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: RELATIONSHIP BETWEEN HEALTHCARE SEEKING AND PAIN EXPANSION IN PATIENTS WITH NONSPECIFIC CHRONIC LOW BACK PAIN
Review round: 2
Reviewer: 2

Output:
Basic reporting: The study needs some corrections:
-	Language review for readability (e.g. lines 88-93 and 286)
-	Content review; there are several examples of confusion between causality and associations (e.g. lines 83-86 and 90-93)
-	Clarification; how was fibromyalgia (excluded) differentiated from wide-spread pain (focus of the results)? If only by diagnose, was it really excluded or was it only people with known rheumatological diagnosis including FMS that were excluded? And if so, how could this influence the results?
- There are some factual mistakes in the text including the defintion of nociplastic pain (see IASP for true definition) and the role of tissue damage in chronic pain (line 58). I strongly encourage the authors to use original references and to reference them directly if they decide to re-submit.
Experimental design: The design itself seems appropriate but results focus too much on secondary outcome. In the current format this not only confuses the reader, it makes the study too weak to publish.

Twice in the study, the authors reference methods from a different study with no further description. If they used the exact same methods, please describe in an abbreviated version how the methods were the same to give the reader an impression of the data. If only some methods were re-used, please refrain from copying in a reference, and describe in full what was done in the current study.
Validity of the findings: The following results and related analysis are relevant:

"Statistically significant differences have been observed in the pain drawing, being higher in care seekers (Figure 1 and Figure 2). One-way ANOVA revealed significant differences for pain drawings (F=14.49, p < .001, η p 2=.250). The post hoc analysis showed statistically significant inter-group differences between patients with non-specific CLBP who do not seek care and patients with non-specific CLBP who seek care with a medium effect size (p = .009, d=-0.64). As well as, between asymptomatic controls and patients with non-specific CLBP who seek care with a large effect size (p < .001, d=-1.19)."

There is a strong need for the authors to address (discuss) the possible influence of the differences between CLBP-groups (pain intensity, age and educational level), including a discussion of the risk for type-1 and type-2 errors.

To the extend that the secondary data relates directly to the primary data, and they provide novel or relevant information, they can be briefly mentioned with an emphasis on the speculative nature. If not, all analysis, results and discussion of secondary outcomes should be taken out of the manuscript.
Additional comments: Speading of pain is a clinically relevant observation used in most areas of clinical reasoning. As such the study takes a good perspective although it is very difficult to understand how this should have any academically relevant association towards who seeks care? This innate problem in the study seems to be main area of concern in the current manuscript since it leads the authors to lose focus. If they believe that there is an academically relevant association between seeking healthcare and the spreading of pain, it remains to be revealed in the manuscript.
The speculative associations of secondary outcomes to the primary only confuses the message and should be left out completely. Two-point discrimination, which has well-documented associations to pain spreading has already been described in patients with CLBP, but the current study does not seem to add anything to the existing evidence, which it could be left out.

I think the authors should consider discussing Landmark et al. (https://journals.lww.com/pain/Abstract/2019/09000/Development_and_course_of_chronic_widespread_pain_.8.aspx) in relation to their background as well as discussion.


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: EFFECTS OF OUTDOOR RANGING ON EXTERNAL AND INTERNAL HEALTH PARAMETERS FOR HENS FROM DIFFERENT REARING ENRICHMENTS
Review round: 1
Reviewer: 1

Output:
Basic reporting: No comment
Experimental design: The research is within the aim and the scope of the journal. The hypothesis is well explained and the findings and the conclusions are consistent with the aim.
The methods used are deeply explained
Validity of the findings: The results and discussion are well written apart from very minor issues.
line 406 and 410 there is a mistake in table numbering
line 468 and 472: In table 3 the worm count is higher in novelty hens than in control while in the text seems to be lower.
Along with the paper, there are minor editing mistakes (see the revision in the text.
In table n.3 it should be better to add the unit at the column Worms in GI tracts
Additional comments: No comment


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

Input:
Title: EFFECTS OF OUTDOOR RANGING ON EXTERNAL AND INTERNAL HEALTH PARAMETERS FOR HENS FROM DIFFERENT REARING ENRICHMENTS
Review round: 1
Reviewer: 2

Output:
Basic reporting: Is fine
Experimental design: Is fine
Validity of the findings: Is fine
Additional comments: This paper is very interesting because it looks at hen health and welfare related to range use and rearing conditions. Although several studies have been done on this topic, the authors used methods to track individual hens, which enables them to say much more about possible relationships on an individual level instead on a flock level. Furthermore, the paper is well written in good English, well structured and contains the necessary information, including some figures and tables. Therefore, my suggestions for changes are only minor.

Specific comments
L48	‘greater exposure to parasites and pathogens’. Compared to what? To indoors? Are you sure outdoors is a greater exposure? When comparing outcomes of different studies on this topic (loose housing as such may be the risk factor and not the outdoor run per se), I would suggest to state it a bit more careful, for example ‘potential exposure to …’
L58	56 days or 54 days, as is written in L209?
L82-84	‘increased risk of disease, heat stress, predation, parasites, vent-pecking, and mortality (Bestman & Wagenaar, 2014; Fossum et al. 2009; Lay et al. 2011; Richards et al. 2012; Singh et al., 2017)’ Which risk factor is mentioned by which author?
L94	‘tracked free-range birds that use the outdoor area more, show less feather damage than hens that’. Add a ‘,’
L102	‘Larsen et al. (2018) found no association between range access and comb color, beak, footpad ‘. Did Larsen et al investigate range access (a yes/no variable) or range use (with a degree of use)?
L111	‘unclear Richards’. I guess a ‘.’is missing between these 2 words?
L112-114	‘Ranging hens may be more susceptible to internal parasite infections such as Ascaridia galli (Kaufmann et al., 2011) as the range can be contaminated by previous batches (Höglund & Jansson, 2011)’. See my comment above (L48). Are you sure Kaufmann et al proved that it was the range or can they, based on their results and the references they use, only suggest/think that is was the outdoor run? I am asking this, because after I searched myself about the risk of the free-range concerning parasites, there seemed to be a lot of suggestion (copy pased by several authors) and nearly no proofs, ending up with the idea that it is not clear whether it is the loose housing or the range that causes the risk. I would suggest to chose a bit more careful phrasing here. The hen house can also be contaminated by previous batches.
L118	‘of viral and bacterial diseases’: diseases or infections? Clinical or subclinical. Had the hens been ill or were only antibodies found without having been visibly ill?
L158	‘bird density was approximately 15 kg/m2’. Can mention the density in hens/m² as well?
L165	 Was there any daylight in the hen house? And what was the light/dark schedule?
L172	At which stocking density in hens/m²?
L203	‘RFID system, see (Campbell et al., 2018b).’ Add a ‘,’ (2x)
L209	54 days. The abstract says 56 days.
L217-2018	1.4 hours, 5.2 hours: 1.4 and 5.2 is how many minutes on top of the 1 and 5 hours? Do you mean 0.4*60=24 and 0.2*60=12 minutes?
L239-240	‘other external signs of injury or illness’. Can you give some examples, ‘such as …’
L247	‘Hens were killed using CO2.’ Is this a standard method? Is decapitation no more animal-friendly than suffocation?
L264-265	‘Following post-mortem examination all birds were CT scanned on the day of dissection. A total number of 307 Hy-Line® Brown layers were scanned using the HiSpeed QX/I (2003) CT scanner’. Remove the information that is already given elsewhere in your paper.
L277	Leave a white line above and below the formula
L280	 Leave a white line above and below the formula
L277-287	Consider to limit the number of decimals. How relevant is for example .64 when talking about 865.64 μg/mm3? Consider to replace such numbers by 866 μg/mm3
L309	why square root transformed?
L320	‘logit’ or ‘log’ and why transfomed?
L332	why square root transformed?
L354 and further	Consider to use no or less decimals when presenting percentages, or only below a certain value. For example 95% instead of 95.15% and 93% instead of 92.6%. In case of % below for example 10% you can consider to use 1 or 2 decimals.
L355	was it on purpose not to show the feet data in a table?
L364-366	Was it on purpose not to show the other health issue in tables?
L384-386	‘There was a trend for an effect of rearing treatments (F(2, 215) = 2.84, P = 0.06) on
bone mass with the post-hoc tests showing the novelty hens had higher bone mass than the control group but not the structural group did not differ significantly (Figure 4).’ Perhaps 1x ‘not’ should be removed?
L394 	Table 3 instead of Table 4
L398	Table 3 instead of Table 4
L426	‘the highest spleen weight, and fewer comb wounds’ Consider removing ‘,’
L477	‘Further studies, particularly on commercial farms, are’ Add a ‘,’
L493	change 	‘facility Campbell et al. (2017) as well’ into ‘facility (Campbell et al., 2017)
L501-505	Can more excercise and thus more energy burning from fat/muscle explain the negative relationship between range use and body weight?
L521	‘al., 2017a), but this’. Add a ‘,’
L558	Change ‘Bestman M, Niekerk TV, Haas END, Ferrante’ into ‘Bestman M, Niekerk T van, Haas EN de, Ferrante’
L594	Change ‘De Koning C, Kitessa SM, Barekatain R, and Drake K.’ into ‘Koning C de, Kitessa SM, Barekatain R, and Drake K. and then place this reference between  ‘Kolakshyapati et al 2019’ and ‘Lambton et al., 2010’.
L681	Change ‘Rodenburg TB, Van Krimpen MM, De Jong IC, De Haas EN, Kops’ into ‘Rodenburg TB, Krimpen MM van, Jong IC de, Haas EN de, Kops’
L711	Change ‘Van de Weerd HA, and Elson A.’ into ‘Weerd HA van de, and Elson A.’
Figure 4	Remove ‘,’ ‘from control, novelty, or structural’
Table 1		Replace and by , : (indoor, low outdoor, high outdoor)
Table 2	Is it possible to insert some horizontal lines to make the table more readable?
Table 2	It seems as there is one data row missing: vent – ranging- High outdoor
Table 3	‘The relative organ weights or worm counts of free-range hens’. Consider to use ‘and’ instead of ‘or’ (2x)
Table 3	Replace ‘and’ by ‘,’ in  ‘(indoor, low outdoor and high outdoor)’
Table 4	Replace ‘and’ by ‘,’ in  ‘(indoor, low outdoor and high outdoor)’


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: EFFECTS OF OUTDOOR RANGING ON EXTERNAL AND INTERNAL HEALTH PARAMETERS FOR HENS FROM DIFFERENT REARING ENRICHMENTS
Review round: 2
Reviewer: 1

Output:
Basic reporting: No comments.
Experimental design: No comments.
Validity of the findings: No comments.
Additional comments: Thank you for clarifying my questions.
I have only one remark left, concerning L226-228. To me as a rviewer it is now clear what you mean with 1.4h, but I am afraid that for a fresh reader the information between brackets in the manuscript now may be confusing. Consider to write the decimal times down as 1h 24min and 5h 12min. Or, if 1.4 is a standard way to write it down, change it back into the old phrasing.

