 431: Please add the species of Christiansens study: “The article on this and that species published in 2014…”
Line 435: remove “flight” since the sentence refers to high lift trucks and not UAV?
Line 445. Remove “previously prepared meadows and fields” and consider changing “with minimal risk of killing” for “without the risk of killing”
Line 447. Adverse or rainy weather also stops mowing, so the negative effect of bad weather on the UAV applicability does not seem as much a big problem to me.

Conclusion
Line 460: remove “submitted”
Line 436: “carrying” better than “carried”

References
Some references use capital letters (line 513, 533, 542) while the rest not. Please correct.
Line 477: year of publication placed wrong in the reference
Line 481,494,511: species name shall be in italics
Line 486: Grey partridge not gray
Line 531: structure of what? Missing word?

Table 1: all the variables shall be explained. What does “number of test flights” means?
Table 2, legend: “…detection of roe deer fawns at 13 locations in the Czech Republic during 2016-2018”


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

Input:
Title: USE OF AERIAL THERMOGRAPHY TO REDUCE MORTALITY OF ROE DEER FAWNS BEFORE HARVEST
Review round: 1
Reviewer: 3

Output:
Basic reporting: This is an interesting contribution dealing with an authentic wildlife management problem in agricultural landscapes. Limited literature (at least official peer reviewed literature) is available on this item. English is clear and professional (...but I'm nor English native myself). Abstract is OK. Literature is ok as well as the provided context. Goals are clear.

Neverteless, I perceived some weaknesses, as follows:
1) Introduction is a bit too long, I suggest Authors to delete or shorten some parts (eg, delete from lines 75 to 84);
2) Tests flights methodology should appear in M&M;
3) Results are somewhat uncomplete. In particular, I would have expected data i) on the time needed to exaustively cover the prospected fields (at least raw data, average time/prospected hectare, standard deviation); ii) on the amount of days (during the study period) in which climatic conditions were deemed unfavourable to prospection; iii) on the number of detected adult deer;
4) I'd appreciate a (short) comment on the expected survival of fawns which were removed from alfalfa fields;
5) Discussion on the limiting factors should be more in deep;
6) Figures are too many and not all are of sufficient quality or interest. I suggest to delete Figg. 1 and 8, improve (if possible) the quality of Figg. 3-5-6, and improve the appeal and self standing character of all figures' and tables' captions. In addition, it should be clearer how Authors may state that spots on Fig. 4 are suggestive of two fawns.
Experimental design: No major comment adding to Section 1. Basic reporting
Validity of the findings: No major comment adding to Section 1. Basic reporting
Additional comments: No comment


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

Input:
Title: USE OF AERIAL THERMOGRAPHY TO REDUCE MORTALITY OF ROE DEER FAWNS BEFORE HARVEST
Review round: 2
Reviewer: 1

Output:
Basic reporting: I am totally satisfied with the corrections made.
The article merits to be published.
Experimental design: I already gave my positive opinion on it.
Validity of the findings: The results are very interesting and promising.
Additional comments: I am totally satisfied with this last version of the paper. There are only very few typographical errors in the quoted literature.

row 518: scientific Latin name in italics
row 548: space among words
551: space between words
556: Scandinavian


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

Input:
Title: USE OF AERIAL THERMOGRAPHY TO REDUCE MORTALITY OF ROE DEER FAWNS BEFORE HARVEST
Review round: 2
Reviewer: 2

Output:
Basic reporting: Authors considered all my comments and improved the manuscript considerably.

The language is professional and the text is largely fluent to read.
Experimental design: All my concerns were appropriately addressed.
Validity of the findings: All my concerns were appropriately addressed.
Additional comments: I only have few minor comments.

line 10: Workswell s.r.o. (a space needs to be added between Workswell and s.r.o.). Also is the "m" correctly placed there?

line 85: Sentence "it is very...this way" is a repetition of line 69-71 and my opinion is that it shall be omitted.

line 93: please add "populations" (Since an increase in roe deer populations is mainly...)

line 136: Please move "medicago sativa L." to line 130, where there is the first mention of alfalfa in the text.

Line 137: Sentence "However, alfalfa...deer fawns" is a repetition of line 82-84 and shall be omitted.

Line 214: I believe that "purpose" is correct, rather than "purposes"

Line 258 and Fig. 5. I believe it makes sense to switch Fig. 5A and 5B as reader may be interested first in the situation before NUC and then after NUC to see the improvement.

Line 324: I believe "application" shall be deleted.

Line 327-329: please be consistent and use either "$" or "USD"

Line 337 ("The search... 5 to 7 a.m.") and line 339-343 ("Morning hours...technology") shall better fit either on line 354 or 361.

Line 404: Perhaps shorter: "Scanned area for flight altitudes 50 m and 70 m was 1428 m2 and 2632 m2, respectively"

line 410: delete "about". uncertainty is given by provided SD

Line 438 and 475-476: Perhaps shorter to join the sentences in one, e.g. "The area of field blocks have increased steadily in the past to reach on average 21-30 hectares in the Czech Republic in more recent time (Figala et al 2001, Reuter and Eden, 2008)"

Line 477: "by using additional battery sets"

line 495. "100 %" rather than "100%"

Line 497-499: Incomplete sentence?

line 505: " ...that are more difficult to detect than larger species such as roe deer."

line 518. Capreolus capreolus shall be in italics
line 551 "Capreolus capreolus" not "Capreoluscapreolus"


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

Input:
Title: USE OF AERIAL THERMOGRAPHY TO REDUCE MORTALITY OF ROE DEER FAWNS BEFORE HARVEST
Review round: 2
Reviewer: 3

Output:
Basic reporting: English substantially improved. Literature references and background OK. Structure OK. Raw data (while few) can be visualized. Match results/hypotheses OK. Still, in Fig. 4 it is not explained on which basis Authors can say that those spots are two fawns. Please, be authentically informative on this particular detail
Experimental design: OK
Validity of the findings: OK
Additional comments: No additional comments


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

Input:
Title: A MULTI-GENERATIONAL RISK ASSESSMENT OF CRY1F ON THE NON-TARGET SOIL ORGANISM FOLSOMIA CANDIDA (COLLEMBOLA) BASED ON WHOLE TRANSCRIPTOME PROFILING
Review round: 1
Reviewer: 1

