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: PROFILE AND CLINICAL IMPLICATION OF CIRCULAR RNAS IN HUMAN PAPILLARY THYROID CARCINOMA
Review round: 1
Reviewer: 2

Output:
Basic reporting: The writing is not of professional standards. I suggest the authors check the English by a competent person in the next version
Experimental design: The study aims to find out circular RNAs associated with Papillary Thyroid Carcinoma (PTC). They first did microarray in three pairs of patient samples from PTC and adjacent normal tissues to identify differentially expressed circular RNAs and then they validated eight of them with RT-PCR in 40 paired PTC/normal samples. Finally, they associated expression of two circular RNAs with clinico-pathological factors. Additionally, they showed the possible miRNA interactions of these two circular RNAs with bioinformatic analysis. I have two things to comment on the experimental design process. Firstly, was it only the two circular RNAs they reported that had any association with clinical factors? If that was the case it would be fine, but the authors never mentioned if they tried to associate other circular RNAs with clinical factors and did not find significant association. Secondly, the authors reported that by bioinformatic analysis they found miR-522-3p/miR-153-5p, which have been implicated in PTC, to have putative target site in one of the clinically relevant circular RNA hsa_circRNA_047771. It would be nice if they could validate the expressions of these miRNAs in their samples and correlate the expressions of hsa_circRNA_047771 and miR-522-3p/miR-153-5p to further develop on this point.
Validity of the findings: The section on functional analysis of circular RNAs with KEGG and GO enrichment analysis looks redundant and does not provide any solid information in context with the potential association of the circular RNAs with PTC. The authors should clearly explain what is the relevance of this study.
Additional comments: none


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: PROFILE AND CLINICAL IMPLICATION OF CIRCULAR RNAS IN HUMAN PAPILLARY THYROID CARCINOMA
Review round: 2
Reviewer: 1

Output:
Basic reporting: The English writing in the manuscript has been sufficiently improved.
Experimental design: In this revised version, the descriptions in the Materials and Methods section has been provided with sufficient clarity. However, there is still a misconception which should be cleared. In the subsection Functional analysis, the authors described that they performed GO and pathway enrichment analysis on the "differentially expressed" target genes of circRNAs. This is a mis-representation. Because from the study it is evident that the authors did not check the expression of the target genes, they only predicted the target genes using in-silico miRNA-target prediction tools. So, in line 133, instead of writing "These differentially expressed genes were then enriched using DAVID", the correct way to represent is "These putative target genes were then checked for functional enrichment using DAVID". This representation should be corrected in the subsequent sections also.
Validity of the findings: The authors have included some explanations on the relevance of the functional enrichment analysis of the circular RNAs in context to cancer in general; though not conclusive in context of PTC. But there remains another major point which the authors did not address in the revised version: they only checked the association of two circular RNAs with the clinical parameters. While I understand that these two circular RNAs had the most fold-change in expression, still I feel that this study will be incomplete if the authors do not check the association of other up-regulated/down-regulated circRNAs with clinical parameters. At least for the eight circular RNAs whose expression changes were validated by qRT-PCR in 40 paired tumor/normal samples, checking the association with clinical parameters should not be too much work.
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: PROFILE AND CLINICAL IMPLICATION OF CIRCULAR RNAS IN HUMAN PAPILLARY THYROID CARCINOMA
Review round: 2
Reviewer: 2

Output:
Basic reporting: This study by Ren H. et al. aimed to profile the circRNA expression and evaluate its functional consequence and clinical implication in papillary thyroid carcinoma.  The manuscript is well written and the hypothesis has a strong basis as growing evidence show the functional involvement of ncRNA’s including circRNA
in cancers.
Experimental design: Overall the experiments designed has met with the conclusion, but may require below modification/evidence.

1.	The scatter plot (Fig. 1B) would convey better if you indicate/label the candidate circRNA (hsa_circRNA_047771 and _007148 ) within the plot itself.
2.	Correct the spelling for “exonic” in Fig. 1D
3.	The output from the GO analysis is very general and so it did not add much to the conclusion, however the KEGG pathway analysis does show some relevant pathways (RAS, NOTCH, VEGF) associated with tumorigenesis suggests it could be improved.  To further strengthen and to obtain more specific functional pathways, if feasible with your data, I would recommend doing Gene Set Enrichment Analysis (GSEA) or similar type analysis.
4.	In Table 3, column hsa_circRNA_047771 under the Row BRAF mutation, the percentage within the bracket (both low and high expression) doesn’t tally up to 100 %? confirm/correct.
5.	Figure 6 is overloaded, if it could be simplified/add a subfigure, with only the significant/major cancer associated MRE network, it will better convey the message.
6.	The hsa_circRNA_047771/miR-522-3p/miR-153-5p role was not sufficiently developed.  Besides, the statement in the discussion (line 231-234) says it is upregulated in PTC's compared to normal, which is not the case in the supplementary Fig. 3?
Validity of the findings: The findings justify the relevance of this study in circRNA profiling of human PTC’s and its clinical implication as a maker.
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: PROFILE AND CLINICAL IMPLICATION OF CIRCULAR RNAS IN HUMAN PAPILLARY THYROID CARCINOMA
Review round: 3
Reviewer: 1

Output:
Basic reporting: The manuscript writing has been improved sufficiently.
Experimental design: The revised manuscript addressed all my previous concerns.
Validity of the findings: All concerns were addressed in this version.
Additional comments: The manuscript has been improved sufficiently from the previous version and the authors addressed all my concerns adequately


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: PROFILE AND CLINICAL IMPLICATION OF CIRCULAR RNAS IN HUMAN PAPILLARY THYROID CARCINOMA
Review round: 3
Reviewer: 2

Output:
Basic reporting: The authors addressed all the questions very carefully. English writing is improved in this revised version of the manuscript.
Experimental design: Everything is clearly mentioned.
Validity of the findings: The data is well organized and validated carefully. Quality of data is very clear and presentable.
Additional comments: The revised version the manuscript addressed all the questions. The quality of the manuscript  is improved. I recommend this manuscript for publication.


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

Input:
Title: PROFILE AND CLINICAL IMPLICATION OF CIRCULAR RNAS IN HUMAN PAPILLARY THYROID CARCINOMA
Review round: 3
Reviewer: 3

Output:
Basic reporting: Ren H. etal., by profiling circRNA expression identified differentially expressed circRNAs in PTC, of which the expression of hsa_circRNA_047771 was negatively correlated with BRAFV600E mutation, LNM and advanced TNM stage, whereas, the expression of hsa_circRNA_007148 was positively correlated with LNM.

The revised version of the manuscript has significantly improved and the authors have addressed most of the critics convincingly.  Even though the functional/mechanistic aspects of the identified circRNA were not well established, I believe this manuscript would still benefit the recently evolving circRNA’s and their role in thyroid tumorigenesis.   Hence, I recommend this manuscript may be published after correcting the spelling error that is on the name of the gene itself (most places-figure legend, figures, tables, results and discussion it was written as 'has_circRNA_' instead of 'hsa_circRNA_').
Experimental design: no comments
Validity of the findings: no comments.
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: MATE AVAILABILITY DOES NOT INFLUENCE MATING STRATEGIES IN MALES OF THE SEXUALLY CANNIBALISTIC SPIDER ARGIOPE BRUENNICHI
Review round: 1
Reviewer: 1

Output:
Basic reporting: #25218
Mate availability does not influence mating strategies in males of the sexually cannibalistic spider Argiope bruennichi
Author(s): Anna-Lena Cory & Jutta M. Schneider

MAJOR ISSUES

(1) 1 experiment with 2 treatments or 2 separate experiments?

(2) Use of the term “[word(s)] availability”.

(3) Experimental Conduction (LINES 173-196): Experimental setup and running of the trials.

- - - - -

LINE 73: “The causes of sexual cannibalism are discussed until today and seem to be diverse.”
> Citation needed. Suggestion = (Wilder et al. 2009).

Wilder SM, Rypstra AL, Elgar MA. 2009. The importance of ecological and phylogenetic conditions for the occurrence and frequency of sexual cannibalism. Annu. Rev. Ecol. Evol. Syst. 40, 21–39.

LINE 112: “…extreme female-biased sexual size dimorphism…”
> How do you define “extreme” female-biased sexual size dimorphism? Suggestion = see (Schwartz et al. 2013, Hormiga et al. 2000, Scharff & Coddington 1997).

Schwartz SK, Wagner WE, Hebets EA. 2013. Spontaneous male death and monogyny in the dark fishing spider. Biology Letters, 9, 20130113.
Hormiga G, Scharff N, Coddington JA. 2000. The phylogenetic basis of sexual size dimorphism in orb-weaving spiders (Araneae, Orbiculariae). Syst. Biol. 49, 435–462.
Scharff N, Coddington JA. 1997. A phylogenetic analysis of the orb-weaving spider family Araneidae (Arachnida, Araneae). Zool. J. Linn. Soc. 120, 355–434.

