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: NUMERICAL ABILITY AND IMPROVEMENT THROUGH INTERINDIVIDUAL COOPERATION VARIED BETWEEN TWO CYPRINID FISH SPECIES, QINGBO AND CRUCIAN CARP
Review round: 1
Reviewer: 2

Output:
Basic reporting: The English Level is good throughout the manuscript. The structure of the article is clear and follow a precise workflow.
The Raw data file provided is clear, but it would be helpful to add some labels that better explain what each data group refers to.
The Figures are simple and clear.

- Line 41: please add birds and tortoises to the list, e.g.
https://royalsocietypublishing.org/doi/10.1098/rsbl.2018.0649
https://www.ncbi.nlm.nih.gov/pubmed/18665721
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3171108/
https://www.ncbi.nlm.nih.gov/pubmed/17227192
https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0065262
https://royalsocietypublishing.org/doi/full/10.1098/rspb.2009.0044

- Line 42: Given that the comparative literature on this topic is large and examples cannot cover all the field, it would be important to provide the readers with some general reviews, I would recommend e.g. Vallortigara, G. (2017). An animal’s sense of number. In “The nature and Development of Mathematics. Cross Disciplinary Perspective on Cognition, Learning and Culture” (Adams, J.W., Barmby P., Mesoudi, A., eds.), pp. 43-65, Routledge, New York.
https://www.tandfonline.com/doi/abs/10.1080/02643294.2012.654772
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5784035/
https://royalsocietypublishing.org/doi/pdf/10.1098/rstb.2017.0120

Some of the reference reported are not appropriated for the sentence to which they refer. For example:
- Line 45: the reference Miletto Petrazzini and colleague (2015) is not correct if referred to shoals comparison and the discrimination of the larger one. The cited study investigated ordinal numerical abilities using a learning procedure with food as a reward. Cite this study in the list of numerical evidence at line 41
- Line 183: The study of Potrich, Sovrano, Stancher & Vallortigara (2015) does not provide evidence about interindividual cooperation and “collective intelligence”. I suggest to move this citation in the evidence of numerical discrimination of groups of conspecifics.
Experimental design: It is unclear to me how data for dyads were analyzed. It seems that individual data for each dyad were used, thus raising a serious problem of pseudo-replication. If not, the authors should explain how data for dyads were computed (one animal selected at random in each dyad?).

I suggest to provide more information about the set up and the procedure used for each condition. For example, in the heterospecific dyads, were the two shoals formed by a mix of the two species (half of qingbo and half crucian carp)?
Validity of the findings: There is a contradiction in the discrimination results of singletons of qingbo fish. In the graph and the manuscript the authors indicated that singleton of qingbo discriminate all the three comparison proposed (8 vs.12, 9 vs. 12 and 10 vs.12), but in the result section (line 130-131) and in the raw data the authors indicated that qingbo fish fail in the 9 vs.12. I suggest to correct these misunderstanding. Moreover, if it is correct that qingbo fish fail in the 9 vs.12, the authors should discuss about this.

It is unclear to me why the authors use the term “cooperation of conspecific” to account for changes in performance in numerical discrimination. A simple and more parsimonious hypothesis is that emotional/motivational variables can account for change in performance without involving anything ‘cognitive’ (see as an example in a different domain, that of object permanence and spatial cognition, e.g. https://www.sciencedirect.com/science/article/pii/S0003347285702321?via%3Dihub.

- Line 159: I would be more cautious in attributing or denying quantitative abilities to any particular species. The authors are testing here quantitative abilities during shoaling and it could be that carps are able to make quantitative discrimination under different testing conditions, i.e. using food or in general under associate learning procedures. Furthermore, the raw data show that a lot of carp subjects spend the entire time close to one group (irrespective of whether it is larger or smaller) without exploring the environment, suggesting that they don’t pay enough attention to the two shoals.
Additional comments: The Discussion is very long and repetitive; I would suggest shortening it.

- Line 55: To my knowledge, Crucian carp is not the species Carassius auratus as the authors indicated in the manuscript, but Carassius carassius.

- Line 58: please provide reference about previous studies on numerical ability in qingbo.

- Line 185-186. It is unclear to which species the authors are referring to in the sentence “However, both species can discriminate at a ratio of 0.75…”, since the results discussed in this paragraph were only about qingbo fish.