Output:
Basic reporting: The writing is excellent. This is a very clearly written and well-organized presentation and a pleasure to read.  The structure of the paper is excellent and conforms to journal standards, figures and tables are useful and relevant, and raw data are provided. The introduction and background emphasize literature to support the long-term study for Cry1Ab toxicity to Collembola. The argument for conducting this research is overstated and does not holistically represent understanding of toxicity of Cry1Ab protein to non-target organisms. An example is the statement “The possibility that Bt plants might have long-term effects on the soil biota remains a major concern (Icoz & Stotzky, 2008).” The statement is more strongly voiced than in the source publication, the publication is dated, and there is substantial literature that challenges the statement. There is little rationale for chronic testing of Cry1Ab based on (1) well-established specificity of the protein which would preclude toxicity to Collembola, (2) extensive prior tests of Collembola which do not support concerns for toxicity (the authors themselves cite long-term studies which they themselves do not consider to be particularly meaningful), and (3) decades of field deployment showing no adverse ecological effects and very low levels of exposure.
Experimental design: This is original primary research within the scope of the journal. The reviewer is exceptionally impressed with the quality, experience and capabilities of the research lab undertaking this study and of the state-of-the art methodology employed for the research. The research question is well defined but as previously mentioned it does not seem clearly relevant and meaningful as a research consideration. This is a concern for this reviewer because justifying this type of advanced studies for Cry1Ab justifies continued skepticism regarding a technology that has been extensively investigated and has a well-established record of safe use. This is a rigorous investigation performed to a high technical and ethical standard with methods that are well described and fully detailed. It is somewhat surprising that the study does not include a positive control to show that the experimental protocol was indeed capable of ascertaining adverse effects, since positive controls are the norm in ecotoxicity testing.
Validity of the findings: Overall the outcomes of this research should be viewed as valid, but since there is no positive control the study conclusions of no effect from prolonged exposure to Cry1Ab could be challenged because there is no evidence (within the study) that the test system was indeed capable of detecting adverse outcomes. The authors to provide data to show exposure of the test organism, but it is not clear as to how the collembolans were prepped for ELISA (that is, was the exposure external or has the Cry1Ab been ingested?). These not a major limitations since the authors cite use of the testing approaches used elsewhere in the literature where adverse outcomes were measured. The conclusions are well stated, linked to the original research question and are limited to the supporting results.
Additional comments: This reviewer wishes to emphasize that the quality of this research is very high and I am impressed with the research capabilities and approaches shown in this work. My concerns are restricted to the rationale for such extensive studies directed toward a protein with such extensive prior analysis.


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

Input:
Title: A MULTI-GENERATIONAL RISK ASSESSMENT OF CRY1F ON THE NON-TARGET SOIL ORGANISM FOLSOMIA CANDIDA (COLLEMBOLA) BASED ON WHOLE TRANSCRIPTOME PROFILING
Review round: 1
Reviewer: 2

Output:
Basic reporting: This paper reports on a three generational study to evaluate possible non-target effects of Cry1F protein on the collembolan, Folsomia candida. This is the first attempt to use RNA-Seq to evaluate the impact of Bt toxin on gene expression of this insect. Although Cry1F protein was continuously digested and was detected in F. candida throughout the study, there were no significant differences in the global gene expression between F. candida fed diets with and without Cry1F. Over the three generations, there were no consistently different co-expressed genes. In addition, Cry1F did not obviously alter the expression profiles of seven sensitive biological markers. The authors conclude that the Cry1F protein had no long-term harmful effects on F. candida.

This paper makes an important contribution to understanding how focusing on gene expression with RNA-Seq can be a powerful tool for evaluating non-target effects of Bacillus thuringiensis (Bt) derived products that are commonly used in insect-resistant genetically engineered (IRGE) crops. These methods probably also could be used to evaluate other types of arthropod stressors, including next generation RNAi-based stressors for IRGE crops. This is an important contribution and step toward using biological markers for such studies.  I hope overtime such studies will become more common and more refined as more specific biological markers are found based on the nature of the tested stressors. The authors should be commended because they focused on major trends and not the few differentially expressed genes (DEGs) (potential false positives) that may be due to random events caused by uncontrolled conditions.

Literature citations, however, could be improved.  More key papers outside of China should be referenced. More specific recommendation are provided below. The paper is very well written and should be interesting and valuable to readers of PeerJ. I highly recommend publication.
Experimental design: By all accounts, this is a first-rate comprehensive study. The experimental design is excellent (flow chart in Figure 1 was useful) and the analyses and interpretation of results are appropriate. The conclusions are supported by the data.
Validity of the findings: no comment
Additional comments: The authors should consider the following recommendations to improve the manuscript.

References:
37: There are numerous references that could be included here with Liu et al.
44-45: Two of these references are not appropriate because they are not herbivores, predators, parasitoids, or detritivores. Goa et al. is a non-target study with zebrafish, and Jia is a non-target study with honeybees. The honeybee study is more acceptable, but technically they do not fit into the categories listed. Again, several other references could be used here.

Here is a partial list of additional references to consider:

Neher, D.A., A.W.N. Muthumbi, and G.P. Dively. 2014. Impact of coleopteran-active Bt corn on non-target nematode communities in soil and decomposing corn roots. Soil Biology and Biochem. 76:127-135; doi:10.1016/j.soilbio.2014.05.019.

G. P. Dively. 2005. Impact of transgenic VIP3a x Cry1ab lepidopteran-resistant field corn on the nontarget arthropod community. Environ. Entomol. 34: 1267-1291.

Svobodová, Zdeňka, Oxana Skoková Habuštová, William D. Hutchison, Hany M. Hussein, and František Sehnal. "Risk assessment of genetically engineered maize resistant to Diabrotica spp.: influence on above-ground arthropods in the Czech Republic." PloS one 10, no. 6 (2015): e0130656.

Bitzer et al. Environ.  Entomol. 34, 1346–1376, https://doi.org/10.1093/ee/34.5.1346

Authors should check all the references to determine if others are inappropriate.

For future studies, the authors should consider using a positive control in addition to the negative control. This would allow them to test the validity of their biological markers.  There are many ways to approach this with acute or chronic stressors, or both.