The manuscript was not complete now; only table 3 was in it and no table captions at all.


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: ALBUMIN PROMOTES PROLIFERATION OF G1 ARRESTED SERUM STARVED HEPATOCELLULAR CARCINOMA CELLS
Review round: 1
Reviewer: 1

Output:
Basic reporting: The majority of the manuscript is well written and clear.  Concerns regarding Discussion are made in General Comments.
Experimental design: Appropriate experiments have been undertaken with correct controls.
Validity of the findings: no comment.
Additional comments: In this study the authors have addressed a simple question regarding the impact of albumin on the proliferation of HepG2/C3A cells.  The justification being that albumin is utilised as a drug delivery system and HepG2/C3A cells are used as a model for screening drug cytotoxicity and side effects involving liver injury.  Fundamentally, the authors conclude from their preliminary findings that albumin alone may promote cell growth in the absence of serum.  The majority of the manuscript is well written and clear. Appropriate experiments have been undertaken with correct controls.

There are however some minor issues which can be corrected to improve the manuscript.

(1)	Use of the word “stagnate” in abstract is not appropriate and should be defined more scientifically.  Do the authors mean that cells cease to proliferate or proliferation is reduced?
(2)	Percentages for the increases in cell growth/cell phases/cell death etc should be provided in the abstract.
(3)	Authors may want to reorder columns in graph 1 D so serum starved is first and albumin is second to reflect order in main text.
(4)	Are cell counts mentioned total cell counts or cells per ml.  It is not clear and should be stated in text and figures.
(5)	Figure legends could be improved by stating main finding of figure rather than what was done.
(6)	It would help if figure 2a and b were labelled with either starved or albumin. The same is true for all figures.
(7)	Whilst there appears to be a small but significant increase in growth after 72hrs with albumin treated cells in figure 2, further time points would be more convincing.  Could the authors justify why further time-points were not provided and provide rationale in main text?
(8)	Figure 3c required axis to be labelled.
(9)	There appears to be no reference to figure 4 in main text.  It would help if there was labelling of A and B in X axis of figure 4.  A more detailed labelling of x Axis of Figure 4 C is required.
(10)	Do the authors think that albumin stimulates G1 arrested cells into proliferation or rather allow a small number of cells that have not yet undergone G1 arrest to continue to proliferate? Maybe a point for discussion.
(11)	The Discussion appears to be mostly a repeat of results and methods with less emphasis on discussion. The authors should consider improving discussion to reflect more this section concisely. The Authors should highlight the pitfalls of their preliminary data and suggest what needs to be done further to improve.
(12)	The Authors should also clearly state why they think their findings are important, providing specific examples.   Is the relevance of their findings geared toward drugs targeting cancer that are actively proliferating rather than normal quiescent cells?  The physiological relevance if any of their data could be discussed.  Maybe the data is only relevant to in vitro models and drug testing, if so, how?
(13)	References are quite extensive for such a short report.


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

Input:
Title: ALBUMIN PROMOTES PROLIFERATION OF G1 ARRESTED SERUM STARVED HEPATOCELLULAR CARCINOMA CELLS
Review round: 1
Reviewer: 2

Output:
Basic reporting: a)	The English language should be improved.
Eg: The title of the first result, lines 201-202: “Albumin alters cell morphology and results in increased cell counts in sérum starved HEPG2/C3A cells” could be rewritten as “Albumin alters cell morphology and results in increased of counts serum starved HEPG2/C3A cells”.

b)	Some texts present in the introduction should be other sections.
Lines (126-128): “Proliferation was evaluated through changes in cell counts and cell cycle analysis whereas cell death was measured by analysing apoptotic DNA fragmentation and membrane permeabilization”. This text should be in the materials and methods section.
LInes (131-134): “Our results contrarily suggest that albumin promotes proliferation of serum starved HEPG2/C3A hepatocellular carcinoma cells by allowing G1 to S phase cell cycle transition. We also show that albumin does not significantly interfere in cell death by apoptosis or necrosis”. This text should be in the discussion section.
Experimental design: Your study did not show the molecular mechanism of the observed effect and presents an opposite result to a previous study (Nojiri, 2014). In the Nojiri study, the expression of proteins involved in proliferation signaling was modified, such as p21. Thus, it is necessary to include the evaluation of cell proliferation signaling pathways to prove your hypotheses.

The albumin concentration applied was 5mg / mL because it is comparable to total protein concentrations in 10% FBS used in cell culture. However, the plasma concentration in healthy humans is approximately 40mg / mL and other studies such as that of Nojiri, 2014 applied a concentration of 50mg / mL. Thus, it would be interesting to experimentally evaluate different concentrations of albumin, especially those higher concentrations to resemble the concentration in human plasma and determine if the observed effect is dose dependent.
Validity of the findings: The results shown need additional experiments to show by which mechanism albumin enhances cell proliferation.
Additional comments: Research question is well defined and relevant, however the results need to be complemented with the evaluation of cell proliferation signaling pathways.


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: TEMPORAL PREDICTABILITY DOES NOT IMPACT ATTENTIONAL BLINK PERFORMANCE: EFFECTS OF FIXED VS. RANDOM INTER-TRIAL INTERVALS
Review round: 1
Reviewer: 1

Output:
Basic reporting: The article is clearly written and nicely presented. It’s great to see all of the individuals depicted in the violin plots. This gives a nice overview of the data.
Experimental design: The overview design of the experiment is clear but I think some more of the technical details should be included for replication purposes.
Line 153 – stimulus. Please include the timing of the stimulus presentation. I assume it’s not the 100 ms SOA described in Figure 1 as 273 ms is mentioned for lag 3 later on so maybe 91 ms? Important to know the details including fixation duration, any blank foreperiod etc. All of those details needed to replicate the methods.
Consider including these timings on Figure 1 rather than the standard 100 ms timings (for example). I think a reader could be forgiven for assuming that Figure 1 reflects what you did rather than a general description of typical tasks.
Validity of the findings: This is a simple and elegant design and the results and discussion clearly follow from this. There should be more research like it!
Additional comments: This paper reports on a simple but useful manipulation of inter-trial interval in a dual-target rapid serial visual presentation, estimating the attentional blink based on two inter-target intervals/lags: short (lag 3) and long (lag 8). Random or Fixed inter-trial intervals are manipulated in a counterbalanced, repeated-measures design. The analysis indicates equal performance between condition, supported by a Bayesian inferential test.
Dear Dr Apthorp and colleagues,
This is a great little project, well placed within the limited literature exploring this sort of preparation within the attentional blink literature, and nicely conducted with appropriate statistics and good use of Bayesian tests to examine null effects. Great to see JASP and Jamovi getting a run! And I appreciate that you talked to the participants about their experience – should be more of this.
I have a few comments that might have some bearing on the results and a few specific things that I think should be adjusted.
I hope you find the ideas helpful.
Best wishes,
Nic Badcock
I’ve arranged my comments in two parts: major comments related to larger suggestions for consideration and specific comments related to a specific line or figure.
Major comments
1.	Order effects. Given that there’s counterbalancing in here, I think it would be valuable to test whether this impacts performance. Practice seems to make a difference after little exposure to these paradigms so I could imagine that the first conditions participants completed (a between-subjects test or mixed-ANOVA) would be most sensitive to the research question. Relatedly, I also note that the mean in Figure 3 for Lag 3 in the Random condition doesn’t touch any of the data and the underlying distribution is somewhat bimodal. I’ve recently seen this in some data I’ve been collaborating on in a completely different paradigm and when we broke it down by order, that bimodal distribution reflected the order of condition completion (lower scores for participants completing the condition first, higher for participants competing the condition second). Potentially order could be coded as different symbols in Figure 3 to get a quick idea of whether this matters but I think the interaction needs to be tested and reported. You might want to consider excluding the individual who’d had a lot of previous experience from this analysis.
Note: I just had opened the data as recommended as part of the review and noticed you have order in there – might actually be opposite to my suggestion, seems to be an advantage of doing the Random condition first. See what you think.
Also: There seems to be a duplicate data file with different names but the ‘peerj-42318-AB_TemporalPredictability_for_R.csv’ file has participant initials in there. I’d strongly recommend not including this for publication from an ethics-anonymity point of view but more generally, I’d only keep this identifiable information in a ‘key’ document that links a random id to the individual – best to avoid keep the data with any potentially identifying info.
2.	Linear mixed effects. There’s a rich amount of variability from trial-to-trial in the Random condition. This could be examined in relation to T1 and T2|T1 accuracy in a linear mixed effects model, which would be a more sensitive test of the hypothesis: with the expectation that shorter inter-trials intervals would be associated with lower accuracy. I appreciate that this may be a novel analysis, often implemented using R, and so may represent some new learning but I think it could add significant value to the work so would be worth the investment.
3.	N-1 effects. This is a final broad suggestion to make sure that we’re not missing anything. In the foreperiod literature (e.g., Los et al., 2001), there’s an influence of the foreperiod from the previous trial such that this seems to set expectations. If the n-1 foreperiod is long and the n foreperiod is short, performance (reaction time in particular) is poorer/slower, but if both are long, performance is good. In thinking about this, it might be nice to explore whether this has a role in the current data set using the above-suggested linear mixed effects approached by based on the timing of the previous trial. In fact, it’s probably worth running the n and n-1 timings within the same model as they could well interact as they do in the foreperiod literature.
Los, S. A., Knol, D. L., & Boers, R. M. (2001). The foreperiod effect revisited: Conditioning as a basis for nonspecific preparation1. Acta Psychologica, 106(1–2), 121–145. https://doi.org/10.1016/S0001-6918(00)00029-9
Specific comments
4.	Line 152, there’s an extra reference for version 3 of the Psychtoolbox that might be nice to include:
5.	Kleiner, M., Brainard, D., Pelli, D., Ingling, A., Murray, R., & Broussard, C. (2007). What’s new in psychtoolbox-3. Perception, 36(14), 1–16. Retrieved from https://nyuscholars.nyu.edu/en/publications/whats-new-in-psychtoolbox-3
6.	Lines 200-202: Given that Bayes Factors are still potential unfamiliar to readers, it might be worth explaining this in more detail in the statistical analysis section of your methods. Including the criterion against which you’re judging the null to be supported would be helpful – I’m still used to seeing it in the other direction (i.e., <.03 is support for the null) but I understand that this is easily flipped. Probably more of a Bayes-advocacy role, helping people new to Bayes understand that the directions can change and sometimes > 3 can be considered support for the null.
7.	Figure 2: Consider relabelling the y-axis to Percentage Correct. It’s just that the T2|T1 is a slight deviation from this. I’m not sure about this one – perhaps depend on the next comment
8.	Figure 2: Relate to the above, I think it’s probably more accurate for the legend to reflect the task. This is the case for T2 Only but not for T2|T1 (which has two | by the way: “T2||T1” rather than “T2|T1”).
9.	Figure 3 title: probably good form to add the citation for the BEST package