THROUGHOUT THE MANUSCRIPT
Be consistent with word/term use.
Specifically: low-availability vs. low mate availability, high-availability vs. high mate availability

LINES 173-196: Experimental Conduction
The specifics of the experimental setup (“types” of arenas) and running of the trials (starting point, front end, back end) were difficult to follow.

25218 - RAW DATA
Column B: density = high-availability, low-availability?
Column F: male_adultweight = units?
Column G: male_testweight = units?
Column M: female_adultweight = units?
Column N: female_testweight = units?
Column T: copulation_duration = units?
Why is V20 (test_no) 1425 (male_id) highlighted (Column M & X)?
Experimental design: As the manuscript is currently written, it is not clear if the research is covering: 1 experiment with 2 treatments (high-availability & low-availability) OR 2 separate experiments: Experiment 1 = High-Availability (4 virgin females), Experiment 2 = Low-Availability (1 virgin & 3 sub-adult females).

If the authors choose to go with 2 separate experiments, I would suggest they split their MATERIALS & METHODS and RESULTS into “Experiment 1” and “Experiment 2” with appropriate titles/headings.
Validity of the findings: It is important to publish negative results, however I think the current manuscript would be more publishable (i.e. stronger) if an additional experiment (or two) and/or relevant natural history observations were added.
Additional comments: LINE 14: “While many studies focused on male competition, the impact of female density has been widely neglected.”
> While many studies HAVE focused on…
> The impact of female density on what?

LINE 18: “Males mutilate their paired genitalia to prevent sperm competition…”
> To REDUCE sperm competition? Do we know that it completely prevents it?

LINE 20: “Males can reduce the risk of cannibalism if they jump off the female in time, but will then transfer less sperm.”
> FEWER sperm, since fewer is used with words denoting countable things (e.g. sperm).

LINE 21: “An alternative solution of THIS trade-off is to copulate longer, commit self-sacrifice and secure higher minimal paternity. THIS self-sacrificial strategy may be adaptive if prospective mating chances are uncertain.”
> Use THIS.

LINE 31: “We found that males needed more time to start mate searching if the mate availability was low.”
AND
LINE 37: “However, we found no evidence that different levels of female cues and mate availability…”
> Both PERCEIVED mate availability?

LINE 33: Remove “often”.

LINE 37: “However, we found no evidence that different levels of female cues and mate availability prompted adjustments to mating decisions in males.”
> Suggestion: However, we found no evidence that different levels of female cues and availability prompted adjustments to male mating decisions.

LINE 39: ONE reason…

LINE 52: “…in response to a high risk of male competition…”
> Consider using STRONG male competition as used later in LINE 110.

LINE 52: “…mate guarding, nuptial gifts, or ejaculates…”
> Consider using AND/OR, since can’t it be a combination?

LINE 54: “…the effects of female density often remain unnoticed…”
> Unnoticed by who?

LINE 67: “Here, it was found that males developed faster if they received volatile…”
> Here? Where? Are you referring to a specific species or study system?

LINE 71: “In many mantises and spiders, males show an extremely high limitation in mating rates…”
> Extremely high limitation in mating rates? Do you have any examples of this?

LINE 73: “Different studies HAVE found that sexual cannibalism depends…”

LINE 87: “…also function as mating-plug…”
> Mating-plugs.

LINE 97: “Females stereotypically attack males during copulation, but males can reduce the risk of cannibalization if they copulate short and try to jump off the female in time.”
> Suggestion: Females stereotypically attack males during copulation, but males can reduce the risk of cannibalization if they copulate quickly and try to jump off prior to female attack.

LINES 117, 146, 148, etc.
> Are these “mating” or “choice” tests?

LINE 133: “On two days a week…”
> Two days a week…

LINE 143: “Females of both maturation states were put in little frames…”
> You list the dimensions. Remove “little”.

LINE 157: “In both treatments, males were exposed to four females, but we manipulated the number of receptive females.”
> Receptive = Pheromone Emitting females?

LINE 165: “…we used individuals from Southern Europe…”
> Specifically France?

LINE 174: “In the time of July 14th until August 16th, 2016, the experiments…”
> Suggestion: Between July 14 and August 16 2016, the experiments…

LINE 175: “…underneath a waterproof pavilion roof.”
> So was this an open-air space?

LINES 176, 181, etc.
> Are these “choice” or “test” arenas?

LINES 175-177: “The tests were conducted in cleaned choice arenas that had a size of 100 x 70 x 50 cm. The floor was covered with packing paper that was exchanged after each test.”
> Suggestion: The tests were conducted in clean arenas (100 x 70 x 50 cm) with the floor covered with fresh packing paper.

LINES 199, 200
> We used 3 times. Consider revising the use of “We used…”

LINE 208: “…which we log-transformed the copulation duration.”
> Log-transformed why?

LINE 225: “In the low-availability treatment, males left the starting point significantly later...”
> Left the starting point = started mate searching?

LINE 227: “…reintroduced to the starting point after 45 minutes…”
> Why?

LINE 244: “The only significant predictor of sexual cannibalism was male size (Table 2, Fig. 2).”
> What was the predictor? Was it larger or smaller males? Specifically mention it in this sentence.

LINES 360-366: Conclusion
> The CONCLUSION seems a bit weak. Consider revising it.

LINE 361: “Male mating effort is often traded off against future reproductive success and should strongly rely on prospective mating opportunities.”
> Consider revising. Something like… Male mating effort is often a trade-off between A and B.

FIGURE 1 CAPTION
> Effects of WHAT on copulation duration? Add more detail to the figure caption so it is able to stand-alone.

FIGURE 2
> X-axis label? Males?

TABLE 1 CAPTION
> Testing the effects of WHAT on copulation duration?

TABLE 2 CAPTION
> Testing the effects of WHAT on cannibalism rate?


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: MATE AVAILABILITY DOES NOT INFLUENCE MATING STRATEGIES IN MALES OF THE SEXUALLY CANNIBALISTIC SPIDER ARGIOPE BRUENNICHI
Review round: 1
Reviewer: 2

Output:
Basic reporting: Overall, the authors' organization of their paper is to be applauded. The figures, tables, and literature references appeared to be well-thought out and appropriate. There were many cases, however, where the grammar needed to be dramatically improved.

Some examples of grammar that need to be fixed include the lines below. However, I eventually made most of these comments in the General Comments section instead. Eventually, I admittedly got a bit tired of fixing some errors, so I politely ask the authors to take the changes I have suggested and be sure to apply it to all areas of the manuscript, and not just the lines mentioned. I also want to point out that there were, in fact, many paragraphs and sentences that were quite well-written.

In the ‘Background’ section:
Line 14: Please change to “While many studies have focused on male competition” (add “have”).

Line 15: I suggest having the 3rd sentence use the same verb tenses. “Here, we aimed to close this gap and tested effects of female availability…” (change aim to aimed)

Line 23: Please change to “We hypothesized that males opt” (change hypothesize to hypothesized). First, it should arguably be in the past tense, because you already made the hypothesis before conducting the study. Second, you used the past tense in the last sentence of this section (“we further predicted”), so it should, at the least, remain consistent.

In the Results summary (at the beginning):
The word choice could use some improvement.

Line 31: I politely suggest changing “needed more time to start” to something like “males took more time to start”.

Line 32: I would also re-structure the 2nd sentence to avoid stating “low mate availability led to”. Instead, state that copulation duration and sexual cannibalism rates were not affected by manipulations in mate availability.

In the Discussion summary (at the beginning):
Line 36: You state that the availability of virgin females facilitates mate searching, but not other mating decisions. Please re-phrase this, as many would consider mate searching to be a type of mating decision. Alternatively, re-phrase “mating decisions in males” to something more specific.
Experimental design: The experimental design appeared to be entirely original and appropriate for the hypotheses that the authors proposed. There were certainly some shortcomings that I pointed out in my General Comments, but in many cases, the authors already appropriately addressed these shortcomings in their Discussion.

I do, however, strongly suggest that the authors make several of the changes that I have proposed in the General Comments to improve the overall framework of their study, given the experimental design of their study.
Validity of the findings: The authors do a good job of providing alternative approaches and explanations as to why they might not have found significant results. In fact, I strongly believe that this paper is exactly why it is important to still make sure that non-significant data gets published and is available to the scientific literature.

Some data analyses comments are made in the General Comments, along with some possible additional explanations or thoughts to consider regarding why you found mostly non-significant differences.

Overall, the authors did not overly speculate. Some changes, however, should be made regarding more clarification that they are studying current context, not the effects of past social experience.
Additional comments: This is the section that has the majority of my comments and suggestions in it. Please take the time to address each of these thoughts, suggestions, or requests. Respectfully, many of them do need to be improved before, in my opinion, the manuscript should be considered for acceptance.

Introduction:

Overall, the first paragraph could use some improvement. The authors do a good job introducing the first sentence. However, the rest of the first paragraph needs citations and additional background information. Otherwise, it almost reads more like a prediction (especially the last sentence of the first paragraph).