- Line 222: The authors demonstrated that singleton of qingbo fish have a threshold of 0.67 (since they discriminate only 8 vs. 12), not 0.75 as indicated.

Some Authors name reported throughout the manuscript are grammatically wrong or some authors are missing (e.g. Bisazza at line 182, Agrillo at line 154, Potrich, Stancher, Sovrano & Vallortigara al line 183). Please check and correct.


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: CHRONIC FLUOXETINE TREATMENT OF JUVENILE ZEBRAFISH (DANIO RERIO) DOES NOT ELICIT CHANGES IN BASAL CORTISOL LEVELS AND ANXIETY-LIKE BEHAVIOR IN ADULTHOOD
Review round: 1
Reviewer: 1

Output:
Basic reporting: The manuscript would benefit greatly by a thorough edit by a native English speaker.
Experimental design: Review the methodology presenting more details of the procedures.
The sample number presented in the methods is not the same as shown in the figure / table captions.
What is the premise for the removal of outliers data?
Validity of the findings: The data presented are not robust.
The author concludes that "juvenile fluoxetine exposure does not elicit
any adverse or maladaptive effects on stress reactivity in young adult zebrafish, "without presenting any stressful stimulus to conclude on stress reactivity, simply performs an analysis of baseline levels of cortisol.
In addition, many of the references presented by the author (Abreu et al., 2014, 2015, 2017, Giacomini et al., 2016ab, Marcon et al., 2016), fluoxetine alters the stress response only in response to the stressful stimulus (e.g. chasing), fluoxetine has no effect per se.
Thus, the present data do not support the conclusion.
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: CHRONIC FLUOXETINE TREATMENT OF JUVENILE ZEBRAFISH (DANIO RERIO) DOES NOT ELICIT CHANGES IN BASAL CORTISOL LEVELS AND ANXIETY-LIKE BEHAVIOR IN ADULTHOOD
Review round: 1
Reviewer: 2

Output:
Basic reporting: The author investigates an interesting and appealing aspect. The article is written in a clear and understandable way, with a good number of literature references and a sufficient field background /context provided. In addition, the structure is well organized with a sufficient introduction, well explained Material and Methods, an exhaustive chapter of Results and Discussion.
Experimental design: Aim and scope are well defined. However, the following points should be clarified:
1) In experimental design the author did not take the effect of fluoxetine immediately after chronic treatment into account, in terms of cortisol level and behavioural performance. This group is important to verify if Fluoxetine has a biochemical and behavioural effect.
2) In order to validate the data, a further behavioural test on anxiety (light-dark) should be done.
Methods are described with sufficient detail.
Validity of the findings: 1)  It would be better to express the data concerning the evaluation of the anxious state (in the Table 1) using graphs/figures in order to facilitate the reader.
2) It is not clear the sentence in line 156-157: "Values were excluded from analyses if two standard deviations above or below the mean". How many values did the author exclude?
3) In Discussion (line 194-197) the author can not affirm that his findings are related to serotonin level changes. The author should provide a reference demostrating that fluoxetine alters serontonin levels in zebrafish brain
Additional comments: Line 114: Check the reference order.


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: CHRONIC FLUOXETINE TREATMENT OF JUVENILE ZEBRAFISH (DANIO RERIO) DOES NOT ELICIT CHANGES IN BASAL CORTISOL LEVELS AND ANXIETY-LIKE BEHAVIOR IN ADULTHOOD
Review round: 1
Reviewer: 3

Output:
Basic reporting: The manuscript by Petrunich-Rutherford proposes an investigation of the effect of chronic fluoxetine treatment of juvenile zebrafish in cortisol levels and anxiety-like behavior in adulthood. The paper has a good scientific background and is well-contextualized regarding the literature; in addition, the language used is clear, unambiguous and professional. However, several points are critical to be considered regarding the methodological approaches used (particularly from the point of view of methodology description, experimental design and statistical analysis). I have also raised a series of questions regarding the necessity of further experiments to adequately test the hypothesis of this study.

Abstract and Introduction
1. The goal and the relevance of the study need to be described in a narrower way. What phenomenon does the study intend to model? In the introduction, the author discusses the use of SSRI in adolescent patients suffering from depression and anxiety disorder. However, this study does not use an animal model of depression or anxiety to mimic what is observed in the clinical practice. The author also discusses concepts about stress, but uses no intervention to evaluate a response to a stressor stimulus.