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: TEMPORAL PREDICTABILITY DOES NOT IMPACT ATTENTIONAL BLINK PERFORMANCE: EFFECTS OF FIXED VS. RANDOM INTER-TRIAL INTERVALS
Review round: 1
Reviewer: 2

Output:
Basic reporting: This is a well-written paper which reports results of relevance in point of AB magnitude stability over ITI manipulation (fixed vs. variable). The paper is clear, concise, and to-the-point.
Experimental design: Just a question: how was the 100 -- 3100 ms jittered? In steps of -- like -- 100 ms? Or what? This should be specified in the method section.  Related to this: given the fixed ITI was included in the variable-ITI range, one may wonder what could have happened by testing just the extremes of the temporal range in the variable ITI condition (100--500 ms and 2600--3100 ms, perhaps in steps of 100 ms...).
Validity of the findings: Although I am positively inclined towards acceptance of the present paper, I am wondering how the authors can conclude anything about subjects' engagement, task difficulty, and motivation (see General Discussion) without a hint of empirical evidence testing these various aspects. The AB effects obtained are quite small, and perhaps the task was not difficult enought to detect the possible effect of task difficulty. Secondly, I am not sure what the authors mean by engagement and less so by motivation.

To give an example of a different manipulation of ITI to explore its possible influence on the SNARC effect, the authors should consider:

Brigadoi, S., Basso Moro, S., Falchi, R., Cutini, S., & Dell'Acqua, R. (2018). On pacing trials while scanning brain hemodynamics: The case of the SNARC effect. Psychonomic Bulletin & Review, 25, 2267–2273.

in which my team and I, besides SNARC, tested several psychological aspects to show that particulay long ITI have in fact an effect on subjects' performance (using RTs).
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: TEMPORAL PREDICTABILITY DOES NOT IMPACT ATTENTIONAL BLINK PERFORMANCE: EFFECTS OF FIXED VS. RANDOM INTER-TRIAL INTERVALS
Review round: 1
Reviewer: 3

Output:
Basic reporting: Background and Introduction do give some context. However, the aim of the study remains opaque. Is it to investigate if an alternative approach to manipulating temporal predictability of T2 (and T1) affects AB performance similiar to those manipulations studied previously? Or is it to compare the influence of random ITI on the AB with its influence on tests of vigilance? Related, information on the rational of the ITI manipulation – crucial for the study – is rather restricted. I think that given the trial foreperiod findings, it can be predicted that manipulating ITI should have similar effects on the AB, and indeed, this has not yet been studied. The section Inter-Trial Interval (ITI) however only considers findings in vigilance tasks and leaves open why one would expect that the AB is affected by an ITI manipulation. Is it simply because AB, PVT and CPT all measure some aspect of attention or because the authors propose they all measure a particular aspect of attention that should be sensitive to an ITI manipulation? Is the key word here then maybe “temporal attention”, and if so, what is the concept of temporal attention in this context? Or does research suggest that AB and vigilance tasks are similarly affected by other variables, such as personality or pre-stimulus alpha, and therefore ITI should have the same effect? Details on how ITI affects reaction time and accuracy in PVT and CPT tasks are also missing. This information is however needed to put the AB results into context. Moreover, in how far it is relevant for the study that the AB task requires unspeeded responses (line 125) remains unclear.

The basic prediction is that the random ITI increases the AB. Given the aim of the study, what would be the implication of finding a difference between the two conditions? What would be the implications of not finding a difference?

The content of the first two sentences of the second paragraph of the ITI section (lines 119-125) is highly redundant.

Line 132: Additionally, … - In how far what follows is an additional aspect of the study remains unclear.

The last sentence in section The Current Study (line 134-135) is highly redundant to the third-to-last sentence (lines 130-132).

How were trials with non-responses to T2 treated in the analysis?

I was surprised to find participant’s initials in one of the provided data files.

Also, assuming that column Accuracy in supplemental table AB_TemporalPredictability contains T2IT1, I miss T1 accuracy data.

Typo line 269: may can
Experimental design: In how many steps was the ITI varied?
Validity of the findings: Discussion
“This finding suggests that randomizing the ITI of an AB task neither impairs nor improves performance” (Discussion, 228-229). The authors expected differences and therefore tested for it, they did not design the study to show that there is no effect. Therefore, all they can conclude is that they did not find evidence that randomizing the ITI affects AB task performance. Also, Bayesian t-tests indicate that evidence for the null is not particularly strong.

„This result expands upon findings made by Badcock et al. (2013) who found that randomising the ISI between trial commencement and T1 presentation did not affect
AB performance when compared to a brief or extended ISI (273ms or 880ms respectively).” (Discussion, 229-231). This contradicts the author’s summary of Badcock et al.’s findings in the Introduction: “Badcock et al. (2013) found that, compared with randomly variable foreperiods, having a predictable foreperiod attenuated the AB, but only for relatively long foreperiods (~880 ms).”

“This finding has useful implications for future research, as it provides confirmation that
the standard AB task, typically used as a measure of transient attention, has similar levels of interest and difficulty to those tasks which employ a random ITI [PVT and CPT]”. (Discussion, 238-240). I do not get this conclusion. How does not finding an effect of a random ITI for the AB confirm similarities with tasks in which – as summarized in the Introduction - an effect of a random ITI is normally seen? Moreover, what is meant by “similar levels of interest and difficulty”?

The authors should consider that even though on average accuracy does not differ significantly between conditions, the variable ITI might still affect performance. For instance, performance might be very poor for the short ITI conditions and much improved for the long ITI conditions, which would not show up in the averaged accuracies. This could be tested in an exploratory analysis.

I can follow the reasoning for using the abridged ITI in the present study. However, it should be discussed that this decision clearly restricts the comparability to the vigilance paradigms with much longer randomized ITIs. It also might be the reason for not finding a difference between the experimental conditions. The authors should also consider the possibility that their null finding indicates that within the AB, the onset of the RSVP stream is more relevant for the distribution of attentional resources and thus task performance than the ITI.

The final paragraph of the Discussion (160-266) needs more context. What is meant by “sensitivity thresholds around the current experimental paradigms”? How would a more accurate representation of a hybrid PVT and AB task help to measure the possibility that task difficulty effects performance? In turn, why should task difficulty not affect performance? What would be learned from measuring RT in a task that is unspeeded?

Conclusion
“Importantly, these findings support the legitimacy of comparing performance on the AB and random ITI tasks as a measure of transient and sustained attention respectively”. I cannot see how the authors arrive at this particular conclusion, and, more generally, the repeated claim that their results have broad implications for research comparing the AB as a measure of transient attention and other paradigms measuring sustained attention. Maybe this will become more clear if Introduction and Discussion are revised around a clearly described study aim.

Other
As previously mentioned, assuming that column Accuracy in table AB_TemporalPredictability contains T2IT1, I miss T1 accuracy data.
Additional comments: I agree with the authors that ITI is a factor in AB paradigms whose potential influence on task performance has been overlooked so far. The statistical analyses run and the description of results is comprehensible. The weakness of the manuscript is the lack of a clearly formulated objective, which is reflected in problems in the Introduction, Discussion and Conclusions outlined above.


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: DIVERSITY OF FALL ARMYWORM, SPODOPTERA FRUGIPERDA AND THEIR GUT BACTERIAL COMMUNITY IN KENYA
Review round: 1
Reviewer: 1

Output:
Basic reporting: This paper general approach was to investigate the S. frugiperda microbiome ad its role in the insect’ success and adaptability to rice and maize cultivars in Kenya, Africa.