Line 51: Is there a review paper that you can cite to back up your statement that most studies focus on the latter? If not, perhaps consider changing the sentence to “although many studies focus on the latter”.

Second paragraph: This paragraph is well-written and includes useful, informative citations and background information. Good job. I would consider ending it with a more conclusive “take-home” message though. Perhaps something like “Therefore, that might be some scenarios in which female density is the only social cue that can be accurately assessed.”

Line 64: Change “All three taxa” to “Some members of these taxa are known”. Otherwise, the reader might think that EVERY single member of the taxa can use chemical cues to assess female quality. Additionally, I would consider using a different word than “quality”. Perhaps, use traits, condition, or status. Too many sexual selection biologists dislike the word “quality”.

Lines 65-66: Please make this a new sentence. No need to follow up a statement about all 3 taxa, then switch to discussing only spiders.

Line 69: Perhaps, more specifically, you mean “whether past chemical information about female availability is also used…”

Line 72: It looks like you forgot to include the 2nd author on the Elgar citation. Please fix.

Line 73: Please fix the verb tense. I think you mean “the causes of sexual cannibalism are still discussed today and are diverse”

Lines 85-86: The phrasing makes it sound like all spider species only have two maximal mating chances. This is, of course, not true, so please fix the phrasing. The same is true for the next sentence in Lines 86-88.

Line 97: Please change “if they copulate short” to “if they copulate briefly”

Lines 100-110: Citations are needed for several of these facts and statements about the study species. Please add them.

Lines 115-124: Good job. In terms of the information previously provided in your Introduction, your predictions make perfect sense. I also appreciate the careful use of words, as you are correct, you are not testing “female density”. You are testing “mate availability”. On a side note, this is comparable to studies that have investigated sex ratios or operational sex ratios – the operational part includes only the mature, reproductive individuals, while sex ratios include all individuals, regardless of their reproductive status.

Materials and Methods:

Lines 129-130: A quick look at Cory & Schneider (2018) suggests that very little details about the lab breeding strategies are described. Specifically, can you please provide the reader with details regarding whether or not the spiders used in your current study are siblings or not? How many different eggs sacs are represented by the spiders in your current study? This is important information that will inform the reader about how transferable and repeatable your results might be.

Line 133: Please remove “On” from the beginning of the sentence.

Lines 134-135: Please look into PeerJ’s editing preferences. I am not certain whether “spec.” is an appropriate abbreviation for species. I am much more familiar with “sp.” (singular) or “spp.” (plural). Either way, in one instance you have it italicized, while it is not italicized in another.

Line 135: I am more familiar with providing water to spiders (and other arthropods) ad libitum through the use of cotton wicks or rolls. Can you please provide more detail about how you provided the spiders with water? Did you simply spray their web? Why 6 times a week and not 7? I am simply curious.

Lines 136-141: As an arachnologist, I am familiar with these terms and the differences between penultimate vs adult male and female genitalia. Many readers, however, will not. Could you please provide some citations that might direct the readers to the original (or more recent) description of the genitalia for your specific species? If such a description does not exist, at least a more general description for orb-weaver spiders or Argiope as a genus would suffice.

Lines 158-159: Make sure it is clear that you are talking about your species only. Recently, a study was found that demonstrated that widow males could copulate with penultimate females and successfully reproduce using this alternative mating tactic.

Lines 163: Please explain how ages were matched. Does this mean some batches of 4 virgin females had an average age of 21 days post-maturity (but were all “matched”), while others had an average age of 10 days post-maturity (but were all “matched”)? Matching does not seem as important to me as making sure that the average age across groups was not statistically different and was randomized.

Lines 166-168: Please provide more details about what is actually being measured here. Specifically, when you state “males copulated with similar large females in both”, it sounds like copulation (no or yes) was the dependent variable. Please re-phrase so it is more clear as to why you needed to run a t-test. (I am confident I understand why, but the phrasing can be improved to better make your point).
Line 168: You are still assuming, though. Therefore, please change the word from “conclude” to “assume” or something less strict.

Lines 169-171: Respectfully, this is a bad habit to start doing, though many behavioral ecologists, unfortunately, do so. To the best of my understanding, you did not statistically incorporate (which is a very difficult thing to do in the first place, because not all females were re-used) the fact that some females were re-used, while others were not. I applaud the authors for being honest. There is a possibility, however, that the re-used females did not make contact with the males in previous trials for a reason. You might be unintentionally biasing the results by continuing to use individuals that produce negative results (in which there could be an array of reasons why).

Lines 186-189: On the same day, they were re-tested? Why not test them on a different day? Further, many would argue that not making a choice, is in fact, a choice. Are you not unintentionally forcing them to make a decision if you give them a 2nd chance any time there is not a decision that is made?

Lines 195-196: I politely, but strongly, disagree with the authors’ operational definition of bigyny here. Why is a male considered bigynous if he left, but simply did not return? Should not bigyny be only used to describe males that actually mate twice? Perhaps you need to re-adjust your definitions. Alternatively, I might politely suggest to change the phrasing to “bigynous mating strategy”. That way, it suggests that they might be bigynous, but you technically do not know for sure. One option will require you to re-do your statistical tests, but from my perspective, some sort of change is necessary.

Lines 207-208: I highly suggest using a survival curve, so that you do not have to log-transform your data. I am not entirely sure if you had a cut-off point (e.g., if they were copulating for more than a set amount of time, you had to end the trial), but if so, this would also allow you to “censor” your data to statistically incorporate that.

Lines 209-210: Please use a citation in your description of body condition. There are a variety of ways to define this, and taking the residuals is one of these ways. Please add an appropriate citation (e.g., Jakob et al 1996).

Results:

Line 221: “Closest female” was never described to be incorporated into your statistical models in your Methods section. Please provide the details as to how you tested this in your Methods.

Lines 228: Your p-value is greater than 0.05, therefore, this sentence is not accurate.

Lines 234-238: I appreciate your disclosure in how you addressed the outlier. It would be tempting to ignore it, or never mention it in the first place. I applaud you for simply presenting both stats results and allowing the reader to decide how to interpret that result. Good job.

Lines 248-250: Again, please re-phrase your definition of bigynous. On a related note, do we know how long it might take a male before he would mate a 2nd time with a new female?

Lines 262-268: I was delaying this observation to wait and see if the authors eventually addressed this. I suggest that the authors strongly emphasize that they are studying decision-making within the current context, not based on past social experience. For example, the Bailey et al. 2010 paper that is cited in the Introduction provided experience with male courtship signals to test the effects. From the best of my understanding, you did not provide experience with different availabilities of potential mates. You manipulated the current context. I highly suggest digging into the literature deeper regarding this difference and to think about (and ultimately, discuss in your paper) whether or not that is why you did not find any significant results for many of your variables. Perhaps they need more experience with these social cues in order to demonstrate plasticity in their mating behaviors. Specifically, look at Kasumovic & Brooks 2010 and read about socially-cued anticipatory plasticity. Also, take a look at one of my own papers, Stoffer & Uetz 2015, entitled “The effects of social experience with varying male availability on female mate preferences in a wolf spider”. I have a series of other manuscripts that have also focused on plasticity in mate preferences in Schizocosa ocreata that would perhaps be of interest as well. While I am not trying to make a shameless plug for my own research, I think this paper could prove to be rather useful for your paper to consider and discuss. It focuses on the female decision-making end, however, and not the male decision-making end. It still, however, should be a good read on examining this difference between past experience and current context.

Lines 278-281: Studies on Schizocosa (Hebets 2003, Rutledge et al. 2010, Stoffer & Uetz 2015, 2016, etc.) might also be good to take a look at regarding asynchrony of maturation.

Lines 290-291: While I understand why you chose your method, I still think it would have been useful to also include a treatment group in which there was only 1 adult virgin.

Lines 294-298: Given that this was the case, do you know what % of your subadults were in fact penultimate females? This is a very important component of your study and could explain why you did not obtain significant differences! (Though I understand your follow-up paragraph) In short, females could prefer virgin females when given a choice, but it could still affect their mating tactics in a different way.

Lines 312-319: Great point. Nice paragraph.

Lines 344-346: Again, when you state sentences like this “the effect of mate availability on male investment”, I immediately assumed they were given experience with different treatments of mate availability and subsequently measured for their mate investments/tactics. I think the phrasing throughout the manuscript needs to make this difference a bit more clear.

Lines 353: If my memory serves me correctly, the Berglund 1995 paper is another great example of a study that technically measure the current context of manipulating female density, not the experience of it. In reality, these organisms would be experiencing these social manipulations, not have zero experience and then suddenly be thrusted into a low-availability or high-availability context. Your study still is very interesting and is necessary to our understanding. I am just giving you other perspectives to think about and address.