Minor edits:
47: consider “. . . plant residues, collembolans are an important group of soil
arthropods detritivores.”
171: should be “was used to detect”
234: rewrite “has no any negative effect”


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

Input:
Title: A MULTI-GENERATIONAL RISK ASSESSMENT OF CRY1F ON THE NON-TARGET SOIL ORGANISM FOLSOMIA CANDIDA (COLLEMBOLA) BASED ON WHOLE TRANSCRIPTOME PROFILING
Review round: 1
Reviewer: 3

Output:
Basic reporting: Authros did not use the existing regulatory data for non-target effects of Cry1F in maize and cotton that was reviewed and summarized by ILSI (http://ilsi.org/researchfoundation/wp-content/uploads/sites/5/2016/05/Cry1f-monograph.pdf), including data on Folsomia and Eisenia as representative soil invertebrates.
Experimental design: The authors describe existing data on the effects of Bt protein on life history parameters (mortality and sublethal effects) of non-target organisms including C. candida, which found little to no direct toxicity.  Nevertheless, they propose that a multigenerational analysis may identify effects that are not apparent over a single generation, and that transcriptomic investigation may provide more sensitive measures of the effects of Cry1F protein.  However, the authors fail to establish a plausible mechanism whereby a non-toxic protein that has no measurable effect on a single generation could have ecologically relevant effects that are only detectable through transcriptomics.  The introduction needs to describe the plausible risk hypotheses the authors are investigating, including (1) how Cry1F exposure, based on the known mode of action of Bt Cry proteins, could meaningfully affect transcription without measurable life history effects, and (2) how changes in transcriptome over generations could be ecologically important for risk assessment.  If transcriptome differences had been found, what would be the ecological harm that could be concluded? Without setting up the study this way, this study encourages fishing expeditions to find differences, which harms the goals of ecological risk assessment.  It would suggest that if we don’t find evidence of ecological harm, we must keep looking, instead of concluding that there is no relevant harm.
Validity of the findings: The authors did not mention in the materials and methods section how the groups of individuals were selected for RNAseq.  In particular, transcriptome is likely to vary significantly as the insects develop; therefore if all individuals selected were not in the same developmental stage (instar) and age within instar, there is expected to be considerable uncontrolled variation.  Please specify how individuals were selected and discuss how differences in age and instar might affect the ability to detect treatment effects, as well as the biological relevance of not a finding treatment signal in the age/instar noise.

The statistical analysis of the data assigns “generation” as a fixed effect in the ANOVA.  However, generation should not be regarded as fixed as individuals could not be randomly assigned to a generation, as they are to a diet treatment.  Likewise, generation is not a random the order of the  generations is not random. Instead, the correct model for the ANOVA is Repeated Measures.  The authors need to replace the two-way ANOVA with a repeated measures ANOVA.

Was the Cry1F diet confirmed to be active against sensitive insects? Both the source of the protein (intended use as a positive control for detection assays, rather than ecotox studies) and the preparation could lead to reduced or removed activity.  Confirmation of activity of diet preps is an essential step in ecotox studies, and results of studies  (especially with no-effect findings) without this confirmation are not valid
Additional comments: Additional specific comments are provided in the pdf file


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

Input:
Title: A MULTI-GENERATIONAL RISK ASSESSMENT OF CRY1F ON THE NON-TARGET SOIL ORGANISM FOLSOMIA CANDIDA (COLLEMBOLA) BASED ON WHOLE TRANSCRIPTOME PROFILING
Review round: 2
Reviewer: 1

Output:
Basic reporting: This is a second review of this paper.  The authors have incorporated recommendations and made edits I suggested in the first review.  The manuscript is improved but the quality of the writing in the edited sections is somewhat inferior to that of the original manuscript.  This is not a major concern because this can be easily addressed with basic editing.

A major stumbling block with this paper, as pointed out with all three reviewers, is the lack of a positive control. The authors have stated that they tried potassium arsenate as a positive control, but it was a poor choice for a multi-generational study. This now is explained in the manuscript.  Also, the authors used ELISA to document high concentrations of Cry1F in the BT diet, which shows the toxin was present.

I stand by my first assessment “This paper makes an important contribution to understanding how focusing on gene expression with RNA-Seq can be a powerful tool for evaluating non-target effects of Bacillus thuringiensis (Bt) derived products that are commonly used in insect-resistant genetically engineered (IRGE) crops.”

Perhaps the authors should consider discussing the challenges with finding positive controls in a multi-generational study.  Presumably a sensitive insect would not survive a multi-generational study.  Should one consider new sensitive insects for each generation to show exposure or should one try to find a species that can survive multiple generations with sublethal effects, or other approaches?
Experimental design: The experimental design is adequate (flow chart in Figure 1 was useful) and the analyses and interpretation of results are appropriate. The conclusions are supported by the data.
Validity of the findings: no comment
Additional comments: Many minor edits are needed, for example:
75: “detective some potential” should be “detect potential”
83: don’t use “thing”
150: “into baker’s yeast granules with” consider “into baker’s yeast granules”


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

Input:
Title: A MULTI-GENERATIONAL RISK ASSESSMENT OF CRY1F ON THE NON-TARGET SOIL ORGANISM FOLSOMIA CANDIDA (COLLEMBOLA) BASED ON WHOLE TRANSCRIPTOME PROFILING
Review round: 2
Reviewer: 2

Output:
Basic reporting: See previous review and response below.
Experimental design: See previous review and response below
Validity of the findings: See previous review and response below
Additional comments: I thank the authors for their thoughtful responses to reviewer concerns and suggestions.  I remain skeptical that multigenerational research into effects of a plant-expressed Bt protein on an insensitive non-target organism. I also remain skeptical that transcriptomic analysis is warranted where no physiological or ecological effects are observed.  Nevertheless, the quality of the research and its contribution to the vast literature on the ecological safety of Bt crops make the manuscript worthy of publication.  The authors have added sufficient additional information to address the major concerns.

My remaining concern is in the incorporation of "generation" into the two-way ANOVA as if it were simply an independent variable.  The authors would be better justified in conducting three one-way ANOVAs, one for each generation.


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

Input:
Title: DOMINANCE IN DOGS AS RATED BY OWNERS CORRESPONDS TO ETHOLOGICALLY VALID MARKERS OF DOMINANCE
Review round: 1
Reviewer: 1

Output:
Basic reporting: The manuscript is overall well-written with regards to the language used. However, while the proposed goal is to address the misuse of the concept of dominance (which in itself is an interesting goal) the authors themselves are inconsistent in the terms they use in their introduction. Specifically, while the authors first appear to distinguish between ‘agonistic’ and formal dominance (line 54), they do not then clearly state which one they refer to. Similarly, at times they appear to use the term ‘dominance’ interchangeably with ‘aggression’ (e.g. line 75-76).

Dominance as an ethological concept refers to an individual’s position within a social group. ‘Dominant’ individuals have higher social positions than ‘subordinate’ individuals. A number of behavioural markers (ritualistic displays, behaviours and outcomes of fights) permit researchers to categorise individuals as ‘dominants’ or ‘subordinates’.
To improve reading, I suggest that the authors re-organize their introduction, to clearly state, form the beginning the various ethological definitions of all relevant concepts (dominance, dominance hierarchy, agonistic behaviours and displays etc.). Then, explain what parameters have been shown to covary with dominance status (age, sex etc) and then explain how these concepts are misused and misunderstood. This way, the ‘gap’ in knowledge will have been logically identified and the aims of the paper can be clearly related to that issue.