2. There is some important issue of concept. In many instances, the author uses generalist terms assuming that there are changes in brain levels of serotonin, but this parameter was not evaluated in this study. The modulation mechanisms behind the mechanism of action of SSRI are complex and indirectly involve other neurotransmitters and receptors. Therefore, without performing a technique to ascertain the levels of neurotransmitters, such as high-performance liquid chromatography (HPLC), for example, it can not be inferred with certainty that there was actually a change in serotonin levels at the fluoxetine concentration used in this study. (see lines 18 and 195).

3. The author hypothesizes that juvenile exposure to fluoxetine would reduce cortisol levels and elicit anxiolytic effects in the novel tank test in adulthood, but does not propose a neurophysiologic mechanism by which the chronic fluoxetine treatment of juvenile zebrafish would lead to biochemical and behavioral changes in adulthood.

4. Please add the phrase about the novel tank test (100-104) in the Materials and Methods section.

5. The phrase "These data suggest that juvenile fluoxetine exposure does not elicit any adverse or maladaptive effects on stress reactivity in young adult zebrafish" (19-21) is generalist, as the study solely evaluates cortisol levels and behavioral parameters. The term "stress reactivity" may not be ideal, since in this study the animals were not exposed to a stressor stimulus (such as acute or chronic stress).

Materials and methods
6. I believe there is a typographical error in line 147.
Experimental design: The manuscript only partially follows ARRIVE guidelines and there is a need for more minute descriptions of the experimental design. Gerlai, 2018 (Reproducibility and replicability in zebrafish behavioral neuroscience research, doi.org/10.1016/j.pbb.2018.02.005) and Masca et al. (2015; doi:10.7554/eLife.05519) discuss important issues of reproducibility and replicability in scientific research, which must be taken into account:

1. In the Animal section (111-115), besides citing a reference, the author needs to add the maintenance details. In this study animals are used at different stages of development, requiring different types of food, maintenance and housing. How were food (commercial food, artemia or paramecium) and maintenance handled at these different stages? Was the density of animals in the aquariums controlled? What were the parameters of water quality used? Was the system water obtained from reverse osmosis? In addition, it is very important to add the approval number of the ethics committee.

2. Studies show that there are differences between males and females (see Rambo et al. 2017, doi: 10.1016/j.physbeh.2016.12.032), both in behavior and in stress response. Why was this not considered in the statistical analysis?

3. The author states that there are no studies of chronic treatment with fluoxetine in juvenile zebrafish (89-91) and, therefore, there is no known optimal concentration for the drug in this case. Moreover, the concentration used was chosen according to studies performed in adult zebrafish, most of them with acute exposure, not reflecting the design of the present study. In this case, why did the author choose not to establish a dose-response curve of fluoxetine treatment?

6. Most references cited in this manuscript use acute or chronic stress protocols to more concretely evaluate animals' responses to stress. In order to evaluate stress response, I think it would be necessary to expose the animals to a stressor stimulus. In this case, why did not the author use this type of intervention in this study?

7. Please add the dimensions of the apparatus used for the novel tank test (133-134).

8. Please add the P value considered as the cutoff for statistical significance in Data Analysis (155-159).
Validity of the findings: 1. An important question is about randomization and blinding. Masca et al. (2015) and Gerlai (2018), examining the problems of reproducibility in biomedical research, state that one of the main reasons for irreproducible research is the lack of blinding/masking. The author describes that randomization was performed to allocate the animals to the treatment groups (control x fluoxetine) (116-117), but were the animals randomized as to the order of testing in the novel tank test? What was the method used for random allocation in the treatment groups? Were experimenters blind to treatment? Were data analysts blind to treatment? These questions need to be addressed and clearly stated in the methods section.

2. Other important issue is that there is a misconduct in the use of degrees of freedom and describing terms of t test statistics. You should not just remove an outlier from a specific parameter; when you consider an outlier animal, you should remove it from all parameters.