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: MATE AVAILABILITY DOES NOT INFLUENCE MATING STRATEGIES IN MALES OF THE SEXUALLY CANNIBALISTIC SPIDER ARGIOPE BRUENNICHI
Review round: 2
Reviewer: 1

Output:
Basic reporting: Great improvement in the grammar and overall explanation of the manuscript.
Experimental design: The authors appropriately address the limits and constraints of their experimental design, while appropriately emphasizing the benefits of their design.
Validity of the findings: Data seems to be appropriately addressed, though some clarification regarding model comparisons (see below) would be helpful.
Additional comments: Line 54-56: When you list two or more items, you should make sure the list includes all of the same type of parts of speech (e.g., a list of nouns, a list of verbs, but NOT a mixture of nouns and verbs). Therefore, change to “or the consumption of time and energy”.

Lines 71-73: If they cannot perceive chemical cues, then how would they detect female density? This of course seems possible, but I suggest the authors suggest how this would be done (using a different sensory modality?)

Lines 263-264: “using the anova command” is a very R-specific way to describe what you did. Generalize what you did and describe the statistical test you conducted, not what command you entered into R. With model comparisons, using the anova command resulted in comparing the models using a likelihood ratio test. That the models were compared using LRT seems, to me, just as important.

Lines 264-266: Perhaps I am misunderstanding what you are stating here. As currently phrased, if you were to retain “the model with the highest p-value”, this makes it sound like the simplified model and the original model each were given a p-value after the anova command. This is, of course, not true, as the LRT results in a single p-value. Of course, if p<0.05, the simplified and original models are different, and one of them is a significant improvement upon explaining variation in the data. I would focus on the latter (that you chose to keep a model if it successfully explained more variation in the data (unless you used AIC comparisons instead, which needs to be noted if the case).
	I am also not sure what you mean when you say that you stopped when you “only found significant differences between the models”. Wouldn’t you stop when there were no significant differences between the models?

Line 280: Please change to “The sample size of surviving males was fairly small, which was why we only conducted a Fisher exact test…”

Lines 368-369: What makes you think this would be especially true if the information is chemical-based?


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: TRIASSIC PENTADACTYL TRACKS FROM THE LOS MENUCOS GROUP (RÍO NEGRO PROVINCE, PATAGONIA ARGENTINA): POSSIBLE CONSTRAINTS ON THE AUTOPODIAL POSTURE OF GONDWANAN TRACKMAKERS
Review round: 1
Reviewer: 1

Output:
Basic reporting: This manuscript presents important new records of Pentasauropus that merit publication if analyzed thoroughly. However, the analysis here is far from thorough. With a  more comprehensive analysis, as discussed below, this paper will become an important contribution.

Spencer Lucas

Thus, the point of the article seems to be to identify a dicynodont with subunguligrade feet with pads as the trackmaker. That may be correct, but the analysis of the footprints to demonstrate this is far from complete. Thus, looking at the illustrated tracks, those that are parts of trackways look to have short stride lengths for such large tracks, making me think that overstepping may be happening—but this is not discussed. Indeed, the dynamic potential of the trackways needs to be realized through some metrics to tell us more about the trackmaker and its posture BASED ON THE TRACKS, not based on some general ideas about dicynodont locomotion. This is a serious flaw in the manuscript as it fails to really bring the tracks to bear on its main thesis. I encourage the authors to revise the manuscript to do just that—really show how the tracks constrain the trackmaker and its posture.

I also think that many of the tracks illustrated in Figs 4-9 tell us very little, and those illustrations could be eliminated. The ones to keep are Figures 4c-d, 5a-d, 6g-h and 8a-d.

I have made some edits to the English grammar and also have these suggestions/comments keyed to numbers on the pdf:

1.	So, how to resolve this conflict? Aren’t the numbers obviously wrong? (as stated by Lucas, 2018 in Tanner edited volume on the Late Triassic published by Springer).
2.	These many measurements would be easier to understand in a table.
3.	Also see Hunt et al. 2018 in the Tanner volume.
4.	This is the problem—some statements are made here but no real analysis is presented. Are the measurements referred to here published? Please greatly amplify this discussion with documentation. Show us how the tracks from Argentina demonstrate/support your conclusions.
5.	But, see Lucas 2018. The numbers are obviously wrong and should never have been published.
6.	What about age assignments published by Lucas 1998, 2010?
7.	This idea has been revised by Hopson 2015 in discussing pelycosaur tracks and their locomotion.
8.	What does this mean?  Please rewrite to clarify.
9.	So, here you are advocating an elephant-like foot, so say that here. Then, what about overstepping?
10.	What does this mean? Please rewrite to clarify.
11.	So, the presence of sesamoids is inconclusive, or did Sidor simply overlook them?
12.	Norian according to Lucas and Hancox (also see Lucas, 2018).
Experimental design: See above--the article fails because it does not bring the footprints it documents to actually bear on the problem it claims to solve.
Validity of the findings: May be valid, but need to be documented (see above)
Additional comments: Please improve this paper as suggested to make an important contribution.


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: TRIASSIC PENTADACTYL TRACKS FROM THE LOS MENUCOS GROUP (RÍO NEGRO PROVINCE, PATAGONIA ARGENTINA): POSSIBLE CONSTRAINTS ON THE AUTOPODIAL POSTURE OF GONDWANAN TRACKMAKERS
Review round: 1
Reviewer: 2

Output:
Basic reporting: This is some important footprint material that increases our knowledge on Gondwanan tetrapod faunas.It is worth to be published in PeerJ after improvement.

Generally the author's arguments for a dicynodont trackmaker of the presented material and for the proposed anatomy and foot posture, are conclusive.

The English needs to be largely improved. The authors should contact a native English speaking person and let him make a complete re-check of the language, before resubmitting the revised manuscript.

The authors give sufficient introduction and background. Literature is fine, a few additional (optional) references I have proposed.

The article structure needs much improvement. Thereby the manuscript can be shortened widely. In my opinion it is too long. This concerns the following parts (see also attached manuscript with comments):

1) Introduction. The general statement about importance of tetrapod ichnology includes too many citations. Most of them maybe cancelled. Reduce to one or two.

2)Material and Methods. Shorten largely; some parts of the photogrammetry technique explanation maybe redundant.

3) Abbreviations. Put these right after introduction. Explanations of acronyms have to occur before they are mentioned for the first time. Then you can delete this in Material and Methods and explanations in brackets there. This part will be shortened as well.

4) Sedimentological observations. Put this part with the subhead under Geological setting.

5) Track preservation. Move the first paragraph (listing of the slabs) to Material and Methods.

6) Description. The authors give extensive measurement details for each track (line 364-387). This should be better placed in a table, which is more easy to read. Also the description part will be shortened largely. Some parts of the description should be moved to discussion (see comments in the manuscript). Some descriptions are confusing and should be re-phrased.

7) Discussion. Shorten the discussion largely.

8) Zoological attribution. Line 427-433. Trackmaker attribution by former workers can be given in one sentence. The authors do not have to mention all Pentasauropus ichnospecies again with all former interpretations.

9) A biochronological/ichnostratigraphical usefulness of Pentasauropus is questionable, because this ichnogenus is not well-established presently as a global marker. Furthermore, the authors stress that the definite ichnotaxonomic assignment of the Los Menucos tracks has to be cleared in future studies. Also Dicynodontipus is known from the Late Permian to the Late Triassic. Against the background of the uncertain age of the unit (?Late Permian-Early Triassic-Late Triassic) and after more recent dating, the authors should be careful.

10) Conclusions. Sentences are too extensive. Some statements were repeated from discussion. Authors should give main points only, in a few short sentences.

11) A stratigraphic section (general overview of formations with track levels) would be good in Fig.1 added to the map. Also give a table with measurements (see comment above).

12) Further, minor points and some language improvement are in the attached manuscript.
Experimental design: No comment.
Validity of the findings: To Conclusions see my comment above (point 10).
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: TRIASSIC PENTADACTYL TRACKS FROM THE LOS MENUCOS GROUP (RÍO NEGRO PROVINCE, PATAGONIA ARGENTINA): POSSIBLE CONSTRAINTS ON THE AUTOPODIAL POSTURE OF GONDWANAN TRACKMAKERS
Review round: 1
Reviewer: 3