The background provided is good. Figures and tables are fine. However, the raw data provided is unclear. Particularly the 'questionaire' sheet, as we have no way of understanding what these values correspond to.
The discussion is fine, although somewhat lengthy with no clear development of the implications of the results. The main question as to whether owners have a good or not understanding of what dominance really is, and whether they can properly assess it in their dogs is not answered. This is partly because the study design and analyses do not allow to do so (see below for more details)
Experimental design: The effort taken in collecting the data is commendable, and so is the dataset gathered. The use of a questionaire, although having limitations, is also fine in the context of this study. However, the overall analysis requires significant improvement in order to allow drawing conclusions that are pertinent for the research question.

The aim was to assess whether owners correctly perceive dominance status in their dogs. In order to do so, the test should be to first objectively define each dog’s dominance status (through ethological/biological measurements) then assess whether the distribution matches owners answers. However, this is not what the authors did. Instead they tested whether the distribution of dogs into dominant vs subordinate categories differed from random. All that this can tell us is that owner-perceived dominant/subordinate dogs perform more or less specific types of behaviours.
Furthermore, as it stands, all items are analysed independently from one another, while in truth you would expect them to be correlated (as at least some of them should be reflecting dominance status). Thus, it would be more appropriate to used GLMMs with binomial distribution (or multinomial logistic regression so that the ‘similar’ category can also be included) to assess how and to what extent the different items predict owner-attributed dominance status.

The supplementary analysis has some potentially valuable results (such as the relationship between behavioural measures of dominance and owners’ reported dominance status), however, the rational for the difference in rank analysis is not provided, and the explanations are not detailed enough to understand what was done and why. Specifically, if a rank difference variable was calculated, then there should be only one value per dyad, so how can “dominants” have higher dominance difference scores?? Furthermore, if the aim was to determine what key variable (i.e. sex, age, neutered status) predicted the size of the score difference, they why create combinations of these parameters? It would be better to include these variables as fixed effects (i.e. age effect as ‘same’ or ‘different’ etc) and include relevant interactions
Validity of the findings: As it stand the validity of the results cannot really be assessed as the statistical analyses need to be improved.
The dataset is commendable, as such with improved analyses the results should be interesting.
Additional comments: Line 30. ‘may be’ instead of ‘maybe’
Line 39. Which ‘psychologists’ and ‘authors’ are you referring to? Please give a reference earlier on.
Line 43. It is unclear what inconsistencies there are. You should contrast how other authors define dominance before making this statement.
Line 54. You state that de Waal distinguishes between agonistic and formal dominance. Is that the stand that you also take? If so, you should keep consistent in the reminder of the ms and specify when you refer to either of these categories. As it stands it is unclear what your stand is.
Lines 63-64. A reference listing like this is not very informative. Can you please, rather explain provide details on what arguments and counter-arguments the different sides propose.
line 75-76. Do you not mean ‘aggression’ rather than ‘dominance’ here? Or are you implying that mixed-sex groups do not have dominance hierarchies?
Line 151-152 appear to be repeats of lines 148-150.
Line 200. The wording is wrong. Binomial tests do not allow you to assess whether a factor predicts another one. It only allows to assess whether the sample distribution statistically differs from a theoretical distribution.


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

Input:
Title: DOMINANCE IN DOGS AS RATED BY OWNERS CORRESPONDS TO ETHOLOGICALLY VALID MARKERS OF DOMINANCE
Review round: 1
Reviewer: 2

Output:
Basic reporting: This manuscript is well-written and has an incredibly comprehensive literature review. The raw data, figures, and supplementary analyses are all clear. My only comment is that the 6-item survey the authors suggest for future work could be more easily accessible. It currently is buried in the conclusion section, but it would be nice to have a separate table that lists the 6 questions they suggest (or some way of flagging which questions they suggest in their current Table 1), and perhaps also the additional 2 questions that might prove informative (i.e., which dogs wins fights and which dog licks the owner's mouth). Building on this suggestion, it would be useful for the authors to provide a bit more explanation of why they suggest these specific 6 questions, either in the table legend or in the text of the manuscript itself. I'm assuming it's because they were marked the highest proportion for the dominant dog, but it would be helpful to clarify this so that people can quickly use, understand, and cite the 6-item survey in their future work.
Experimental design: This paper covered original research that is both highly relevant and meaningful, and the method used was appropriately rigorous. However, my one comment is the research question could have been better defined in the introduction. Although the immediate goal of the survey (to determine owner's perceptions of dominance in their dogs) was clear in the method section, the way this linked to a broader goal was not always clear.

In particular, at the beginning of the introduction (lines 29-36) it sounded like the authors were arguing that owners do not have accurate perceptions about dominance in dogs. This left me wondering what a survey with owners could tell us about dominance in dogs since their evaluations on the survey would necessarily be based on misperceptions. Then the following paragraphs talked about other theories of dominance from prior work. All of this is certainly interesting and important literature review, and I'm not suggesting the authors remove this. Instead, I would suggest the authors add one paragraph at the end of the introduction that clearly lays out the goals they have for this survey, both in terms of what we can learn about (1) owner perceptions and (2) actual dominance in dogs (e.g., the age effect the authors discuss, in addition to other research they review in the introduction).
Validity of the findings: An impressive amount of data was analyzed in this study, and the authors did a fantastic job of explaining their analyses and correcting for multiple comparisons throughout. See my note in the "Basic Reporting" section, though, for a way to make the "Difference in ranks" analyses more transparent in the main text. The conclusions are well-stated and link the results of the current study to a wide range of previous research. Any future researcher examining dominance in dogs will find this paper an incredible resource.

My only suggestion here is that the authors more directly mention or address the issue that this is one culture's (i.e., Hungary) impressions of dominance in dogs, so it might be interesting to see how these inferences about dominance differ across other countries.
Additional comments: No additional comments


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

Input:
Title: DOMINANCE IN DOGS AS RATED BY OWNERS CORRESPONDS TO ETHOLOGICALLY VALID MARKERS OF DOMINANCE
Review round: 1
Reviewer: 3

Output:
Basic reporting: Paper is mostly well structured and written. The authors write in such a manner that a broad audience can follow and understand what is presented. The cited literature is appropriate as is the presentation of results.

My primary problem with the paper is the discussion (discussed further in my general comments). I feel it could be cut down and more focused on interpreting the results instead of restating them. Use the structure of the conclusions as a guide.

The figures are simple, they could benefit by removal of the background lines. Also, I personally find the tables that are in the supplementary to be more useful than the figures presented. I would consider switching which go in the main text and which go in the supplementary text as, as least for me, it's more important to see the numbers.
Experimental design: A simple study design with a robust sample size. The authors are appropriately cautious with their analyses and though they multiple binomial tests, they use a Bonferroni correction to adjust for p-inflation. As I mentioned in my general comments, it would have been valuable to have the dogs rated by multiple people. A limitation that requires addressing.