1.	They compared the microbial composition, abundance and diversity of S. frugiperda larva and adult specimens collected from maize and rice from growing regions in Kenya.
2.	They analyzed the bacterial diversity between larvae and adults.
3.	They determined this insect population prevalence within maize and rice cultivars.

Authors identified mainly Proteobacteria and Firmicutes. They detected differences in bacterial diversity between larvae and adults, where some but not all prominent bacterial groups in larval stage are lost during metamorphosis, thus suggesting being transmitted.
They detected differences in bacterial diversity between larvae and adults, where some but not all prominent bacterial groups in larval stage are lost during metamorphosis, thus suggesting being transmitted. They detected several entomopathogenic bacterial clades and one entomopathogen fungus. DNA haplotyping of this insect population indicated higher S. frugiperda prevalence of the ‘rice’ strain.

Since microbiome plays an important role among insects’ prevalence and adaptability to different environments, this study is important. Based on this study results, I do recommend this manuscript for publication.
Experimental design: All experiments were well designed and performed in concordance to this study aims.

Main concern
Why the authors did not analyze larvae and adults from sorghum cultivars??
Validity of the findings: Data analyses from experiments were performed adequately to overcome this study purposes.
Additional comments: Suggested editing of this manuscript are:

Abstract

Where the insect were collected?
Ln 28. Here, authors stated “larval and adult specimens of S. frugiperda collected from four maize growing..”, whereas in Ln 126 (introduction section) they described collecting insects from rice in addition to maize. In the M&M section (ln 130) they stated “Spodoptera frugiperda larvae were collected from infested maize fields in Kenya.
Ln 29. Change “non-bacterial” by “fungic”

Introduction

Ln 52. Delete underlines before “et al., 2016”
Ln 53. Delete “Goergen et al., 2016”
Ln 53-54. Do authors need nine references to support the first paragraph? They should be able to select up to three.
Ln 65. Change “Further” by “Furthermore”
Ln 67-68. Do references really support the statement: S. frugiperda readily develop resistance to most chemical insecticides and transgenic cultivars?
Ln 69. The “Yu 1991” reference is out of place.
Ln 70. Change “In light of this,” by “In resume,”
Ln 93-96. Do authors need eight references to support the first paragraph? They should be able to select up to three.
Ln 126. Here, authors describe collecting insects form rice in addition to maize, whereas in the abstract (ln 28) they stated “larval and adult specimens of S. frugiperda collected from four maize growing..”

Material and Methods

Ln 130. S. frugiperda larvae were collected only from infested maize fields?

Results
Ln 189-190. Move paragraph “In addition, samples were collected from these 4 sites plus two additional sites for mtDNA haplotyping” to the Insect collection subsection of the Material and Methods section. Please specify if those samples were from rice cultivars.

Ln 192. Add “where” before “the median length”
Ln 204. Change “note” by “noticed”
Ln 210. Change “observed” by “detected”
Ln 2015. Change “fungi” by “fungus”

Discussion
Ln 266-267. Here the authors again state that all samples were collected from maize plants.
Ln 232. Delete “Other”
Ln 284. Change “non-bacterial” by “fungic”

Conclusions
Ln 310. “Symbiotic bacteria play a key role in the biology of insects.” is not a conclusion.


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

Input:
Title: DIVERSITY OF FALL ARMYWORM, SPODOPTERA FRUGIPERDA AND THEIR GUT BACTERIAL COMMUNITY IN KENYA
Review round: 1
Reviewer: 2

Output:
Basic reporting: The manuscript contains genomic analyses conducted on specimens of Spodoptera frugiperda aimed at identifying and quantifying the main bacterial groups.
I believe that the work is not acceptable in the present state, because of some major concerns:
1) The introduction creates the expectation of an original study aimed at identifying potential microbiological control agents, but no observation is conducted on bacteria identified in the insect's microbioma to establish such potential. The objectives of the work should be clearer and in line with what has actually been done. This requires a significant revision of the introduction.
2) It is not clear which species have been identified. Does the work focus on bacterial taxa or information on species are available? Some species is mentioned in the discussion as a potential biocontrol agent. Please clarify.
3) Lack of comparison with the microbiome of a reference species, to make original this work referred to the Spodopter frugiperda
4) Fungi (eg. Metarhizium, etc.) are included in this work.; were they detected with bacterial 16S analyses. This has to be clarified.
5) Tha actual originality of this work should be clearly stated, based on the significance of the results.
Experimental design: Revise this section on the basis of previous comments.
Clarify methods of detection of fungi and bacteria.
Validity of the findings: Revise results and discussion on the basis of previous comments and according with the actual study objectives.
Additional comments: Because of major concerns I recommend to deeply revise this manuscript.


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: CHARACTERISATION OF GLUT4 TRAFFICKING IN HELA CELLS: COMPARABLE KINETICS AND ORTHOLOGOUS TRAFFICKING MECHANISMS TO 3T3-L1 ADIPOCYTES
Review round: 1
Reviewer: 1

Output:
Basic reporting: Basic reporting standards are mostly met; see comments under 3) Validity of the Findings.
Experimental design: Experimental design standards are mostly met; see comments under 3) Validity of the Findings.
Validity of the findings: This manuscript examines GLUT4 trafficking in HeLa cells and compares it to that in 3T3-L1 adipocytes, and then goes on to use the HeLa cells for an siRNA-based analysis and screen. The data to establish the HeLa cell system are similar to that recently published by Camus et al., J Cell Biol. 2020 Jan 6;219(1). pii: e201812135 and so are not particularly novel, but they do further establish and expand upon the utility of the HeLa cell system for studies of GLUT4.  The siRNA data are convincing, although one wishes that the work were taken a little further.  Because the guidelines for PeerJ do not require assessment of impact or novelty, this should not preclude publication.

Specific comments:

In Fig. 2, the rate of increase in GFP fluorescence in the TIRF zone is rather slow, compared to previous studies reporting that maximal or near-maximal GLUT4 translocation occurs within 5-10 min after insulin addition, and that a steady state is reached by 15-20 min.  Detailed kinetic were published in Govers et al., Mol Cell Biol. 2004 Jul;24(14):6456-66 and Bogan et al., Mol Cell Biol. 2001 Jul;21(14):4785-806.  There is some discussion of this point, but the wording is unclear.  Line 248 reads: “The slower rate of translocation…” without stating what translocation is slower than.  Presumably, translocation is slower than that previously reported, but the text needs to state this more explicitly.

The manuscript uses the term “GSVs” on line 126 and subsequently in several other places, but this term is not defined. The “GLUT4 Storage Vesicles” are thought to be cell type specific, and can be detected in fat and muscle cells, and not in other cell types.  Even the recent Camus et al JCB paper reports that CHC22 is involved in a “GLUT4 pathway” that is present in HeLa cells, but is careful not to claim that it mediates budding of the GSVs (or GSC) itself.  It would be appropriate to replace this term with “GLUT4-containing vesicles” – which is more noncommittal about what these vesicles actually are.

The static vs mobile GLUT4 vesicle analysis is somewhat confusing, based on the description in the Methods section and on the data shown in Fig. 3.  It appears that in 3T3-L1 adipocytes, insulin caused no change in the number of static vesicles in the TIRF zone, and caused a transient increase in the number of mobile vesicles observed.  By contrast, in HeLa cells, the numbers of both static and mobile vesicles decreased.  These observations do not support the idea that HeLa cells and 3T3L1 cells behave similarly.

Of note, the adipocyte-specific GSVs are small (60 nm) and can be difficult to visualize by TIRF microscopy, and GLUT4 traffics to the plasma membrane in both GSVs and, subsequently, in larger (150 nm) endosome-derived vesicles in 3T3-L1 adipocytes (Xu et al., J Cell Biol. 2011 May 16;193(4):643-53 and Chen et al., J Cell Biol. 2012 Aug 20;198(4):545-60; the Chen reference identified the GSVs as Rab10-positive).  It may be that most of the vesicles observed in the Lizunov et al J Cell Biol 2005 article (and possibly subsequent papers) are the larger, endosome-derived vesicles, which constitute the ‘second wave’ of GLUT4 translocation and are more readily detected using TIRF microscopy.  These considerations further complicate the interpretation of the static vs mobile GLUT4-containing vesicles in the TIRF zone in HeLa cells.  It is unclear what sort of vesicles are present in HeLa cells, and to which type of vesicles they are being compared in 3T3-L1 adipocytes.

It is somewhat disconcerting that Figure 5 does not examine GLUT4 translocation to the plasma membrane, despite that this has been the main focus of figures up until this point.  Does insulin stimulation affect the colocalization of GLUT4 with ERGIC53 or GSM130 (or alter the GLUT4/DNA ratio) in HeLa cells?  If so, are these effects blocked by GOSR1 or Ykt6 siRNA treatment?

Having identified GOSR1 and Ykt6 as proteins that affect in GLUT4 in HeLa cells, it would be logical to then examine these in 3T3-L1 adipocytes.  Indeed, a main point of the manuscript is that HeLa cells can be used for preliminary studies, which would then be tested in a more physiologic cell type.  Thus, the conclusion that these proteins are normally involved in GLUT4 translocation would still have to be characterized as preliminary.

Other items:

Methods:

Line 149 do the authors mean 3 micromoles siRNA rather than 3 micromolar siRNA?

For the FACS analysis – how were background fluorescence intensities determined?  If the flow cytometer measures fluorescences on a logarithmic scale, then were cells not expressing GFP or not stained for cell surface HA used to assess background?  Was the geometric mean or median used to quantify fluorescence intensities?

References:

The Garvey et al 1993 reference is repeated twice.
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: CHARACTERISATION OF GLUT4 TRAFFICKING IN HELA CELLS: COMPARABLE KINETICS AND ORTHOLOGOUS TRAFFICKING MECHANISMS TO 3T3-L1 ADIPOCYTES
Review round: 1
Reviewer: 2

Output:
Basic reporting: The manuscript is well written and clear throughout, with 1 or 2 exceptions.
1. Please check for use of standard abbreviations in the methods - e.g. minutes, min
2. Please check consistency of units in methods - mg/mL, ug.ml-1

Literature references are appropriate.