Output:
Basic reporting: -English: is good, but a native speaker must review.
- Intro & review: A good review of Los Menucos Group is made. However, the occurrence of Pentasauropus in North America is missing.
Hunt-Foster R.K., Lockley M.G., Milner A.R.C., Foster J.R., Matthews N.A., Breithaupt B.H. & Smith J.A. (2017). Tracking dinosaurs in BLM Canyon Country, Utah. Geology of the Intermountain West, 3: 67-100.
Lockley M.G. & Hunt A.P. (1995). Dinosaur tracks and other fossil footprints of the western United States. 338 pp. Columbia University Press, New York. Pages 77 and 80.
A paper published last year from the type locality of Pentasauropus of the Elliot Formation (Lesotho) must be cited.
Bordy et al., 2017. The Subeng vertebrate tracks: stratigraphy, sedimentology and a digital archive of a historic Upper Triassic palaeosurface (lower Elliot Formation), Leribe, Lesotho (southern Africa). Bollettino della Società Paleontologica Italiana, 56 (2), 181-198.
Pentasauropus-like tracks occur in Poland. See Klein H. & Niedzwiedzki G. (2012). Revision of the Lower Triassic tetrapod ichnofauna from Wiory, Holy Cross Mountains, Poland. New Mexico Museum of Natural History and Science Bulletin, 56: 1-62. Figure 52E.
- Structure: A table with the measurements is needed to make it easy to interpreter the data. Improvements in the captions and figures should be made (see comments in the PDF).
- Figures: Figures are very good and have a good quality. However, a paleogeographic map showing the distribution of Pentasauropus is necessary (including the new references listed above), and a scatch comparing the juvenile with the adult tracks would greatly improve the manuscript. The different morphologies from each slab should be indicated in the caption and/or in the image.
The captions can be improved, see more comments in the PDF.
Experimental design: The manuscript is original, with a large number of footprints never studied before and in the scope of PeerJ.
Validity of the findings: - The research is relevant and improves the knowledge of dicinodonts and made a good description of 60 footprints classified as Pentasauropus from Argentina (which was only briefly reported). The studied material allowed the recognition of ichnotaxon variability, involving the differences of juvenile and adult, locomotor dynamics and foot anatomy.
- The techniques used are very good, with three-dimensional representations and interpretative drawings of the studied material.
- Methods are well described.
Additional comments: See PDF for more 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: ASSESSING THE CAPABILITY OF FOURIER TRANSFORM INFRARED SPECTROSCOPY IN TANDEM WITH CHEMOMETRIC ANALYSIS FOR PREDICTING POULTRY MEAT SPOILAGE
Review round: 1
Reviewer: 1

Output:
Basic reporting: - English is ok
- Extremely poor sue of literature and very weak knowledge of the background is provided
- this is ok
- yes but this is not the problem
Experimental design: - this is ok
- can be considered as such
- limited
- this is ok
Validity of the findings: - The experimental design is weak (single T, and single analysis was performed) and thus not solid conclusion can be drawn.
In  this field numerous of studies have been published with a greater amount of data; thus the limited data that have been used within this study do not allow to support the conclusions reported in this article. The analysis (PCA,PLS) have been performed with inadequate number of samples; this is in my opinion a main concern regarding the impact of this study
In other words this study can be considered as VERY preliminary and more data are needed.
Additional comments: Further experimental work is needed before this study can be considered as solid. The limited number of samples examined are not enough (4 points for microbiological analysis and 4 points for FTIR). The absent of any information regarding those microorganisms that are the main spoilers (pseudomonads) are also a weak point.


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: ASSESSING THE CAPABILITY OF FOURIER TRANSFORM INFRARED SPECTROSCOPY IN TANDEM WITH CHEMOMETRIC ANALYSIS FOR PREDICTING POULTRY MEAT SPOILAGE
Review round: 1
Reviewer: 2

Output:
Basic reporting: Suggestion to rewrite the abstract considering the following:
The abstract structure includes copies of same text in both methods and results without distinction between the two sections.
Line 29: what were the statistics for this significance and what is the direction and strength of correlation
Lines 30-31: PCA does not measure strength of models
Line 31: add prediction stats and avoid the subjective "good predictions"
results relating Fourier transform infrared spectroscopy and traditional methods are not presented in the abstract
Introduction
The objectives of the study are not clear. The authors quote a similar study by Ammor et al. (2009) exploiting FTIR spectroscopy 55 along with chemometric analysis for non-destructive prediction of meat spoilage. Does this study aim to repeat these findings but using other spoilage measures?
Experimental design: What was the sample size?
was there replications of the experiments?
What is the citation for Halal Ethical Guidelines? Are these standard scientific protocols?
Lines 75-76: which media and which incubation conditions- what morphology and typical colony types categories?
Lines 96-97: it is not cleae if PCA or is it discriminant analysis is used.
Validity of the findings: Lines 134-136: value of FTIR spectra from isolated pure cultures of different members of Enterobacteriaceae is not clear as isolated cultures would not show differences in meat quality
Lines 141-148: the principal components have not been clearly explained and what is implied by the various groupings of the variables
Lines 200-205: it is not clear from PCA results how these findings are supported by this research and these may not be valid findings from the results unless this is made clear.
What is the sensitivity and specificity of this new approach.
Additional comments: Line 75: change "Total plate counts (TPC) Enterobacteriaceae" to "Total plate counts (TPC) and Enterobacteriaceae"
Line 100: Is this Partial least squares regression? Should be written in full at place of first mention
Lines 174-179: these do not really imply new findings as it is quiet general knowledge that bacteria multiply in foods such as meat over time
Line 216: do the authors mean "promoting" rather than "glorifying"? use different choice of wording.


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 OYSTER RIVER PROTOCOL: A MULTI-ASSEMBLER AND KMER APPROACH FOR DE NOVO TRANSCRIPTOME ASSEMBLY
Review round: 1
Reviewer: 1

Output:
Basic reporting: This is really interesting paper that I feel could yield good results however this manuscript feels rushed to me and I don’t think it is ready for publication yet.

Some of this is pedantic detail regarding the manuscript; the English needs to be be improved (e.g. “Troublesome, this biology is lost from all downstream analysis and inference”) as could the flow of the manuscript. There is considerable repetition (for example, at the start of the Discussion and the Conclusions) and p-values are routinely quoted to more significant figures than I believe (e.g. Section 3.2.1 p=0.0001322 for a comparison of the distributions with only 15 data-points per distribution). There are more serious shortcomings to the manuscript however that I think require considerably more attention.

I understand that the manuscript is a short presentation of a potentially useful tool but I do not feel that the authors give sufficient context in the introduction. In particular, there is no attention given to previous work on attempting to benchmark and reconcile the differences between potential de novo transcriptome assemblers, no attempt made to provide a list or or reference to the current suite of tools or any discussion of previous work highlighting how sensitive these tools are to a sensible and carefully chosen set of parameters (although the authors do correctly note that the apparent ease of use of these tools belies the complexity of the process).

On the plus side, the manuscript is well set out and the author has shared their code, the data they use is public and the assemblies will be made available, which is all good news.
Experimental design: The research question is well laid out, however the lack of summarisation of previous efforts to benchmark de novo transcriptome assembly makes it a little unclear how significant the problem they are trying to address is.

A good effort has been made to assess the performance of their new protocol in the context of other stand-alone assemblers, but I am concerned about several aspects of the process:

1) The main standalone tool they compare the Oyster River Protocol (ORP) to is Trinity, which itself forms one of the three assemblers that comprise the ORP. As such, it is perhaps not surprising that the ORP might outperform it.

2) The author doesn’t really give any justification for why he chose these three tools for the ORP as opposed to any other of the myriad of different tools. Did he choose them because they were popular, or known to give good results, or because they have very different underlying algorithms and so might be sampling different regions of search-space?

3) The data preparation for the stand alone Trinity runs is not the same as for the ORP assemblies. Specifically, the data for the stand-alone Trinity runs doesn’t include the error correction step that is included in the ORP from a previously published tool (RCorrector). As a result, it is unclear whether the improvement observed for the ORP over Trinity is a result of combining the assemblies or the error correction. In particular the % read -mapping Transrate score - which is where the ORP sees the most dramatic improvement over Trinity - may well be be strongly affected by the error correction step.

4) The manuscript doesn’t give very much detail on how the BUSCO transcripts were assessed. For example, the manuscript often includes statements such as “Trinity assemblies contained on average 86% (sd = 21%) of the full-length orthologs, 154 while the ORP assembled datasets contained on average 85% (sd = 16%) of the full length transcripts.” without carefully describing what this actually means. Are these isoform matching with 100% identity over 100% of both the ISOFORM and BUSCO transcript length? I highly doubt that the matches are this perfect (from my own experience with de novo transcript assembly). We really need considerable more detail here on how these things are compared and considered matching or not.

5) The inclusion of multiple kmer lengths is interesting, but no real effort is made to determine whether the differences between the individual assemblies is due to the different kmer lengths or the different algorithms of the tools. For example, how does an ORP assembly compare with three Trinity assemblies, with kmers of 25,55 & 75, combined in the same fashion?

Overall, however, the manuscript is lacking the depth and detail I think it needs to really fill the knowledge gap and without this details reproducing these results would be difficult despite the easy and availability of the software install and the dataset.
Validity of the findings: In its current form, the manuscript has not convinced me that the ORP represents any significant improvement over Trinity, the existing stand-alone de novo assembly tool tested. As such, I think that the conclusion the the ORP represents an improvement over existing tools is not yet sound.
Additional comments: This work details a new multi-assembler approach to improving de novo transcriptome assembly.  This is a really good idea that is worth exploring and I really want to see a strong detailed exploration of the concept and how much we might gain from combining de novo transcriptome tools, but I’m afraid that these have not been adequately illuminated here. A little more time, exploration of the tools and more careful analysis and I think it would make a good paper. I'd be happy to review future revisions of the paper and would like to see ORP bradened to include more assembly tools.