There are no ethical concerns and the methods are well described.
Validity of the findings: Data is clearly labelled in Excel sheet and makes sense with how it's formatted. Statistics make sense. As stated above, my primary problem with this paper is the discussion section where not enough interpretation is done, nor is the focus appropriate. More details including line comments can be found in my general comments.
Additional comments: Review of “How do owners perceive dominance in dogs?”

Abstract:
Clear, concise, and easy to follow.

Introduction:
Line 30: Change “maybe” to “may be”. Also, are they unsure or do they simply misunderstand? It’s been my experience that people feel strongly that they know what dominance is, but they are often wrong.
Line 34: Interesting example, though if you’re to speak about abusive training technique I would say “negative reinforcement and positive punishment” and adjust the sentence as Casey et al. looked at both those in their studies and positive punishment has clear abusive connotations.
Line 49: A good point and one that can be leveled against the animal personality literature as a whole as “Dominance” has been used to describe personality domains across primates as well.
Line 89: Adjust to: Pongrácz et al. (2008)
Line 94: Interesting, what does it say about dog dominance that that more dominant individuals copied people more?
Line 99: Perhaps adjust to “In this study we asked owners…”
Overall a well cited and clearly structured introduction.

Methods and materials:
Line 136: Please indicate the version of R used.
I don’t have any other comments. This section is clearly laid out and the statistics make sense and are fairly conservative thanks to the authors’ dedication to Bonferroni corrections.

Results:
Line 178: I don’t find these descriptive statistics overly informative, I wonder if they’d be better as a table in the supplementary materials.
Line 183: The way this is reported is rather awkward. I would like to see it adjusted in a way that mean and SD can be reported as: (mean ± SD = 16.1 ± 8.6) or something equally as readable.
Line 202: Typos: but „dominant”
One problem with the study is that multiple owners weren’t asked to assess the dogs. Given how short the survey is, it seems like this would have been beneficial to reduce the chance that answer are reflective of one owner’s feelings rather than an accurate measurement of observed behavioral patterns.

Discussion:
Line 239: In your introduction you mention that owner assessments of dominance are not novel, perhaps a more tempered first line if the introduction is called for.
Line 240: A hierarchy of two animals isn’t much of a hierarchy and given that you asked owners to only look at two dogs when some had multiple, how accurate is the hierarchy? This makes for a more artificial hierarchy.
Line 243: This is first you specifically say that personality traits were examined but you don’t identify which items or mention this is in the methods. Please bring this up earlier in the paper to make it clear.
Line 244: I would reduce the amount of repetition of percentages and the exact results and focus on interpretation.
Line 263: This is a tough one, if they don’t understand dominance how can you be sure your other rates did as well? Perhaps they just answered based on misinformation and perhaps the accompanying answers are based on this as well, hence the match between dominance status and other items.
Line 273: What is the importance of this difference? Is there an interaction between these differences that explains this? I don’t find this overly compelling because essentially you’ve got a survey where, it’s psychometrically valid, dominance should go with specific behaviors.
Line 286: Again, what does this suggest, what’s the importance?
Line 340: Adjust to: Bonanni et al. (2010)

I’d like to see the discussion cut down and focused on the meaning of these findings. There’s too much time spent reviewing the results again and too little focus on the interpretation and relationship with the rest of the field. I don’t get a feeling for the overall message of the paper and importance. The authors also spend no time considering the validity of their survey. Without behavioral methods I’m not convinced that the survey is actually measuring dog dominance even though the authors spend a lot of time interpreting it in relation to behavioral studies. First, I think the author should discuss how valid their survey is based on their results, then pick the most relevant points from their results to interpret with the broader field, then the limitations of their methods.

Conclusion:
Line 382: I feel like this is the line I was looking for throughout the discussion. I would start the discussion with this focus and argument in support of your survey and work from there.
Line 386: Where was this in the discussion? This conclusion is so well written and focused, I think you should layout your discussion like this.


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

Input:
Title: DOMINANCE IN DOGS AS RATED BY OWNERS CORRESPONDS TO ETHOLOGICALLY VALID MARKERS OF DOMINANCE
Review round: 2
Reviewer: 1

Output:
Basic reporting: The manuscript is much improved regarding the clarity of the the aims and methods and validity of the study. I am happy to say all my previous concerns have been addressed by the significant changes made by the authors.
I have made some minor editorial changes and a few comments on the tracked change docx provided.

I re-state the few comments I still have here:

lines 199-204: Repeat of previous paragraph (Lines 182-188). Please, combine.

lines 210-212: As this analysis aims to test the age-related hypothesis, please detail it in its own paragraph with explicit mention of the hypothesis investigated. Also, some further details on what this test does would be good, as it not a test as well known as typical t-tests and others.

line 269-270: Again, as this tests a separate hypothesis (i.e. age related hypothesis) the results should be better highlighted/detailed.

lines 302-306: Could this not be combined with the descriptive statistics paragraph? Or left for the discussion?

Figure 1: What are the colors of the figure coding for?
Experimental design: No more issues
Validity of the findings: No more issues
Additional comments: No additional comments


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

Input:
Title: DOMINANCE IN DOGS AS RATED BY OWNERS CORRESPONDS TO ETHOLOGICALLY VALID MARKERS OF DOMINANCE
Review round: 2
Reviewer: 2

Output:
Basic reporting: The authors addressed all of my concerns in this section. However, I have a few wording suggestions given the extensive nature of these changes (i.e., many of the sections are essentially fully re-written). In particular I want to flag that there are a number of typos, grammatical mistakes, and some confusing wording that have been introduced in this new version. I have flagged several of these below, but I think a more careful editing pass will need to be done on the paper before it is fully ready for publication.

First, the new Results subsection (Items most convincingly linked to perceived dominance) could be worded more clearly as "Seven items are suggested for future work based on their occurrence (% of 'Similar' responses were lower than 24.7 and % of 'N/A' responses were lower than 16.1, see Descriptive statistics) and their significant link with dominance status, independent from the sex of the dogs: bark, eat first, reward, walk first, resting place, defend group, and aggressive. These items are highlighted in Table 1."

Line 51: The words "rather on" should be removed.

Line 86: The wording of the sentence starting on line 86 is hard to follow

Line 108: Extra period

Line 110: comma can be removed here

Line 386: Typo here - some text seems to be missing (or there is extra text that was not deleted)
Experimental design: The changes here are very helpful for clarifying the goal of the study, especially the two new sentences added to the beginning of the abstract and the new sentence added to the last paragraph of the introduction starting on line 332.