Manuscript is structured appropriately, and the figures of good standard.

The results are well written, and provide a good overview of the hypotheses for each subsequent experiment.
Experimental design: The Authors describe a series of comparisons of GLUT4 trafficking in 3T3-L1 adipocytes and HeLa cells. The studies reveal similarities between these cells lines in specific aspects of GLUT4 trafficking. Data included in this manuscript are useful to the field in that they provide a rationale and evidence for the utility of HeLa cells for screening for novel regulators of GLUT4 trafficking. Using HeLas cells, the Authors report that GOSR1 and YKT6 as novel regulators of GLUT4 traffic (in HeLa cells).

The manuscript clearly describes the rationale for the study.

Methods - In general very well described.
Please define the permeabilisation buffer.
Also, include the specific test used to define statistical significance - e.g. figure 2. For tests comparing two values, one where there there is no error associated, did the Authors use a one-sample t-test?

Can the Authors clarify the statement "generated stable clones of HeLa cells" (Results and Discussion). Were single clones of HeLa cells expressing HA-G4-GFP isolated, and the experiments performed in a cloncal cell line? This is not clear from the methods.
Validity of the findings: The data presented include controls and replicates as needed.

Comments:

Figure 1C. The descrption of these data does not talk about the 3T3-L1 response, in comparison to the HeLa cell data. This is a key difference between the 2 cell lines and should be highlighted here. Is the difference in resposne due to higher basal in HeLa cells, or a greater response in L1 cells?

Figure 2. The writing around these data is a little unclear:
"The slower rate of translocation ... GLUT4 in the plasma membrane". What is the translocation "slower" than? Is this in comparison to othe reported T1/2?

One additional piece of data that would be very informative would be a comparison between TfR (endosomal recycling) and GLUT4 in the two cell lines. For example - colocalisation between GLUT4 and TfR in unstimulated conditions. This would adress (to an extent) whether HeLa cells have a bone fide specialised GLUT4 compartment or whetehr GLUT4 traffics with TfR in HeLa cells.

While there are some differences in the responses between cell lines, the Authors generally lean towards pointing out similarities in the Abstract. For Fig. 3, while the data show that the number of static/mobile vesicles are similar, there are stark differences in the responses of both the static and mobile GLUT4 vesicles between L1s and HeLas. The Authors acknowledge this by saying that "insulin-dep mobilisation of GLUT4 to the cell surface is an inherant property of .. cell types". It would be useful to include indictaion that there are differences in GLUT4 trafficking between cells in the Abstract/introduction.

Regarding VAMP2 KD data.
- The VAMP2 KD appears to be minor - what is % KD here (Fig. 1 supp)?
- The major effect is increasing cell surface GLUT4 in unstimulated cells. Is this the same or different from what is described in adipocytes? Also, Figure 4A shows that VAMP2 KD chnages the GFP loclaisation  - has this been reported before?
- Other papers have described redundancy in the Vamp requirement in adipocytes - this study should be quoted - Variations in the requirement for v-SNAREs in GLUT4 trafficking in adipocytes. Ping Zhao, Lu Yang, Jamie A. Lopez, Junmei Fan, James G. Burchfield, Li Bai, Wanjin Hong, Tao Xu, David E. James. Journal of Cell Science 2009 122: 3472-3480; doi: 10.1242/jcs.047449

GOSR1 and YKT6. Do the Authors suggest this is a specific effect on GLUT4, or an effect on all protein trafficking to/from the ERGIC.

Can the Authors translate these findings to L1s? As suggested, HeLa cells may be used as a screening cell line, but it would be interesting to know if these proteins regulate GLUT4 in adipocytes/muscle cells (cells that express GLUT4). This is not essential.

As a suggestion, would a table of the experiments performed in this study and how the cell types compare be useful?
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: CHARACTERISATION OF GLUT4 TRAFFICKING IN HELA CELLS: COMPARABLE KINETICS AND ORTHOLOGOUS TRAFFICKING MECHANISMS TO 3T3-L1 ADIPOCYTES
Review round: 2
Reviewer: 1

Output:
Basic reporting: No comment
Experimental design: No comment
Validity of the findings: No comment
Additional comments: The authors have addressed my critiques and I think the article is now acceptable for publication.

As an aside, I respectfully disagree with the authors’ response to a comment from to the other reviewer, who suggested examination of the overlap between the GLUT4 reporter and transferrin receptor (TfR) in HeLa cells.  The authors stated that McGraw’s group showed a clear distinction between GLUT4 and TfR in CHO cells and that, in their view, repeating the experiment in HeLa cells will likely show that GLUT4 and TfR “will not significantly overlap” and that the data will not add much to the paper.  In fact, the Lampson…McGraw MBoC 2001 and Johnson…McGraw JBC 1998 papers reported substantial overlap of GLUT4/IRAP and TfR in CHO cells.  Our work (Yu JBC 2007) found that in 3T3-L1 adipocytes, TUG overexpression drew GLUT4 out of a TfR-positive compartment, whereas TUG disruption caused both 1) ablation of this GLUT4-positive, TfR-negative compartment and 2) redistribution of GLUT4 (but not TfR) to the cell surface.  Thus, although CHO (and, likely, HeLa) cells may have a “specialized GLUT4 compartment” to some degree, this compartment is not well developed, compared to that which is present in adipocytes.  In 3T3-L1 cells, the compartment becomes more prominent at day 2-4 of differentiation, such that GLUT4 targeting to TfR-negative membranes is increased, basal GLUT4 sequestration is increased (e.g. Bogan MCB 2001 and other articles), and the fold-translocation response is greater.  This results at least in part from upregulation of sortilin (as shown by Kandror’s group) and possibly other proteins (e.g. Usp25m, KIF5B).  Sortilin is expressed to some extent in HeLa cells, yet it is likely that there would still be substantial overlap of GLUT4 and TfR, even using current microscopy techniques.  This is contrary to the authors’ suggestion.  Whether the GLUT4-positive, TfR-negative staining in HeLa cells accounts for 5% or 25% or 50% of the overall GLUT4 staining may be useful to know, and could be assessed in future work.  The information could help to improve screens using this system.  Given the difference in the magnitude of the translocation responses, it is likely that the GLUT4-positive, TfR-negative compartment accounts for a much lower fraction of total GLUT4 in HeLa cells, compared to 3T3-L1 adipocytes (in which it is ~50% of total GLUT4).

The above considerations do not detract from the main point of the authors’ manuscript, which is that HeLa cells can be useful for preliminary or high-throughput studies of GLUT4 traffic.  However, it is important to avoid pushing the issue too far.  The abstract emphasizes similarities between HeLa cells and adipocytes without noting the difference in the magnitude of translocation response.  The authors may wish to note this to provide a more balanced view, and I leave this to their discretion.


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: CHARACTERISATION OF GLUT4 TRAFFICKING IN HELA CELLS: COMPARABLE KINETICS AND ORTHOLOGOUS TRAFFICKING MECHANISMS TO 3T3-L1 ADIPOCYTES
Review round: 2
Reviewer: 2

Output:
Basic reporting: Morris et al. characterise GLUT4 trafficking in HeLa cells. They report similarities to GLUT4 trafficking in adipocytes and highlight the HeLa cell model as a useful tool in studying GLUT4.

The Authors have addressed all comments.
Experimental design: The Authors have addressed all comments.
Validity of the findings: The Authors have addressed all 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: RECENT ADVANCES ON THE ESTIMATION OF THE THERMAL REACTION NORM FOR SEX RATIOS
Review round: 1
Reviewer: 1

Output:
Basic reporting: In this manuscript, the authors propose a new method to estimate the temperature-sex reaction norm at constant temperature for reptile embryos with TSD. They provide a flexible model for estimate the temperature-sex reaction norm using maximum likelihood, as well as a method of estimating the temperature-sex reaction norm using a Bayesian method. They include clear, step-by-step instructions aimed at reducing barriers for authors analyzing TSD data.

Overall, I enjoyed the manuscript and believe it will be useful to future researchers, in terms of the clarity of methodological explanations, the new flexit model they propose which is superior to the commonly used logistic model, as well as the Bayesian MCMC method for model fitting. However, there are some small issues with language, formatting, and references (described herein, and in “General Comments”), as well as also some larger issues with regards to the data used for their analyses (see under “Validity of Findings”, that must be addressed before Acceptance.

1.	The manuscript is written in a logical and easy-to-follow way, and is useful in nature. However, the quality of the language used is at times informal (for example, Line 264, see elaboration under “General Comments”) and appears unfinished due to the use of bullet points (see Line 253-258 and elsewhere, under “General Comments”). Further, the language used is inconsistent; the authors state that they will use “TSD pattern” to describe the temperature-sex reaction norm on line 66, but afterwards continue to refer to the reaction norm (see “General Comments” for exact lines). I suggest the manuscript be refined and edited, in terms of language, before Acceptance.

2.	The authors provide an excellent general overview of the TSD phenomenon in their introduction. That being said, the thermosensitive period and its complexities should be discussed in more detail; the middle-third of incubation, though commonly used, is a poor proxy for the TSP for several reasons – the authors hint at this, but do not describe it in detail, and lack references to key papers describing the relationship between thermosensitive period, temperature, and development rate (see “General Comments” under lines 115 – 116, Line 516 for more detail). Accurately delineating the thermosensitive period is key to estimating sex ratios (whether at constant, or fluctuating temperatures) and this should be stressed, especially given the usefulness of Girondot’s embryo growth package, which integrates development rate with temperature.