3. I believe the term “main effect” should not be used for t test. Please review this concept and change the manuscript.
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: CHRONIC FLUOXETINE TREATMENT OF JUVENILE ZEBRAFISH (DANIO RERIO) DOES NOT ELICIT CHANGES IN BASAL CORTISOL LEVELS AND ANXIETY-LIKE BEHAVIOR IN ADULTHOOD
Review round: 2
Reviewer: 1

Output:
Basic reporting: See below
Experimental design: See below
Validity of the findings: See below
Additional comments: I reviewed again the manuscript entitled: "Chronic Fluoxetine treatment of juvenile zebrafish (Danio Rerio) does not elicit changes in basal cortisol levels and anxiety-like behavior in adulthood". The rebuttal of the authors is quite satisfactory and I think that the article now is ok for the publication.


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

Input:
Title: CHRONIC FLUOXETINE TREATMENT OF JUVENILE ZEBRAFISH (DANIO RERIO) DOES NOT ELICIT CHANGES IN BASAL CORTISOL LEVELS AND ANXIETY-LIKE BEHAVIOR IN ADULTHOOD
Review round: 2
Reviewer: 2

Output:
Basic reporting: When the author uses the term “velocity”, would it be “mean speed”? In this case the correct term must be corrected in the text and figures.
Experimental design: No comment.
Validity of the findings: I am worried that by excluding such a large number of outliers you might lose some important results. How have you evaluated the distribution of your data? Does the data fit a normality curve?
Furthermore, group sizes are different in behavioral analysis and determination of trunk cortisol. When you consider an animal outlier, you should remove it from all tests.
Additional comments: The manuscript by Petrunich-Rutherford proposes an investigation of the effect of chronic fluoxetine treatment of juvenile zebrafish in cortisol levels and anxiety-like behavior in adulthood. The explanations made by the author are valid and the changes improved the manuscript, however, some points still need to be better described (particularly regarding statistical analysis).


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

Input:
Title: MODULATING PAIN THRESHOLDS THROUGH CLASSICAL CONDITIONING
Review round: 1
Reviewer: 1