The instruction for installing and running the software (http://oyster-river-protocol.readthedocs.io/en/latest/aws_setup.html) work and I could set up the tool on my local box and verify that it runs. Unfortunately I don't have access to sudo on my clusters large memory machines, and we have no way of charging AWS tie to grants, so I couldn't test it on live data. Perhaps, given the complex web of dependencies involved, it might be an idea to distribute this as a conda package or a docker instance?


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 OYSTER RIVER PROTOCOL: A MULTI-ASSEMBLER AND KMER APPROACH FOR DE NOVO TRANSCRIPTOME ASSEMBLY
Review round: 1
Reviewer: 2

Output:
Basic reporting: The manuscript is well written and well organized.

The transcriptome assemblies resulting from each of the assemblies are made available as supplementary data, but there are many other supplementary data files and figures that would be useful to include that were not available, such as the TransRate reports for each of the assemblies, and ideally a summary data table that includes all TransRate scores and score components (ie. the individual metrics computed by TransRate that are leveraged in computing the final overall assembly score).  Busco scores for all assemblies should be included as well.

Although multiple transcriptome assemblers were leveraged, the figures focused almost entirely on comparisons between the single assembler Trinity and the author's results from ORP.  It would be useful to include comparisons to the other assemblers as well in a similar format, and provided as supplementary materials if not deemed worthy as main figures.
Experimental design: The ultimate goal of ORP is to generate a final assembly that contains the best assembled transcripts among all leveraged assemblers and to generate a final mixed assembly that is of higher quality than any of the individual input assemblers.  Specifically, the ORP method involves running multiple assemblers, scoring each transcript, and clustering together transcripts that likely correspond to the same isoform of each gene.  Based on TransRate contig scores, the single highest scoring transcript within each group is selected to represent that group in the final ORP reported assembly.

Comparisons among the final assemblies involve the TransRate final assembly scores, percent of reads represented by each assembly, and core ortholog representation as per Busco.  These are all useful metrics, but it would be of interest to include additional metrics that reflect rates of chimerism and correct representation of alternatively spliced transcripts.  In general, accuracy evaluations should rely on two metrics: sensitivity and specificity.  Most metrics leveraged by the author nicely reflect sensitivity, but there is little devoted to specificity.   Targeting specificity is difficult to do with data sets lacking reference data sets, but there are many well-studied model organisms that can be leveraged for this analysis, and among the data sets leveraged by the author, mouse is included - which has a high quality reference genome and reference annotation set where evaluation of correct alternatively spliced isoforms could be explored.   Simulated data sets are also very useful for evaluating performance metrics, given that the reference transcripts (and splicing isoforms) are known entities - and in resulting assemblies, incorrectly assembled transcripts, chimeras, or missing splicing isoforms can be readily identified.

It would benefit the manuscript to include an orthogonal measure of assembly quality such as provided by the DETONATE software.  Ideally, this would show that ORP compiled assemblies have improved DETONATE scores as compared to the assemblies provided as input to ORP.

While ORP attempts to stringently cluster transcripts into isoform-level groupings as a way of minimizing the loss of relevant splicing isoforms, there's no evidence shown of the impact of this method on splice isoform representation in the final ORP assembly.  This deserves much more attention in the manuscript.  Also, in the case of isoforms that are fused as chimeras, selecting a single best representation from a cluster containing chimeras and non-chimeras would expect to contribute to loss of important transcripts.  For example, given a cluster {AB, A, B} where AB is a chimera, selecting a single representative transcript with the best score would presumably yield A or B and hence exclude an important transcript in the final output.  More attention to resolving clusters of transcripts containing chimeras is warranted.

Since ORP is centered around combining results from multiple assemblers, it begs the question of what assemblers would be generally best suited to use with ORP.  The author leverages Trinity, Shannon, and Spades.  Trinity and Shannon have been previously demonstrated to be highly effective transcriptome assemblers, but to my knowledge, Spades has seen comparably little use for transcriptome assembly; instead Spades has been more targeted towards genome assembly.  There does appear to be an rnaSPAdes method included with Spades that's more specifically targeted to transcriptome assembly, but I could find little information about it being used in practice nor how effective it is for resolving spliced isoforms, which is one of the primary goals of transcriptome assembly and one that most differentiates it from other assembly challenges.   One of the goals of ORP is to leverage multiple kmer lengths as part of the assembly, and Spades is run twice using different kmer values as one way to accomplish this.  Using multiple kmer values to generate an assembly, leveraging the benefits of small and large kmers, is a feature of existing transcriptome assemblers Oases and IDBA-tran, neither of which were used by ORP nor cited. It isn't clear as to why the author would rely so heavily on Spades when other highly effective transcriptome assemblers are readily available and already implement ideas that are central to ORP's goals.   ORP would ideally be shown to benefit from using the very best assemblers as well as to be resilient towards including results from assemblers that are sub par.
Validity of the findings: The primary finding is that the author's ORP yields assemblies that are generally higher in quality than the Trinity assembler alone, primarily leveraging TransRate and BUSCO for assembly quality assessment.  In terms of the bulk statistics, this is a valid conclusion, but it does not specifically address reducing chimeric contigs and ensuring representation of alternatively spliced isoforms, both very important aspects of transcriptome assembly and also often negligible with respect to the bulk statistics focused upon here.

Overall, the results section is overly directed towards comparisons to Trinity when there were several other assembly methods being used by ORP.  For each assembly quality metric, it would be useful to see how ORP compares to each of the individual assemblies, including Trinity.  If there exist cases where any individual assembly used as input to ORP ends up outperforming ORP, it would call into question the value of applying ORP as a general protocol.
Additional comments: I commend the author on the development of ORP.  Generating high quality transcriptome assemblies is an important challenge, and ORP encapsulates many of the ideas that are highly relevant towards achieving this goal.  I have several constructive recommendations towards ensuring that ORP demonstrates that it achieves its aims:

1.  ORP should ideally outperform each of the individual methods used as input. Performance should include reduced chimerism and retention of alternatively spliced isoforms that are well supported by the rna-seq data.

2.  ORP should be resilient to including 'bad' assemblies along with 'good' assemblies as input.

3.  ORP should demonstrate improved assembly quality across metrics that address both sensitivity and specificity (eg. leveraging DETONATE along with TransRate, and leveraging high-quality model organism reference data sets).

Additional comments:

As a protocol, ORP should apply equally well to de novo transcriptome assemblies as to genome-guided assemblies.  Using reference model organism data sets, one could explore applying ORP to transcript sets resulting from cufflinks, stringtie, or other genome-guided methods such that ORP will yield the best quality transcript set.

Most data sets in the manuscript appear to lightly benefit from ORP, whereas there are a few assemblies that greatly benefit from ORP, especially in the context of percent reads mapping.   It would be useful to comment on this and whether the suboptimal Trinity assemblies in these cases were reflective of some aspect of the data, and whether the alternative assemblers provided much better assemblies than Trinity in these cases.  This would be much easier to assess if the results from all methods were easily accessible in tables and figures in supplementary materials.

The composition of the final ORP assemblies appears to mostly derive from Spades assembled transcripts.   I imagine this could happen for several reasons.  For example, (a) there could be clusters of transcripts where Spades yields the best representative transcript with the highest score, (b) there are clusters where there are tied scores and Spades is chosen preferentially, (c) Spades generates many transcripts that are unique to Spades and these are automatically propagated to ORP.   It would be useful to know how the composition of the final ORP assembly reflects which transcripts were selected as best within clusters vs. alternative justifications for their inclusion in the final assembly.

It would be useful to include in main figures or supplementary materials examples of genes that are better represented by the output of ORP as compared to any one individual assembler, having captured proper splice variants and/or resolved chimeras.


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 OYSTER RIVER PROTOCOL: A MULTI-ASSEMBLER AND KMER APPROACH FOR DE NOVO TRANSCRIPTOME ASSEMBLY
Review round: 1
Reviewer: 3

Output:
Basic reporting: See below.
Experimental design: See below.
Validity of the findings: See below.
Additional comments: # Review of Oyster River Protocol

In this paper, Dr. MacManes describes a new computational protocol for de novo transcriptome protocol.  Briefly, the protocol first runs three different assemblers (Trinity, Shannon, and SPAdes) with multiple parameters, combines the resulting assemblies and extracts ortholog groups using a custom modification of OrthoFinder called OrthoFuse, and finishes using a custom version of Transrate to select the highest contig score from each ortholog group.  Dr. MacManes demonstrates that there are very few downsides to using this protocol over e.g. a standard Trinity protocol: many metrics are either the same or improved.

This is an important paper for a wide range of biologists, because (as is correctly noted in the paper) de novo transcriptome assembly is very widely used in research into the biology of many (most!) different eukaryotic organisms. The completeness of reference transcriptomes is important for all the downstream analyses, including annotation, recovery of homologs, and inference of differentially expressed genes.