3.	Figures and tables could benefit from further description; I found the captions somewhat vague. Further, they should be integrated into the text more comprehensively. For example, on lines 484, the authors state that comparisons of fit using Maximum Likelihood and Bayesian MCMC are shown in Table 5, yet the Maximum Likelihood estimates are not shown in Table 5. In Table 2, the caption states “bold lines indicates the selected model”; there are no bold lines in Table 2. In table 4, the caption states “selected models are indicated in bold font”, but there is no bold font. I also suggest the authors elaborate on their table and figure captions to include Species and Locations.
Experimental design: No comment.
Validity of the findings: 1. While I find the methods the authors have used intriguing, and understand that the purpose of the paper is to present these new methods, I found the source data they were using to be low-quality when compared to other datasets in the literature being used to calculate the temperature-sex reaction norm in turtles. See, for example, Wibbels et al 1998 (reference below this section), where sample sizes were 19 or greater for each temperature, in this species. For example, at every temperature in Pirambu Beach and Orissa, as well as the majority of temperatures in Playa La Destiladeras, there are fewer than 10 embryos used. At 26.3C in Pirambu beach, only two embryos were sexed; I do not believe that, with certainty, you could say this temperature produces all males. I understand that sample sizes can be low for protected species (as you said on line 340) but these sample sizes are extremely low. I give two suggestions, either of which would improve the quality of the paper:

a) Give significant justification and explanation for the sample sizes used and explain to the reader how this would affect analyses (Bayesian MCMC vs. MLE).

b) If possible, could you use and/or compare this with another dataset, perhaps from another species in which there are high sample size estimates of the temperature-sex reaction norm, to see how your new method compares with MLE?

2. Within your text, could you justify the pooling of data from various sites? For example, Nancite and El Verde Camacho are over 2500km apart as the crow flies, I am not sure why it makes sense to pool these data; in a study of freshwater turtles, Ewert (2005; reference below) found significant differences in temperature-sex reaction norm between populations that were closer geographically.

3. Relating to #2 (above) - La Escobilla and Playa La Destiladeras have very few temperatures represented. Note that in Table 1, La Escobilla has 32C represented twice (perhaps a typo?), giving a total of three temperatures. Please clean up or clarify this table. Can you justify the use of sites with four or fewer temperature treatments?

3. Concomitant with comment #2 under Basic Reporting, proper delineation of the TSP is very important to accurately estimating sex ratios (even at constant temperature) under TSD. As we know, temperatures can fluctuate to a high degree (relative to the TRT) within an incubator. Is it possible to modify your model with an understanding of the TSP in L. olivacea from stage-shift experiments, increasing the resolution from simply "middle third of incubation" (E.g. Massey et al 2019 for the Snapping Turtle)? This is just a suggestion, I would be curious to see how it affects your modeling of the temperature-sex reaction norm.

References:
Ewert, M. A., Lang, J. W., & Nelson, C. E. (2005). Geographic variation in the pattern of temperature-dependent sex determination in the American snapping turtle (Chelydra serpentina). Journal of Zoology, 265(1), 81–95. doi:10.1017/s0952836904006120

Wibbels, T., Rostal, D., & Byles, R. (1998). High pivotal temperature in the sex determination of the olive ridley sea turtle, Lepidochelys olivacea, from Playa Nancite, Costa Rica. Copeia, 1998(4), 1086-1088.
Additional comments: Line 46: It may be more comprehensive to mention that some fish exhibit TSD (see: Ospina-Alvarez, N., & Piferrer, F. (2008). Temperature-dependent sex determination in fish revisited: prevalence, a single sex ratio response pattern, and possible effects of climate change. PloS one, 3(7), e2837.)

Line 47: “termed” the “thermosensitive” period. Termed is preferable to named as “thermosensitive period” is jargon. Thermosensitive rather than thermo-sensitive has been used elsewhere in the manuscript and is more commonly used in the literature.

Line 51: Present in some turtles is more accurate. Snapping Turtles, for example, have Type II, whereas many softshell turtles have GSD.

Lines 72-74: I am confused by this statement; if latitude (local incubation conditions) explained differences in TRT breadth, why do geography (which encapsulates latitude) and local thermal conditions (which encapsulates local incubation conditions) not explain variation in TRT breadth?

Line 81: I recommend changing pertinent to empirical.

Line 92 – 93: The citation format here and elsewhere are incorrect.

Line 113: “The” second-third. Suggestion: many authors refer to it as the middle third.

Line 115 – 116: The assumption that the middle third of incubation corresponds exactly to the TSP is likely invalid in general, and likely varies with temperature; see several stage-shift experiments, see:

Merchant-Larios, H., Ruiz-Ramirez, S., Moreno-Mendoza, N., & Marmolejo-Valencia, A. (1997). Correlation among Thermosensitive Period, Estradiol Response, and Gonad Differentiation in the Sea Turtle Lepidochelys olivacea. General and Comparative Endocrinology, 107(3), 373–385. doi:10.1006/gcen.1997.6946

Yntema, C. L. (1979). Temperature levels and periods of sex determination during incubation of eggs of Chelydra serpentina. J. Morphol. 159, 17–28.

Line 125: it may be useful to provide citations of studies that have shown mean temperature is a poor proxy for TSD patterns at constant temperature. Reword as: “The use of mean incubation temperatures obtained from natural nests to feed a model to estimate TSD patterns has been shown to produce inaccurate results.”

Line 126: “A further misconception is the difference between resolution…”

Line 143: It is unclear whether you are using data from nesting beaches (in-situ) or are using common-garden data (incubated in lab at constant conditions). Please clarify.

Lines 146 – 148: Embryo growth reaction norms from different locations are highly likely to differ. Did you estimate different embryo growth reaction norms for all populations, or just Pacific Mexico vs. Pacific Costa Rica?

Line 152 and elsewhere: The bullet points make the manuscript difficult to read and look unfinished; can these be sectioned into paragraphs or subheadings rather than bullet points?

Line 180: Can you elaborate on how the Hulin model is challenging to fit?

Line 220: Pieau published this work in 1998 in the Journal of Experimental Biology; it would be useful to cite this paper for other authors.

Line 242: “To fit the TSD pattern using maximum likelihood…” (Delete unnecessary words; previously, you state that you will use “TSD pattern” to describe temperature-sex reaction norm – keep your wording consistent.)

Line 252: “serves various purposes”

Lines 253 – 258: I suggest removing the bullet points and replacing this with a paragraph for clarity and flow. Further, please explain each point in more detail for the reader – e.g. how is it that datasets become comparable?

Line 264: Language is informal and not cohesive with the rest of the manuscript (“if you slightly shift a parameter”…) Consider changing to passive voice: “if a parameter is slightly shifted” to make the paragraph more cohesive.

Line 286: Same as above. Change to “In short, if the standard error of the maximum likelihood parameters are known…”

Line 326: Akaike (1973) is the citation for the original concept.

Lines 372 – 375: I suggest numbering these bullet points and/or giving the symbol for each parameter for readability.

Lines 381 – 382: I suggest removing these bullet points and rephrasing them into a paragraph for cohesion and to add a finished quality.

Line 496: I suggest changing “original methods used were rather crude” to something less harsh; the advanced tools we have now were not necessarily available to authors at the time. Perhaps state “significant methodological advancements have been made since early estimates using…” or something similar.

Line 516: Please include other references here; other authors have noted that nonlinear development rates affect duration and onset of TSP (Georges, 2005); further, others have noted that incubation temperature affects variation in the TSP (Merchant-Larois et al., 1997; Yntema, 1979).

Line 520: The citation indicating that large-scale empirical validation is lacking came before the studies for which you cite improved models. For example, Massey et al. (2019) and Carter et al. (2019) both had extremely large empirical data sets. I would recommend changing your last sentence to something like, “but further analyses of in-situ empirical data would benefit the field.”

Line 522: I disagree that existing statistical methods have inhibited authors analyses and study of TSD; for example, in preparation for Massey et al. (2019), several models were tested (Hill, alpha-logistic, etc.), all of which produced similar results – this did not cause inhibition in any way. However, I enthusiastically agree that in this paper, the R code provided will simplify the process of analyses for authors. I would re-word this paragraph to affirm that a lack of accessible methodology creates “barriers” for authors, and highlight your contribution thereafter.

Lines 529 – 542: I recommend placing this in paragraph form for flow and cohesion.

Line 541: Switch “think” to “suggest”.


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: RECENT ADVANCES ON THE ESTIMATION OF THE THERMAL REACTION NORM FOR SEX RATIOS
Review round: 1
Reviewer: 2

Output:
Basic reporting: This manuscript presents a detailed explanation of one method for fitting a function to constant-incubation data for reptiles with temperature-dependent sex determination. The writing is excellent and 99% clear, with a few locations where the wording could be improved (noted in General Comments, below). Figure 5 would be better if presented with a different color scheme that is more friendly for grayscale viewing and colorblind readers.
Experimental design: This paper reports on the various modeling/statistical methods available in an R package, using sea turtle data in a worked example. It does not pose or answer a scientific question, per se. As such, the editors should determine whether this manuscript fits within the Aims & Scope of the journal or would be considered a case study. If necessary, the paper could be re-worked relatively easily to focus more on the ecological question of TSD in olive ridleys and less on the statistical methods.

That being said, this paper is an excellent treatment of its subject and much needed in the TSD modeling community. The statistical descriptions are detailed, rigorous, and clear for the non-statistician. One could reasonably argue that the detailed explanations of general methods, like AIC, are not strictly needed, since they have been described elsewhere, sometimes to excess. However, my opinion is that it's useful for reference purposes to have all of the relevant models and stats in a single paper, and I would favor keeping them in.
Validity of the findings: Data and data sources are provided, along with R code for running the example. The R functions run as expected.

The authors do state conclusions about the various turtle rookeries, but they are really just an afterthought to the methodological focus. Again, though, this section could be re-worked if the manuscript needs to be more question-focused to fit the journal.
Additional comments: I have some minor comments, so I added line numbers to the manuscript for ease of referencing.