Output:
Basic reporting: - The manuscript is very clearly written, and even though the material is quite complex, it was relatively easy to read.
- At several points, the reference style needs to be adjusted. For instance, line 59 or line 145. Avoid nested/double parentheses within references (and in general).
- The introduction provides a nice overview of the literature. When it comes to describing the literature on conditioned allodynia, however, some more information and details would be warranted. For instance, the two studies by Madden et al show conflicting findings, but no explanation is given on why this may be. What could explain the discrepancy? The current study is a follow-up on these studies, but it is not clear to the reader what the current study adds to these two previous studies; how it is different; how the current study will resolve some of the conflicts (if applicable)? I would suggest elaborating a bit more on what gaps there are and how the current study will address these gaps.
- Something that was unexpected when reading the manuscript was the location of the stimuli (both the CS and US). There is no mention of the locations, hence I expected a standard location such as the hand or arm. The authors choose for the back, but this is not explicitly mentioned in the paper, and is only apparent from the figures. I would suggest providing a rationale in the text, and also mention it in the abstract.
- Related, it would be good to mention in the abstract that the CS was presented at a similar location to the US and that the stimuli had the same intensity (but different other properties).
- The rationale for choosing at-pain-threshold stimuli is not clear enough. I believe you choose these stimuli because they are either experienced as painful or non-painful – at chance level (theoretically). This is mentioned in the discussion, but is a crucial aspect of the current study that is not addressed clearly enough. This also explains why you choose the percentage of trials experienced as painful as the primary outcome (correct me if I’m wrong). This should be stated much more clearly and explicitly, because this will not be clear to most readers. I would suggest being more explicit about this aspect, and presenting clear hypotheses regarding the primary outcome.
- The figures are clear and support the main findings.
- Some more specific questions about the introduction:
* When talking about non-nociceptive signaling, do you refer to somatosensory/tactile stimuli or more in general?
* Line 76 – should this be USpt?
Experimental design: - The methods and design are generally sound and well described and fit with the aims and scope of PeerJ.
- As mentioned above, in my opinion the research question and how it fills knowledge gaps could be clearer and more explicit (see Basic Reporting).
- I am impressed with the extent of the pilot testing, and how that has shaped the design further. It is refreshing to read about this part of the study, that is often not mentioned in publications and kept behind closed doors.
- I struggle a bit with the choice of the USpt in relation to the USp and USnp. The way I understand it, the USpt has the same intensity as the USp and USnp, but has a different duration (3 pulses versus 7 or 1). Did the authors verify that indeed USp was experienced as painful and USnp as non-painful during the calibration phase for instance? And how was the USpt rated prior to conditioning? Or in other words: can we assume that they are really experienced the way it is presented (as either painful or non-painful)? And related, could the participants distinguish between these three stimuli?
- Related to the point above, if the participants cannot really distinguish between the stimuli (since they’re the same intensity, on same location where tactile acuity is quite variable and bad) – can you really conclude that pain thresholds are modulated, or would it rather mean that the manipulation (i.e., the conditioning) was successful?
- Some specific questions about the methods:
* For the sample size calculation, one of the papers from Madden et al was chosen (the one that showed a difference). Can the authors explain why? Was this paper more similar to the current study?
* Were any participants excluded because they were unable to discriminate between the vibrotactile stimuli? I could imagine, since tactile acuity on the low back is very variable across participants.
* Line 143: Individual differences – I would suggest choosing a different title here (e.g., Affect) or just name it Positive and Negative Affect
* line 278 – USt should be USpt
Validity of the findings: - Statistics are generally sound, and the authors have looked into several different controls (e.g., effect of contingency awareness, mislocalization of tractor).
- Some specific questions about the results:
* Line 326: change in intensity rating for CS+ was greater than for CS-. It is unclear whether simple effects were tested for this result.
* Fig5 gives intensity ratings for acquisition and test phase, but not for baseline. Could the authors add this information? Can be referred to at line 321.
* The authors sometimes use CS+/USp and sometimes just CS+. I would suggest being consistent in naming (including in figures).
* Line 333-334 – A main effect should not be interpreted in the presence of an interaction effect.
* Table 2 – it is unclear what mA refers to. Also, it is unclear why a table was chosen instead of a figure (as for the other results). I would suggest using a figure to show this data pertaining to the primary analysis.
* Fig5: Was the SPARS rating transformed to another scale (instead of -50 to 50)? Or does this figure show us that all intensities are perceived above 0 and hence as painful? If the latter would be the case, that may be a problem.
* Recalibration – Although I understand why you choose to recalibrate, it would be good to show some data on how the pain thresholds changed over time. The authors mention the recalibrated values were no different across CS+ and CS-, but it would be nice to see some data on how much they changed.
- Even though I agree that this is an important question and has relevance for chronic pain, I do not agree that the present study investigates ‘whether classical conditioning could be one of the mechanisms underlying the development and maintenance of chronic pain’ (line386-387). This is too strong, and I would suggest rephrasing this.
- When thinking of how this is relevant for chronic pain, I miss some discussion on what this means and how this would actually contribute to development and maintenance of chronic pain, above and beyond other pain-related constructs that can be conditioned (e.g., fear of pain). Related, what mechanisms could be involved in this phenomenon?
- General remark: the authors call it conditioned allodynia, but I am not sure whether this is accurate when assessing pain thresholds (that are theoretically at the border of non-painful and painful).
Additional comments: Overall
The authors present a well-designed study asking the question whether pain itself can be conditioned. Previous studies have shown that all sorts of pain-related constructs can be conditioned, but less is known about pain itself. There has been some work on this topic with conflicting results; some from the same group, which has been a basis for the current study. I believe the topic and question are highly interesting and very relevant for the field of pain and chronic pain. The question has been systematically addressed, well described, and results are quite clear.


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: MODULATING PAIN THRESHOLDS THROUGH CLASSICAL CONDITIONING
Review round: 1
Reviewer: 2

Output:
Basic reporting: The present manuscript uses a clear und professional English language. It terms of the used language the manuscript is well-written und easy to follow. However, due to the structure, particularly in the methods section, the manuscript is in part confusing, because it refers to methodological aspects before they are actually explained. For example, in the section “Stimuli”, line 119 it is mentioned that a procedural adaption was made “to account for perceived control…”. However, why participants should have the impression of perceived control is explained much later in the section “Procedure”. This happens in a couple of instances. I would strongly recommend to explain the procedure much earlier in the methods section, followed by all the details in the sections “Stimuli” and “Measure and Outcomes”, to make it easier to follow the descriptions.

Although the introduction is well-written and refers the relevant literature, it is imprecise when it comes to the description of what should be conditioned. This means, the authors state that “pain itself can occur as a CR”, but “pain itself” is very vague because pain comprises very many very different aspects from pure nociceptive signaling, to the conscious perception of pain with several aspects, and motor responses, autonomic responses etc. It should be clearly stated what aspect of the pain experience is supposed to be the CR. In addition, it would be very helpful if also the CS, US, UR are precisely named. Based on these clarifications the discussion could also be adapted. In addition, the authors should clearly refer to classical conditioning, because in the context of operant conditioning several attempts were made to condition perceptual processes and underlying neural correlates.