My only further recommendation is that it would be helpful to frame the introduction (from the beginning) more in terms of the owner/public's interpretation of dominance early on. The way the introduction is currently written still sounds like it's being set up to test big picture theories of dominance, rather than the owner's perceptions. This could be as simple as adding a sentence or two somewhere in the first paragraph of the introduction (similar to the new sentences the authors added to the beginning of the abstract in this new version).
Validity of the findings: All my concerns in this section were addressed in the revision.
Additional comments: No additional comments


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

Input:
Title: DOMINANCE IN DOGS AS RATED BY OWNERS CORRESPONDS TO ETHOLOGICALLY VALID MARKERS OF DOMINANCE
Review round: 2
Reviewer: 3

Output:
Basic reporting: There are problems with the writing throughout this paper and they become increasingly apparent on a second read through. Perhaps it's a stylistic choice or one relating to a software program but there are single sentence paragraphs in some places and walls of text in others. The introduction and the discussion both needs to be cut to down. Given the study is only survey based, which I believe still makes it worthy of publishing, the authors need not write such a long manuscript and draw such concrete conclusions.
Experimental design: The authors have done a better job at acknowledging the limitations of the study but the focus of the paper drifts away from what the questions are. There are problems with the tables and figures that need to be addressed.
Validity of the findings: As before, I find the conclusion to be well written and direct and feel that the rest of the paper would be greatly improved were this clear, concise, and direct approach taken throughout. The discussion itself needs to be cut down and conclusions drawn need to be made more clear. I found that much of the discussion included reiterating the results or focusing on small parts of them rather than bringing them into the broader literature.
Additional comments: Second review of: How do owners perceive dominance in dogs?

Abstract:
Line 13. This is a well-written and compelling first sentence, I think it’s great.
Line 21: Perhaps instead of “have a certain personality” could say “share certain personality traits…”

Introduction:
Lines 31-43: While interesting, I think this this paragraph is too long given that it doesn’t focus on canids. I’m not convinced that domestic dog dominance resembles the more linear and structured dominance of primates, which makes me inclined to think this paragraph needs shortening and possibly moved to later as the paper isn’t about primates.
Line 44: This is the paragraph that belongs as your opening paragraph.
Lines 71-75: This paragraph needs to be better connected to the rest of the introduction. Currently it reads as an abrupt topic change and a short one at that.
Lines 108-110: I can’t tell if it’s how the paper has been set up but this currently reads as a disconnected paragraph. The statement that dominance is not a personality trait, which I agree with, needs to be supported by a citation.
Line 132: Currently reads as awkward, perhaps adjust to, “In this study, we surveyed people that owned multiple dogs. We investigated the relationship between the dogs’ ranks, behaviour, and demography.”
Lines 133-138: I think this can go in the methods for explaining why you chose the behaviors you did.
Overall, I think the introduction could be cut down and more focused. Also some oddities with how the paragraph are broken up, may be something about the word processing program used but should be fixed.
Line 143: Instead of ‘show’, perhaps use display or perform.

Methods:
Line 197: Why did you not include age and dominance status in the same model? You have a strong sample size and dominance and age don’t have to correlate, why not include them both?
ST 1: What do the highlighted portions of the table indicate? Quite a few of the significant correlations have a fairly low r value. The second column where “Pearson correlation. Sig. (2-tailed)” is repetitive, include that information in the table title or elsewhere so you can delete that column. I’m unsure where the dominance correlations are coming from as they’re not presented in ST1, or perhaps they’re under a column that’s not labelled dominance or are they coming from Table 2 in the main text? This needs to be clarified.
SF1: While simple, I find this figure confusing. Perhaps a simple 2x2 table with the reported percentages would make this easier to understand.
Check the supplementary materials for typos. For example, “dominance” is written “domiance". I appreciate the inclusion of the code, it’s a very nice touch for transparency.

Results:
I found the results to be clear and easy to follow but there are errors and confusing aspects of the tables and figures I’d like to see corrected.
Figure 1: It’s not clear what the different colors indicate.
Figures 1 and 2: Please include titles for all axes.
Perhaps instead of # use boldface, I find the use of # to be distracting and using a symbol that indicates numbers may be confusing to others. Stylistic, I know, but I think it may help for readability.
Across the in-text and supplementary results the columns need to be adjusted so all the title fits, either through the file format or shortening the column names.
Table 3: Mention of bold type but I don’t see any bolding. Were there no significant associations after correction or was this is an error?

Discussion:
The discussion goes into multiple topics but it’s not entirely clear how these minor details add to the overall aim of the study. Again, I think the discussion needs to be shorter and more focused.
Line 299: I would prefer to see a full paragraph instead of a hanging line. Though again, this may be something about how the document has be done.
Line 302: There’s a fifth option: the survey design encourages a selection/answer.
Lines 314-326: I found this paragraph to be difficult to follow as it reads as multiple disconnected sentences. I believe the message is meant to be that there are trends seen across studies but this isn’t clear and could be done much more concisely. Perhaps instead review how your results match those of others and explain how this suggests external validity and be specific. Validity that you’re measuring dominance or that the trends you’re finding suggest your results are valid in a wider context?
Lines 333-371: This wall of text needs to be broken up, as it stands I struggled to follow the writing. This is a common theme with this paper and one that needs to be addressed. Many of the discussion sentences feel disjointed and unfocused.
Line 383: I don’t care for this reference back to the introduction, it asks a lot of the reader. Either include the information here or delete the line.
Line 395: I appreciate the work that has been done to be open about the limitations of the study.


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

Input:
Title: OPTIMIZING DE NOVO GENOME ASSEMBLY FROM PCR-AMPLIFIED METAGENOMES
Review round: 1
Reviewer: 1

Output:
Basic reporting: The manuscript is very well written.  The figures and tables are easy to follow and all of the data are publicly available on JGI IMG.
Experimental design: The research background and objectives are well defined. The methods have been described in detail.  The molecular and computational approaches are state of the art.
Validity of the findings: Roux et al. used a large number of PCR-amplified metagenomic datasets to optimize genomic assembly pipelines for low biomass samples. I think that this will be really useful for deciding on ways to improve metagenomic assemblies of low biomass samples.  It does a great job and examining the effects of amplification on the libraries and benchmarking computational methods to mitigate these effects to improve assembly.
Additional comments: No additional comments


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

Input:
Title: OPTIMIZING DE NOVO GENOME ASSEMBLY FROM PCR-AMPLIFIED METAGENOMES
Review round: 1
Reviewer: 2

Output:
Basic reporting: The article is clearly written. Sufficient relevant background information is provided and appropriately cited. The article's organization is standard. I appreciate that the figures included in the article are relatively simple, but complete results are available in the supplemental files. The table and figure legends are complete. Figure quality provided for the review is sufficient and I assume there will be no degradation of quality in the published version.