Since this paper focuses on methods for fitting TSD data, not just for sea turtles, I think the title would be improved by removing taxon references.

L97-103: Lists of arguments are difficult to read. Consider putting them in a more organized format (table, etc.).

L295: Consider replacing 'high volume' with 'many' because the former has several definitions that could be semantically confusing.

L334: Different authors have presented different perspectives on the delta-AIC value that should be considered equivalent (from 2 to at least 6). It would be good to cite at least one of those perspectives here or, alternatively, note the justification for the choice of 2 as a delta-AIC cutoff in this paper.

L337: Elaboration of this point would be beneficial, as well as justification for the Hurvich-Tsai formula. It would also be useful to note that the AIC value itself is not strictly informative in terms of absolute model fit.

L343: It's not atypical for common species, either, which might be even more important to note.

L358: It's not 100% clear what a ‘dataset’ would be in this case. Results from multiple incubation studies with the same species? All studies that use same/different set of incubation temps? Etc.

L397: If you’re already starting with max likelihood values, it would be reasonable to assume that your estimates aren’t complete nonsense (otherwise, no method would be trustworthy). In that case, why would you also need to burn in your chain? See, e.g., the Geyser essay in the Brooks et al. 'Handbook of MCMC'.

L411: Shift data description to the Methods section.

L427: Given how well-documented the rest of the stats methods are, a quick reference to where the 98% value comes from would be useful here.

L442-460: Much of this paragraph is repeated from the Methods section and is unnecessary here.

L519: The results from this paper don't seem to indicate that an incorrect TSD pattern is a likely explanation for not detecting substantial variation among populations.

L520: Provide an example of this being done in the literature.


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: LET’S TALK ABOUT PAIN CATASTROPHIZING MEASURES: AN ITEM CONTENT ANALYSIS
Review round: 1
Reviewer: 1

Output:
Basic reporting: a few small points :

line 211 – ‘participants’ not ‘patients’
line 250 lacks a verb e.g. ‘amongst which were the constructs…’
line 263 should be ‘by’ not ‘with’.
line 495 ‘from’ not ‘as’.
line 632 ‘It is unlikely that it will ever be.’

line 496-7 Is it appropriate to have ‘available on request’ rather than as supplementary?
Experimental design: no comment
Validity of the findings: no comment
Additional comments: Review of Crombez et al:  Lets talk about pain catastrophizing measures: an item content analysis.

This paper reports a study in which 94 on-line participants rated the relevance of 58 items from six standard pain catastrophizing measures to six pain constructs, including pain catastrophizing.  They found that two measures did not measure pain catastrophizing and that none of the measures pain catastrophizing alone.  Most of the questionnaires measured either pain worrying or pain distress more than they measured pain catastrophizing. This is an important paper and I strongly support its publication.

The strong points of this paper are:
1.	It addresses an important question. Pain is a central scientific and practical domain and the results of this study are important scientifically and practically. Scientifically, the proliferation of constructs that cannot be distinguished operationally is of little value and may be damaging (Dixon & Johnston, 2019). If measures are contaminated by other constructs, tests of theory are invalid and may lead to the design of inappropriate interventions and experimental conditions.  Practically, decisions may be made and services planned on the basis of misleading information:  as the authors point out, patients scoring high on pain catastrophizing may be referred to mental health services whereas this would not occur if the measure was more correctly labelled ‘pain distress’ or ‘pain worry’.
2.	The findings are clear and important for methods and theory in this field.
3.	The methods are excellent.  They advance on our earlier work (Johnston et al., 2014) by the use of more elaborated statistics. In addition the manipulations checks and tests for the level of engagement of participants in the task are excellent.
4.	The Introduction and Discussion present the issues clearly.
5.	The Results, including the rationale for decisions that had to be made, are presented very systematically and clearly.
6.	It is very useful to have both the overall analysis and the analysis for each questionnaire.  In particular, the PCL measure looks extremely poor.

Some queries
1.	The definitions are crucial in this kind of study and merit some discussion.  The authors offer a definition of pain catastrophizing as ‘to view the present pain of pain-related problems as considerably worse than they are.’  I do not work on the pain catastrophizing so cannot comment on whether  that is representative of definitions in that area, but other definitions of ‘catastrophising’ would not require the deviation from the ‘actual’, e.g. defining it  as assuming  or anticipating the worst.  Given that it is difficult to say what pain ‘actually’ is, might these definitions be more useful?  Second, the study used dictionary definitions for other constructs and, as shown in Bell et al (2017), this may not fully represent the construct as used scientifically. Third, the definition should appear earlier in the Abstract – in either Background or Method rather than Conclusion – and earlier in the Introduction to the paper.
2.	The difference between content validity and discriminant content validity gets blurred e.g. lines 84-90 could make the separation clearer. The Abstract Background says ‘we investigated the content validity of self-report measures of pain catastrophising’.  This omits to say that a key part of this paper is detecting whether these measures have discriminant content validity that distinguish them from other pain measures.  The contrast between these two questions is sometimes not clear at various points in the main paper.
3.	It would be useful to present a discussion of the choice of statistical analysis – including the pros and cons compared with Johnston et al 2014 Methods.
4.	If possible, a discussion of the number of participants required would be useful – and might be important in discussing a comparison of the requirements of this method compared with Johnston et al 2014.
5.	Line 64-72.  The argument here could be argued more strongly and clearly.  In particular the first point is barely made – why should the pain catastrophizing measures correlate with the CEQ?
6.	Should the Figures be histograms rather than graphs as the order of items and intervening points have no meaning?
7.	In the Discussion in lines 568 onwards, the challenge of measurement of pain catastrophising depends on the definition and it would be useful to discuss this – see point 1 above.

Small points
1.	It would be useful to indicate in section 2.2.3 where the full list of items was available.
2.	Line 211 – ‘participants’ not ‘patients’
3.	Line 204 – is it useful to mention that the CPG has discriminant content validity (Dixon, Pollard & Johnston, 2007)?
4.	Line 250 lacks a verb e.g. ‘amongst which were the constructs…’
5.	Line 263 should be ‘by’ not ‘with’.
6.	Line 495 ‘from’ not ‘as’.
7.	Line 496-7 Is it appropriate to have ‘available on request’ rather than as supplementary?
8.	Line 632 ‘It is unlikely that it will ever be.’

Bell, C., Johnston, D., Allan, J., Pollard, B., & Johnston, M. (2017). What do Demand‐Control and Effort‐Reward work stress questionnaires really measure? A discriminant content validity study of relevance and representativeness of measures. British journal of health psychology, 22(2), 295-329.
Dixon, D & Johnston, M (2019) British Journal of Health Psychology (2019) © 2019 The British Psychological Society www.wileyonlinelibrary.com Editorial Content validity of measures of theoretical constructs in health psychology: Discriminant content validity is needed.  BJ Health Psychology.  https://rdcu.be/bA14H
Dixon, D., Pollard, B., & Johnston, M. (2007). What does the chronic pain grade questionnaire measure? Pain, 130(3), 249–253. https://doi.org/10.1016/j.pain.2006.12.004


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: LET’S TALK ABOUT PAIN CATASTROPHIZING MEASURES: AN ITEM CONTENT ANALYSIS
Review round: 1
Reviewer: 2

Output:
Basic reporting: This article is well-written and well-structured, meeting the highest standards of basic reporting throughout. The authors use clear, professional English which makes the study and their discussion of results easy to follow. The authors provide adequate consideration of the previous literature, including appropriate references. The authors clearly state their results and provide useful tables.

I have only minor suggestions to improve the basic reporting of this well-written paper:

1)	Can the authors provide the exact scoring they used to calculate Pain GRADE (i.e., what constitutes low/high intensity and disability)? Although this approach stems from the original Von Korff et al paper, the extant literature has used slightly different calculations and approaches. In particular, while the authors chose to have Grade 0 reflect ‘pain free’ participants, other studies have had this grade reflect ‘no chronic pain’ (i.e., pain < 3 months is included).
2)	Can the authors provide a brief description of the frequency of use of the selected pain catastrophizing measures? My understanding is that the PCS is by far the most commonly used, and some of the measures have not been used for a number of years. This summary would be useful to situate the results within an understanding of how the selected catastrophizing measures are typically used in the field today.
Experimental design: The manuscript presents novel, original work that is both within the aims and scope of PeerJ and will be of significant interest to the field of pain science. The research question - whether pain catastrophizing measures indeed measure pain-related catastrophizing, as defined by cognitive-affective theory, is clear and important. The work is timely and fills an identified knowledge gap.

The experimental design is largely appropriate. However, there are two significant methodological issues (detailed below) that are problematic for the conclusions drawn by the authors, and which should be addressed in the manuscript before publication.

1) I find the authors' conceptualisation of 'pain-related distress' problematic. This is important given that one of the primary conclusions of the study is that pain catastrophizing measures are better represented by pain-related distress (and pain-related worry). This is likely to be a well-cited and repeated finding that drives future research in this area. Specifically, unlike concepts such as worry, rumination, and vigilance, the concept of 'distress' is not well-defined in either cognitive-affective theory or in pain science more broadly. The term 'distress' is often used as an umbrella term to capture elevated arousal states including fear, anxiety, catastrophizing, and (hyper)vigilance, both in the affective science literature and in pain science. Partly in reflection of this, the definition that the authors provide for pain-related distress ("Distress related to pain or pain-related problems") is insufficient. The fact that the definition includes the very word that it is trying to define exposes this problem. Relatedly, the measure that the authors chose to represent pain-related distress was actually an affective distress measure, with no relation to pain (i.e., "in the past week, how tense or anxious have you been"). These issues have implications for the authors' work in a few ways. First, given that the term 'distress' is used in a catch-all way, and that the definition provided to participants is so general, is it perhaps unsurprising that the participants rated catastrophizing items as also reflecting the more general construct of distress. Further evidence for this is the fact that worry and distress items were not rated as different, either. Second, readers of this paper will likely start using the term 'pain-related distress' in place of catastrophizing in their own work, believing it is an adequate (i.e., well-defined) replacement of pain catastrophizing. Given the issues described above, I don't believe this is the case. Overall, this issue is perhaps why the authors chose to recommend use of the term ‘pain-related worry’ instead of distress, in place of catastrophizing. But I believe this issue needs to be better addressed throughout the manuscript, including explicit discussion of why the current findings are not necessarily sufficient to suggest using the term pain-related distress are a replacement term as of yet.