Along these lines, there are a number of conclusions in the paper that are quite interesting and important:
* mapping rates and transrate assembly scores are significantly improved in the ORP assemblies.
* Busco completeness scores are generally the same or improved in the ORP assemblies.
* the different assemblers appear to be better at recovering transcripts from different abundance fractions of the reads (which I find really interesting and unexpected, at least in the degree to which it seems to occur).  This observation will likely lead to methodological advances in standalone assemblers, which is excellent.

The work in general seems to be of high quality and many of the conclusions are solidly grounded in the results and discussion.

However, there are a number of puzzling choices and omissions in the paper as it stands.  I detail these below.  There are also many minor typos and grammatical errors that I will communicate directly to the author.

Questions and major comments:

* line 52 - I would imagine that other kinds of biases might result from the approach chosen in the paper; any thoughts as to downsides of the ORP approach here? (See comment on evaluating against a quality ref transcriptome, below.) e.g. in genome assemblies, maximizing N50 leads to misassemblies; might that happen here?

* unweighted assembly content is not systematically evaluated in this paper - most of the evaluations such as mapping rate and transrate score are biased by underlying abundance distribution of the transcripts, i.e. more abundant transcripts will produce more reads. I would suggest doing some kind of unweighted assembly comparison (one with higher resolution than BUSCO; next point) to see if more overall content is produced. This could be what Figure 4 intends to show, at least partly, but see below.

* it seems that no evaluation is done against a quality reference transcriptome, e.g. mouse RNAseq -> mouse. This is a major omission; the major reference-based approach used, BUSCO, only looks at a small subset of the transcripts, and does not evaluate transcript content systematically. It would be valuable to use a reference-based measure of transcriptome quality here, such as the Trinity paper used.

* The choice of taxonomic descriptions in table 1 is bewildering - I don't see any reason (biological or other) for the descriptions chosen.  Maybe stick with 'animal' and 'plant'?

* More, section 4.2 incompletely addresses an interesting question - there are many features of genomes/transcriptomes that are more likely to challenge transcriptome assembly than taxonomic membership, e.g. tissue sampled, tissue complexity in terms of number of cell types, presence of RNA editing, recent genome duplications, polymorphism rate, etc. It's probably beyond the scope of this paper to explore these issues, but I'm not sure how it merits the incomplete discussion it's given in the current paper. Perhaps a single sentence "ORP performed equally well across all of the organisms chosen at random" would be better, or something like that.

* The paper states that no value is seen in including reads beyond 30m, which is intuitive and matches our experience. However, most transcriptome assembly projects sequence multiple tissues and then combine them in a de novo assembly.  Is the recommendation for the ORP to subsample each tissue data set to 30m and then combine them? Or what? Some comment (along with a specific reference to a paper with an overall suggested workflow for making use of the assembly) would be welcome.

* Along those lines, the extra computational requirements of the ORP seem likely to be significant when combining multiple tissue RNAseq data sets. This is one of the reasons that Trinity recommends using in silico normalization, which was explicitly *not* used in this paper. The discussion should be expanded to include this.

* I'm puzzled by the division between Results and Discussion in this paper.  I'd suggest either combining them or having the primary results be separate from the discussion of what the results mean; typically the division I use is "here are the facts I see" (for results), and "here is what I think they mean".

* I had trouble understanding the section starting at line 188, and figure 4, at least at first. If I understand correctly, this paragraph is discussing both swissprot match percentages AND transcript "origin" percentages. The latter can be studied because orthofuse doesn't actually merge transcripts, it picks a "best" transcript from each orthologous grouping - is that right?  The former (swissprot analysis) is of objective interest because it speaks to what genes are being recovered by which assembler.  Or... maybe that's not what the paragraph is saying. It would be nice if this could be clearly split into discussions of homology/orthology/recover of known genes, and recovery of transcripts (known or unknown).  As it is I think it's muddled in its message. Figure 4 seems clear enough but I'm not sure if it's talking about transcripts (my guess) or swissprot matches.  (In retrospect, it seems like the beginning of section 3.2.3 is talking about transcript origin wrt the final assembly, while the bottom of that section is talking about swissprot).

Minor comments:

* surely there is a review citation of some sort for the statement that "transcriptome sequencing has been influential"? p2 line 24
* Throughout the paper, clickable links in the PDF are used (The code ... is available _here_.) these should be replaced with URLs, perhaps in footnotes or references.


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 OYSTER RIVER PROTOCOL: A MULTI-ASSEMBLER AND KMER APPROACH FOR DE NOVO TRANSCRIPTOME ASSEMBLY
Review round: 2
Reviewer: 1

Output:
Basic reporting: The overall quality of the language and the structure of the paper is much improved and it now reads much better.
Experimental design: I am happy to see that the paper has been considerably improved based on the three reviewers comments. The paper now demonstrates more conclusively not that there is a small, but clear, improvement in the quality measures of assemblies constructed using the ORP over stand-alone Trinity assemblies.

I'm still not entirely convinced that the relatively small improvement is worth the extra compute time and resources, but I am convinced its real and that some researchers will find ORP useful. The tool has been made easy to use and the difference between a 16h run of Trinity and a few days with ORP is really not a huge difference in the scheme of things.
Validity of the findings: I am happy with the paper in this category.

I would suggest that the authors include a statement reminding the reader that the one-sided Wilcoxon rank sum test is a non-parametric statistic and, as such, makes no assumptions about the nature of the distributions its testing. I think this is worth stating because its far from clear that the underlying distribution of things like the Transrate Scores are and that using a parametric test (such as a t-test) would not be justified.
Additional comments: The author has adequately addressed all the previous points I raised. I do still have a few minor (mostly cosmetic) comments that I'd like addressed further before publication. I'd recommend accepting the manuscript if these comments are addressed.

1) The author has included a justification for why he chose these three tools for the
ORP as opposed to any other of the myriad of different tools. I'd like to see a reference to the description of the characteristics of these tools in the introduction put in here so a reader who did not read the introduction knows where to look for this summary information!

2) All Figures: I don't like the axes used in many of the figures - axes should span the entire range of the data and have a defined intercept.

3) Figure 1: no axis labels - I realise the axes are not really meaningful for MDS plots but the labels should at least state what is being plotted - MDS parameter1 vs MDS parameter 2.

4) Figure 2 & 3: no axis labels (see above) - also might be better shown as a single tall figure with one column and two/three rows with the panels one above another and a shared x axis.

5) Figure 4: The captions doesn't really walk me through the figure so I'd like that expanded. Also, there are one very tiny lack points plotted on top of and underneath the violin plots - are these the data themselves, or some specific added dashed lines? What do these points mean? If they are intentional, they should be descried in the caption and made more obvious (thicker lines, large markers). If not, they should be removed.

6) Figure 5: I really don't like Joy plots (yes, I know they are the rage and people think they look cool, but...). Different panels of a figure in a scientific should not overlap - they should have their own labelled axis with a scale on. Only for a situation where the figure with separate panels would absolutely not fit on a page could I see an argument for Joy plots in a serious paper over just multi-panel plots - and we're not so tight on space here. These plots need re-drawing and multi-panel distribution plots.

7) The conclusion section is completely lacking in quantitative detail and repeats much of what has been said earlier in the paper. I'd suggest either removing it completely or, if you want to keep it (or the journal require it), then be precise and quantitative in it. Where does ORP do better and y how much does it do better.


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 OYSTER RIVER PROTOCOL: A MULTI-ASSEMBLER AND KMER APPROACH FOR DE NOVO TRANSCRIPTOME ASSEMBLY
Review round: 2
Reviewer: 2

Output:
Basic reporting: In the revised manuscript, the author provides access to additional assembly metrics and data sets.
Experimental design: In the revised manuscript, the orthogonal method 'DETONATE' was leveraged to assess assembly quality, and results appear to be consistent with the TRANSRATE assessments.
Validity of the findings: There is good evidence for the ORP protocol to produce assemblies that are of higher quality than running Trinity alone.

Figure 1 is confusing and I question whether it is valid, as one would expect assemblies to cluster together according to organism type rather than by assembly method, and perhaps within each organism type, there might be some relationship among the methods with some being more similar to others.  Instead, the distance metric appears to be showing a large batch effect where the primary difference is the program being used.  Quite surprising...
Additional comments: I commend the author for the revisions, as the manuscript is greatly improved from the earlier submission.

In Table 2, the ORP method should be included as one of the comparators.  If there are important genes that are found in the concatenated assembly but missing from the ORP assembly, it would be useful to explore why and comment accordingly.

Please check the revised version carefully for additional grammar and spelling errors.


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: CD80 AND CTLA-4 AS DIAGNOSTIC AND PROGNOSTIC MARKERS IN ADULT-ONSET MINIMAL CHANGE DISEASE: A RETROSPECTIVE STUDY
Review round: 1
Reviewer: 1

Output:
Basic reporting: Written English used in the text is quite okay, but some sentences are unclear, i.e. page 3 line 5: "patients still return to full-dose ..." - what does this mean? do patients take steroids on their own?
Abstract: were detected CD80 ... does not make sense ... so please modify these essential parts and look for others.