Data are available through JGI and contigs through an NERSC download site. This seems adequate, although I personally go to the SRA for read datasets.
Experimental design: The question of how PCR amplification of metagenomic libraries influences the quality of assembly is important and worth multiple studies, given the plethora of error correction, filtering, trimming, and assembly tools available. The authors, several of whom are at JGI, make use of the bbtools suite, which is reasonable; the study was not meant to be an exhaustive review of methods. Rather, there were three specific steps tested, in a total of 12 different combinations, on 169 PCR-amplified metagenomes from 127 different samples. The methods are adequately described so that the work is reproducible, and more importantly, can be implemented with other datasets. There is one exception to this; custom perl scripts are mentioned, but not included in the supplemental materials. Please consider doing this, or providing a GitHub link.
Validity of the findings: The results and their interpretation are sound and meaningful. The authors suggest that one of the 12 pipelines can serve as a default first approach, and this is justified by the results as a whole. They also clearly describe the extent of the improvement under different numbers of amplification cycles. This may lead to stronger efforts by PIs to keep amplification to a minimum.
Additional comments: A very useful, timely analysis that will be of interest to a wide community.

Specific comments:
Supplemental Table 1 is unfinished "Ask Christa to release on Portal". Please make sure these asides are addressed and removed.

Lines 173-174: Why expect human contamination? Is this a standard JGI step?

Line 182: I believe that Table S3 is referenced before Table S2; reorder numbering if so.

Lines 221-226: I don't follow the reasoning behind ignoring translocation errors--how can both assemblies be correct in light of the diagram in the QUAST manual (http://quast.bioinf.spbau.ru/manual.html#sec3.1.2). I can see that it might be unlikely to tell which of the two is correct...could you clarify?

Line 223: somewhere earlier than line 422, you need to define what you mean by 'best" assembly, e.g., largest combined assembly in contigs over 10kbp. 'Standard' is defined at line 214 and 'alternative' at 313. Might be helpful to have them in one place, or come up with a label other than 'alternative', since you start with 11 alternatives.

Lines 336-339: Oddly worded--"the amount of additional errors remains lower than the additional number and size of long contigs..."? I think you mean that the relative increase of assembly errors (2x) seems an acceptable trade off for the improved assembly metrics (24x) or something similar. Since this is one of the few caveats to the alternative method, it should be explained quite clearly.

Various places: 'cohen' or 'Cohen'?


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

Input:
Title: OPTIMIZING DE NOVO GENOME ASSEMBLY FROM PCR-AMPLIFIED METAGENOMES
Review round: 1
Reviewer: 3

Output:
Basic reporting: No comment
Experimental design: No comment
Validity of the findings: No comment
Additional comments: The overall outcome of this paper is to demonstrate that metagenome assemblies from PCR amplification dependent library preparation results in highly uneven coverage across assembled contigs as compared to PCR-free library preparation protocols. Further, the authors report that the uneven coverage of de novo assembled contigs for PCR dependent libraries is primarily a result of selective amplification of short insert size amplicons and not impacted by other amplicon characteristics (e.g. GC content). The manuscript further tests the effect of read correction (i.e., strict vs related), read selection (i.e., deduplication vs none), and assembly approach (i.e., metaSpades vs single-cell Spades) to determine the combination that may allow for improved de novo assembly. The authors conclude that relaxed read correction with deduplication followed by the use of single-cell Spades resulted in improved assembly with significantly higher proportion bases included in contigs > 10kbp as compared to strict read correction, no deduplication and use of MetaSpades. Further, the authors compare de novo assembly from a select few samples that were generated with from PCR free and PCR amplification based methods and demonstrate that efforts to improve assembly length can be associated with a moderate increase in miss-assemblies.

The paper is well written and easy to follow, experimental plan is well designed and reported and the overall conclusions presented are supported by the data and associated analyses.

Below are some important points for the authors to consider:
1.	The primary assembly metric driving the experiments is increased length of metagenomic assembly, which will presumably aid in the genome binning process. While the authors do also consider the issue of miss-assembly, they do not report how the choice of PCR cycles, read correction, deduplication, and S-C Spades affects the genome binning process and the quality of recovered metagenome assembled genomes (MAGs). This should be straightforward to do for the Lake Mendota samples and compare how the different de novo assembly pipelines tested affect MAG quantity and quality for PCR amplified metagenomes as compared to the PCR free method. This might also involve some experimentation with binning approaches. I feel that this extra effort would add significant value to the manuscript overall and would be of significant benefit to the research community.
2.	While assessing the assembly errors, the authors focus on comparing assemblies resulting from various data processing steps for PCR amplified metagenomes to that of the PCR free metagenomes. I appreciate this is an important step, but what also matters is what are the implications of PCR cycles, relaxed read correction, deduplication, and S-C Spades on annotation. For instance, how fragmented are genes post for the "best performing" assembly approach for PCR based metagenomes compared to the PCR free metagenomes? While this might not be the ideally suited for illumina only assembly, Mick Watson blogged about a nice approach to assess annotation quality in this blog (http://www.opiniomics.org/on-stuck-records-and-indel-errors-or-stop-publishing-bad-genomes/ ). May be a similar analysis to this might be of value to the paper. However, at the minimum the impact of the proposed approach to enhance metagenome assembly on gene annotation must be included.
3.	It would be extremely helpful if the various scripts and command lines used for evaluating assembly quality, coverage bias analysis are available. For instance, there are at least a couple mentions of “custom perl scripts” in the data analysis portion and would be helpful to release this. If it is not too onerous, it would be even more helpful if the authors could deposit the scripts, command lines used for the entire workflow on github project dedicated to this manuscript.
4.	I tried accessing the results from different assembly pipelines at this url: http://portal.nersc.gov/dna/microbial/prokpubs/BenchmarksPCRMetagenomes/ but could not access them (i.e., site not available).
5.	URL’s for some the raw data (samples: CGHWP, CGUX) are not provided. All the data is on https://genome.jgi.doe.gov/ but for some reason I was unable to connect to the JGI genome portal. I tried accessing this URL and data from a couple different computers (incase of firewall etc issues) but was not able to do so. Could the authors please look into this.
6.	It is possible that while all the data will be made available via urls to the jgi portal, will readers be able to download it in bulk? It would be very helpful if the raw data and assemblies were made available via NCBI.
7.	Please also provide additional information on the samples, such as the DNA concentration prior to library preparation.


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

Input:
Title: OPTIMIZING DE NOVO GENOME ASSEMBLY FROM PCR-AMPLIFIED METAGENOMES
Review round: 2
Reviewer: 1

Output:
Basic reporting: No comment.
Experimental design: No comment.
Validity of the findings: No comment.
Additional comments: The authors have revised the manuscript to my satisfaction.