2) Another major conclusion from the study is that pain catastrophizing measures are not well-defined/specified *in comparison* to the other constructs considered in this paper (i.e., pain severity, pain-related worrying, pain-related vigilance, pain-related distress and pain-related disability). In the abstract the authors state: “The results revealed that items from pain severity, pain-related worrying, vigilance, distress and disability scales were distinctively related to their respective construct. This was not the case for the items from the pain catastrophizing measures.” The issue with this conclusion is that the other constructs were not submitted to the same procedure as the pain catastrophizing measures. Specifically, for the other constructs the authors selected only one measure that was ‘considered appropriate for the respective construct’. Thus, they selected the single most appropriate measure these constructs, while for pain catastrophizing they selected a number of measures, many of which were developed decades ago and have not been in mainstream use for a while. The authors’ additional analyses showing that even the ‘best’ 5 catastrophizing items did not diverge from measures of worry/distress points toward is useful for the authors’ conclusion, but I feel that overall it is overstated. This is especially the case given that pain-related worrying items were not distinct from pain-related distress, indicating issues with even the ‘most appropriate’ single measures of these constructs.
Validity of the findings: There are a number of strengths of the authors' methodological approach that leads to robust, valid findings. For example, they used methods to detect careless responding, which is important for an online sample. They also confirmed that there were no differences in the way participants reporting persistent pain rated items, in comparison to those without pain issues. I have only one issue with the conclusion that the authors draw from their work, described below.

The authors make a strong recommendation at the end of the manuscript – to reclassify pain catastrophizing measures as measures of pain-related worry. The results of this study, ie., that pain catastrophizing items were often rated as reflecting pain-related worry, provides compelling evidence to support his suggestion. However, there are other considerations that I believe need to be addressed, as caveats. The concept of ‘worry’ is very well defined in the cognitive-affective literature, particularly in relation to work on anxiety disorders, and the available pain catastrophizing measures do not necessarily reflect the complexity of ‘worry’ as it is understood more broadly. For example, worry is typically understood as a process of repetitive negative thinking about an event in the future. This is distinct from rumination, which is understood as a process of negative repetitive thinking about events in the past. The available pain catastrophizing measures, including the PCS (which is the most widely used), do not typically distinguish between these. An alternative recommendation that the authors could have made, particularly pertinent for the PCS, is to consider and utilize the constituent subscales instead. Specifically, the PCS has three subscales: rumination, magnification, and helplessness. I was surprised that the authors did not consider including these constructs in their study design – specifically examining how participants would rate the subscale items as reflecting their respective subscale definitions. This data would have been particularly useful for considering the alternative approach of focusing on subscales. Can the authors address this issue and include some consideration to this as an alternative way forward (including the necessary studies for examining the utility of this approach)?
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: LET’S TALK ABOUT PAIN CATASTROPHIZING MEASURES: AN ITEM CONTENT ANALYSIS
Review round: 2
Reviewer: 1

Output:
Basic reporting: The manuscript remains very well written and I appreciate the authors' need for brevity in not adding too much additional information.
Experimental design: The authors have refocused their suggested terminology to pain-related worry, and have provided more caveated discussion about the pain-related distress term. I appreciate that the authors revised some of their language to ensure that they were not making the claim that the other constructs were better defined that pain catastrophizing.
Validity of the findings: Although I still think that the authors could make an explicit statement about the use of the PCS subscales (rather than talking more broadly about 'constituent components'), I believe that the findings of the paper are valid and robust and meet high standards.
Additional comments: The authors have clearly taken a great deal of time to respond to all of the reviewer and editor's comments. I believe this paper will make an important contribution to the field.


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 COMPLETE CHLOROPLAST GENOMES OF SEVENTEEN AEGILOPS TAUSCHII: GENOME COMPARATIVE ANALYSIS AND PHYLOGENETIC INFERENCE
Review round: 1
Reviewer: 1

Output:
Basic reporting: The paper presents relevant data for the field of wheat improvement. The writing needs considerable improvement beyond the duty of a peer-review editor. I attach a copy of my review with notes on the first few pages only.

Some figures need to be improved:
Figure 1, some of the text in the figure is unreadable
Figure 4, the subcategories for each of the gene are not distinguishable.
Figure 5, the labels are unreadable because of low resolution/quality.

I tried to retrieve the accessions from GenBank but they are not available.
Experimental design: The experimental design is appropriate. The authors surveyed a relatively large number of accessions representing the native range of the species.
Validity of the findings: The conclusions reached by the authors are supported by the data
Additional comments: No additional comments


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

Input:
Title: THE COMPLETE CHLOROPLAST GENOMES OF SEVENTEEN AEGILOPS TAUSCHII: GENOME COMPARATIVE ANALYSIS AND PHYLOGENETIC INFERENCE
Review round: 1
Reviewer: 2

Output:
Basic reporting: The paper needs to be text edited in multiple sections.

For instance, in the abstract the authors wrote " IR expansion and contraction with identical structure among 17 Aegilops tauschii accessions were not inﬂuence chloroplast genomes in length. " (not sure what the authors mean in this sentence).
And also "The ndhF gene in AY46 accession appeared the highest ω value". (probably showed or had instead of appeared").
And also "The speciﬁc spreading route of Ae. tauschii revealed in this work, reﬂects the frequent cultural exchange through the silk road from one point of view." (not sure what the authors mean by "one point of view").

Examples like this occur across the manuscript and really constrain the all message of the paper. Figures and tables appear to be good but note that population labels of Figure 1 are very hard to see. Please consider increasing labels and changing colours.

Raw data is shared. Thank you for that!
Experimental design: The aims and experimental design appear to be rigorous but as stated before, the language is sometimes ambiguous. Methods are clear.
Validity of the findings: The Discussion needs to more clear. I don't really understand the main conclusions of this paper. For instance:

- Because the ndhF gene had the highest ω value, the authors conclude that with might be involved in the adaptation to high altitude ecological environment. This is quite speculative. It might also be involved in something else. Note that this gene evolves at a very fast rate in most plants.

- The phylogenetic relationships support that Ae. tauschii in the Yellow River region might be directly originated from Central Asia rather than Xinjiang. Please explain this better.

- The authors also conclude that "The speciﬁc spreading route of Ae. tauschii revealed in this work, reﬂects the frequent cultural exchange through the silk road from one point of view. We conﬁrmed that Ae. tauschii derived from monophyletic speciation rather than hybrid speciation at the chloroplast genome level." I'm really lost in these two sections. First, I don't see how the authors can link this to the silk road. Perhaps, the authors are right but they need to explain this much better. But from the current text/figures/tables, I cannot really support this conclusion. The second sentence, is simply wrong. There is no result in this study that can confirm or reject the presence of hybrid speciation, and even less at the chloroplast level (unless the authors can sustain that the chloroplast is biparentally inherited in Ae, and in that case they have to re-write previous conclusions).
Additional comments: This study has some potential. It appears to be methodologically solid but there are too many unclear and ambiguous sentences that prevents me from accepting this manuscript. I'm also worried about the conclusions drawn by the authors. I hope my suggestions help them to prepare a new version.


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: FINE-MAPPING OF QTGW2, A QUANTITATIVE TRAIT LOCUS FOR GRAIN WEIGHT IN RICE (ORYZA SATIVA L.)
Review round: 1
Reviewer: 1

Output:
Basic reporting: 1、In the part of results “One QTL primarily contributed to grain shape, 182 especially to LWR, locating in the RM240−RM14034 region, namely, qGS2. Another QTL 183 simultaneously influenced TGW, GL and GW except LWR, flanking from RM14056 to the 184 terminal end of chromosome 2, namely, qTGW2”. The author should sequence the DNA of GS2 between the NILs and confirm whether qGS2 is the allelic of GS2, and the results must be submitted as attachment. Discarding the influence of qGS2 by getting rid of qGS2 fragment is essential to further studding the function of qTGW2 because GS2 positively regulates grain shape.
2、Besides of Tw35277 and Tw35395 in Table S1, sequences of other markers used in the study should also be provided, especially for the novel designed, which enable researchers to assess each QTL exactly and quote the paper accordingly.
3、The physical distance of Tw35293 and Tw35395 was 15kb in the Fig.3, while it was descripted as 103kb in the result, which is correct？
4、In the fig.3, the fig.A indicated that the physical distance is 400kb between the markers of Tw35293 and Tw32437, but in the fig.B, I found that 250kb and 1.9Mb was marked among this region. In total, the fig.3 should be redrawn because it is hard to understanding.
5、The loci of qTGW2, qGL2 and qGW2 were all located on the terminal end of the chromosome 2, just as showed in the Fig.2, why is the title named “Fine-mapping of qTGW2, a quantitative trait locus for grain weight in rice (Oryza sativa L.)”?
6、 “A total of 27 QTL were detected”. Here, “QTL” should be “QTLs”.
7、The first appearance of QTL should be descripted as quantitative trait loci.
Experimental design: no comment
Validity of the findings: no comment
Additional comments: This manuscript reported a novel QTL qTGW2 that regulate the development of grain size, which is a key agronomic trait controlling rice yield and quality and paid wildly attention both in rice biology and in rice breeding. The fine-mapping of qTGW2 is beneficial to isolate qTGW2 further and then to clear the molecular mechanism of rice seed development. Therefore, this study is advised to be significant. The questions must be answered in detail and the figures should be improved strongly.