I do have a problem with CD80 staining. We tried for 6 months to stain renal biopsies for CD80 and did not find any consistency (maximum 1 + staining). The recent Kidney Int paper by a French group highlights this issue (The costimulatory receptor B7-1 is not induced in injured podocytes). Thus, I am rather restricted towards further research on CD80, since the "main publications" are coming from similar working groups with clear connections.

Moreover, abatacept is not effective at all as has been shown by a recent JASN paper!

Data in the results part would benefit from sub-headings.
Experimental design: Primary research is well defined.

I would like to see confidence intervals which give us more impact than p-values. Moreover, multivariate analyses are missing, for example you should adapt your findings for baseline proteinuria.
Validity of the findings: The validity might be robust for people who believe in the important role of CD80.
I don't do so, thus I would not consider CD80 as a reliable marker. The presentation of data is accurate, but would benefit from sensitivity / specificity analyses (area under the curve). Moreover, confidence intervals should be given and adjustment for baseline factors to prove whether observed changes remain significant.
Additional comments: Dear Authors,

The paper is written in an acceptable English, however, changes should be made.

I have a few further comments:
1. p3l8: every patient should have renal biopsy, otherwise you can't diagnose your patient.
2. MCD prognosis is in almost 100% favorable in Europe, so I would not consider it as a malign disease. If FSGS is found in a second biopsy, the underlying histology is always FSGS.
3. phase I MN, what is this?
4. The course of steroids in your cohort is absolutely unclear. Which dosage, standardised tapering. 8 weeks is not a timeframe which is suggested by KDIGO, if you have other definitions, you need to state them accordingly. This is clearly missing.
5. Do you re-biopsy your patients once they relapse? This will likely show the same histology or am I wrong?


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: CD80 AND CTLA-4 AS DIAGNOSTIC AND PROGNOSTIC MARKERS IN ADULT-ONSET MINIMAL CHANGE DISEASE: A RETROSPECTIVE STUDY
Review round: 1
Reviewer: 2

Output:
Basic reporting: Please check general comments.
Experimental design: Please check general comments.
Validity of the findings: Please check general comments.
Additional comments: In this paper, 55 patients with biopsy-proven MCD and 26 patients with
 biopsy-proven idiopathic membranous nephropathy (IMN), were detected
 CD80 and cytotoxic T lymphocyte antigen-4 (CTLA-4) levels in serum,
 urine and renal tissue. Authors claim that glucocorticoid treatment
 may result in complete remission for only MCD patients with strongly
 positive CD80 expression and negative CTLA-4 expression in glomeruli,
 or higher urinary CD80 level and lower CTLA-4 level.

 Major flaws:

 - Authors stated that 55 patients with biopsy-proven MCD and 26
 patients with biopsy-proven idiopathic membranous nephropathy (IMN)
 were studied. Maybe it should be more appropriate to design a study
 where groups are numerically balanced. Please explain this issue,
 since it could lead to a statistical unbalance.

 - In the Materials and Methods section, secondary antibodies employed
 in Immunohistochemistry were not specified. Please fix this issue.

 - Conclusions are too vague, please render them more accurate.

 Minor flaws:

 - In order to put your data in the context of literature, please
 consider citing D’Addio et al. 2013 Plos One (CTLA-4 Ig), La Rocca et
 al. 2000 Cell Transplant (kidney-pancreas tx) and Fiorina et al. 2001
 Diabetes (kidney-pancreas tx in T1D).


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: CD80 AND CTLA-4 AS DIAGNOSTIC AND PROGNOSTIC MARKERS IN ADULT-ONSET MINIMAL CHANGE DISEASE: A RETROSPECTIVE STUDY
Review round: 2
Reviewer: 1

Output:
Basic reporting: No comment.
Experimental design: No comment.
Validity of the findings: No comment.
Additional comments: Dear Authors,

Many thanks for the revision and replying to my queries in detail.

There is one reference which should be modified:
16	arin EH should be Garin EH ..

Best regards,
Andreas Kronbichler


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: CD80 AND CTLA-4 AS DIAGNOSTIC AND PROGNOSTIC MARKERS IN ADULT-ONSET MINIMAL CHANGE DISEASE: A RETROSPECTIVE STUDY
Review round: 2
Reviewer: 2

Output:
Basic reporting: After revision the paper has significantly improved in quality.
Experimental design: The description of methodology used is improved
Validity of the findings: the paper gives a significant contribution to MCD patients
Additional comments: The paper is now well written.


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: DAYTIME DRIVING DECREASES AMPHIBIAN ROADKILL
Review round: 1
Reviewer: 1

Output:
Basic reporting: Overall the general writing style is acceptable, however it certainly could use some improvements. I have attached a track-changes word document to this review with suggested changes to some of the writing style and manuscript structure.

The literature is referenced well, however I strongly feel the authors would very much benefit from reading through the recent text on small animal road ecology (see Andrews, K. M., Nanjappa, P., & Riley, S. P. (Eds.). (2015). Roads and ecological infrastructure: concepts and applications for small animals. JHU Press.). Some of the grander statements made in the manuscript are not accurate, and as such need to be re-worded, removed, or at a minimum better cited (again see track-changes word .doc file).

The general format is professional, however (as mentioned above) some changes to structure and style would improve this manuscript further.

This is not a really hypothesis driven manuscript, rather it is more exploratory in nature (e.g., what amphibians are dying on roads, where, why, and how?). This is not a negative, exploratory research certainly has it place. Overall, the findings spur concepts for conservation action, which seem to be the direct purpose of the research.
Experimental design: The questions are well defined.

"This study mainly investigated four questions. : (1) Which which species are the most vulnerable to vehicle collisions? , (2) Does does road mortality have a significant impact on amphibian breeding populations? , (3) What what are the temporal and spatial road kill distribution patterns for the surveyed amphibians, and? (4) What what factors contribute to amphibian road mortality? Based on our study, we can also provide targeted information (identified hot moments and hotspots of amphibian road kill) for implementing mitigation measures through the temporal and spatial distribution patterns in amphibian mortality."

However they are rather exploratory rather than hypothesis-driven.

The methods used to collect and analysis the data all seem appropriate.
Validity of the findings: Although not particularly novel to the field, the data collected seem sound and the findings clear. The conclusions drawn are highly locally specific, but due seem to follow the general trend observed in similar studies.
Additional comments: At its core, this is an acceptable manuscript. The data collection is clear, and the analysis seem fine. I do feel that it may benefit from more of a hypothesis/prediction based approach, rather that simply an exploratory one. Also throughout the manuscript over-generalized and grandiose statements are made that are not wholly accurate. These need to be address (please see tracked-changes word document). Furthermore, some of the writing style and structure should be improved before this manuscript can be accepted for publication


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

Input:
Title: DAYTIME DRIVING DECREASES AMPHIBIAN ROADKILL
Review round: 1
Reviewer: 2

Output:
Basic reporting: The article from Zhang et al. is well written and the results are in general well supported. The literature is current and well used although some comparisons are likely not relevant.
Figures are useful but legend should be presented.
Raw data were included.
Experimental design: The authors have clearly defined their objectives and made their study relevant to the field of road ecology. While the study and analyses were made rigorously, some of the details surrounding the methods could be clarified. The design is also temporarily biased since the road was surveyed 14 times in 2015 and 2016 (7 in June, 7 in August each year) as opposed to 49 times in 2017 (7 times per month in 7 consecutive months). While the authors are trying to separate their results based on these two sampling results, it has some implications on their road mortality data that are not discussed.
Validity of the findings: The validity of the findings is clear and while some of the results should be addressed more, the authors present straightforward conclusions to their study. Assessing road mortality hotspots is not novel but we know little about this threat in China and in particular in Chinese National Park adding to the value and originality of this study. As I said, some of the results should be discussed more in order to fine-tune the conclusions but overall, the results are salient and do not suffer from stretched conclusions
Additional comments: Daytime driving decreases amphibian road kill
By Zhang et al.
Ref 27511v1
This manuscript represents an interesting addition to the extensive literature on road effects on wildlife, in particular associating road mortality hotspots to abiotic factors such as traffic. Roads are known to increase population mortality but most of what we know is coming from Europe or North America. By investigating road mortality in a Chinse National Nature Reserve, this study provide additional information on the detrimental effects of roads on species in this part of the world. The design and analyses are robust and my main concerns are only two-fold:
1- The design is temporarily biased since the road was surveyed 14 times in 2015 and 2016 (7 in June, 7 in August each year) as opposed to 49 times in 2017 (7 times per month in 7 consecutive months). While the authors are trying to separate their results based on these two sampling regimes, it has some implications on their road mortality data that are not discussed.
2-while not significant, there is clearly a decrease in mortality across the 3 sampling years, a fact that is not discussed at all.
3-I suggest that the objective to assess impact on populations be removed as the method does not allow for confidently address this

Additionally, I have some minor editorial suggestions that I have added on the manuscript itself.

Best regards,
David Lesbarrères