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

Input:
Title: THE VACCINATION & HPV KNOWLEDGE (THINK) QUESTIONNAIRE: A RELIABILITY AND VALIDITY STUDY ON A SAMPLE OF WOMEN LIVING IN SICILY (SOUTHERN-ITALY)
Review round: 1
Reviewer: 1

Output:
Basic reporting: English used throughout the manuscript was clear and unambiguous, professional

The literature references describe a partial evidence produced in Italy on this issue. I suggest to consider these papers both for the introduction and the Discussion sections

Napolitano F, Napolitano P, Liguori G, Angelillo IF. Human papillomavirus infection and vaccination: Knowledge and attitudes among young males in Italy. Hum Vaccin Immunother. 2016 Jun 2;12(6):1504-10
Santangelo OE, Provenzano S, Firenze A. Knowledge of sexually transmitted infections and sex-at-risk among Italian students of health professions. Data from a one-month survey. Ann Ist Super Sanita. 2018 Jan-Mar;54(1):40-48.
La Torre G, De Vito E, Ficarra MG, Firenze A, Gregorio P, Miccoli S, Giraldi G, Unim B, De Belvis G, Boccia A, Saulle R; HPV Collaborative Group, Semyonov L, Ferrara M, Langiano E, Capizzi S, Nardella R, Marsala MG, Bonanno V, Ferrara C, Guidi E, Bergamini M, Lupi S. Knowledge, opinions and attitudes of Italian mothers towards HPV vaccination and Pap test. Tumori. 2015 May-Jun;101(3):339-46
Guzzetta G, Faustini L, Panatto D, Gasparini R, Manfredi P. The impact of HPV female immunization in Italy: model based predictions. PLoS One. 2014 Mar 11;9(3):e91698.
La Torre G, De Vito E, Ficarra MG, Firenze A, Gregorio P, Boccia A; HPV Collaborative Group. Is there a lack of information on HPV vaccination given by health professionals to young women? Vaccine. 2013 Oct 1;31(42):4710-3
Pelullo CP, Di Giuseppe G, Angelillo IF. Human papillomavirus infection: knowledge, attitudes, and behaviors among lesbian, gay men, and bisexual in Italy. PLoS One. 2012;7(8):e42856
Sopracordevole F, Cigolot F, Gardonio V, Di Giuseppe J, Boselli F, Ciavattini A. Teenagers' knowledge about HPV infection and HPV vaccination in the first year of the public vaccination programme. Eur J Clin Microbiol Infect Dis. 2012 Sep;31(9):2319-25
Pelucchi C, Esposito S, Galeone C, Semino M, Sabatini C, Picciolli I, Consolo S, Milani G, Principi N. Knowledge of human papillomavirus infection and its prevention among adolescents and parents in the greater Milan area, Northern Italy. BMC Public Health. 2010 Jun 28;10:378

The structure, figures and tables are professionally edited. Raw data are shared.
The structure of the article has an acceptable format of ‘standard sections’
Figures 1 and 2are relevant to the content of the article, of sufficient resolution, and appropriately described and labeled.

Appropriate raw data has been made available, in accordance with PeerJ Data Sharing policy.
Relevant results are related to hypotheses formulated in the aim of the study.
Experimental design: The object of the primary research is original, since according to my knowledge, there is no validated questionnaire to study the specific issue raised by the authors

The research question is clearly define, relevant and meaningful. The knowledge gap being investigated is identified in the Introduction section
The investigation has been conducted rigorously and to a high technical standard.
The authors used a cross-sectional approach, and this is correct to answer their research question.
Methods are described with sufficient information to be reproducible by another investigator. In the statistical paragraph the authors are requested to give a reference for justifying the Internal consistency.
Validity of the findings: The findings are interesting, and the authors at the end of the manuscript clearly state there is the need to replicate the study with a larger sample for test-retest reliability issue.

Data is robust, statistically sound, & controlled.
The data on which the conclusions are based are provided in an Excel file. I made my own analysis with SPPS. While I found 0.882 for the first dimension (between Q9 and Q13), 0.784 for the second dimension, and 0.732 for the last dimension, for the whole for the whole questionnaire I found a value of 0.816. Please check your results carefully.
In the discussion, clearly state the impact of the use of this questionnaire from a public health perspective.
Conclusion are well stated. However, as already said above, some references at the national level are lacking.
Additional comments: This is a very interesting paper concerning the validity and reliability of a questionnaire in measuring HPV knowledge and HPV vaccination. The paper is of interest for an international reader and could be useful to other researchers involved on this topic.
Some minor amendments are required, as stated above and following also the following points:
-	Line 72-74. The authors stated “Data have been collected through a cross-sectional survey conducted at the “Paolo Giaccone” University Hospital in Palermo (Sicily).”. this statement must be moved to the Methods section
-	In the Methods the authors need to describe how and when the participants were selected. Sample size calculations are not reported. If the authors think these are not applicable in this case, please explain in details.


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

Input:
Title: THE VACCINATION & HPV KNOWLEDGE (THINK) QUESTIONNAIRE: A RELIABILITY AND VALIDITY STUDY ON A SAMPLE OF WOMEN LIVING IN SICILY (SOUTHERN-ITALY)
Review round: 1
Reviewer: 2

Output:
Basic reporting: The manuscript is interesting and deal with an important topic of public health. The aim of the study is clear. Discussion and conclusion are related to the aim of the article and are well structured. The English used is simple but clear.

Although there are few comments and suggestions I would like to do.
In the abstract, section method, the age of women interviewed is not specified.
In the introduction the author talks about "bivalent, tetravalent and "enavalent" vaccine (at lines 45, 47, 50), I suggest to give a little introduction of these vaccines and specify the genotype of HPV addressed. The reason why the survey is conducted among women over 25 should be clarified (lines 63-65).
Experimental design: This primary research article is in line with aims and scope of the journal.
Research question is well defined, although the aim is to assess knowledge and attitude not awareness. This consideration is for both aim of the study and the method section.
Materials and methods is complete and described with sufficient details. The reliability and validity is properly evaluated.
Validity of the findings: Data are good and well presented.
In the results a table about socio-demographic characteristics is missing. It would be better to give more explanations about the enrollment of women aged above 25 years, considering the vaccine is recommended only at a young age before sexual activity and exposure to virus. It could be added consideration about the importance of a correct culture about vaccine among the whole population that influence at a different level parent's and adolescent choice.
It would be useful to report the internal consistency of the whole questionnaire and single sub scales in a table.
Additional comments: The article is mainly well-structured, clear and correct in all its sections.