Figure 3 and 4 appear somewhat redundant. I’d suggest omitting Figure 3 because all relevant information is contained in Figure 4 as well.
Experimental design: The reported research is within the scope of the journal, filling a current gap in the literature in psychological pain research.

Although the research question is well-defined and accordingly introduced in the introduction, I think it need a bit more precision as described above, i.e. being more precise about the CR because “pain itself” is a ambiguous term. The research question tackles a long-standing discussion on whether conditioning/learning can alter only processes related to pain such as fear or avoidance behavior or whether the perception of the pain/underlying nociceptive signaling can be altered by classical conditioning. Since classical conditioning plays a very important role in the context of pain and pain becoming chronic, this research question is highly relevant.

The authors used a sophisticated method to induce classical conditioning of pain perception. The methods are adequate and described in a manner, which would allow replication. The only part that I did not understand was how the readjustment by the participants of the applied stimuli between blocks (and phases) of the experiment were used for adjusting the applied stimulus intensities. It remained unclear to me (possibly due to the structure of the methods sections, see above) whether the stimulus intensities were actually adjusted to the intensities participants adjusted themselves.
Technical and ethical standards are kept.
Validity of the findings: The analysis is sound and the results appear robust. The statistics are appropriate and sufficiently. Only the descriptions of the main hypothesis, section “Effects on Pain thresholds” need some more explanation. The percentages when the stimulation was rated as painful are given, but because percentages can be misleading absolute numbers (in the manner: xy number of trials out of xy) are necessary.

For the secondary analysis reported in section “Effects on pain intensity”, the values reported in the second paragraph, lines 365-371, do not fit the values reported in Figure 5. In Figure 5, on the y-axis the values shown do not fit the descriptions of the SPARS rating scale given in the methods section.

Although the results are interesting and convincing, I have a conceptual issue with the reporting of the result and the interpretation given by the authors. The authors state that they aimed at modulating pain thresholds by classical conditioning. However, with the procedure they used in combination with the results reported, make it for me hard to understand, how the conditioning should have affected the pain thresholds directly. By the conditioning procedure, pain perception was modulated in that stimuli of equal intensity were more often rated as painful when combined with the CS+, compared to when combined with the CS-. This should in the end increase perceived pain, resulting as a consequence in a modulation of pain thresholds, but the CR appears not to have been the pain threshold itself, making the interpretation of the results complicated. I’d recommend sticking with a technical description of what was modulated precisely with the procedure and describing in the results section the given ratings (painful vs non-painful) instead of referring to pain thresholds. To get a more precise idea of how and if at all the thresholds were condition, the authors should add more information on the self-adjusted stimulation intensities by the participants, in particular when doing this in reference to the CS+ and CS- separately at the end of the procedure. Here, it is only mentioned in the discussion that these self-adjusted intensities did not differ, but I could not find data on this in the results section.

Table 2 is confusion as the title says “Mean stimulus intensity ratings” but in the table values in mA are given.

To understand the procedure better, a rational should be given, why a self-adjustment procedure war used for the calibration of stimulus intensities, while all other stimuli were applied without self-adjustment and thus control by the participants.
Additional comments: Line 325-326: it is unclear what is meant by “change in intensity ratings”. Please explain.

When describing the procedure in the discussion, the authors describe the procedure as simultaneous classical conditioning. I’d recommend to use the term “delay conditioning”  as a commonly used description of such a procedure.

Instead of citing a doctoral thesis (Bräscher 2014), I recommend citing the paper: Bräscher, A. K., Kleinböhl, D., Hölzl, R., & Becker, S. (2017). Differential classical conditioning of the nocebo effect: increasing heat-pain perception without verbal suggestions. Frontiers in psychology, 8, 2163.


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: TRANSCRIPTOME SEQUENCING AND COMPARATIVE ANALYSIS OF ADULT OVARY AND TESTIS IDENTIFY POTENTIAL GONADAL MAINTENANCE-RELATED GENES IN MAUREMYS REEVESII WITH TEMPERATURE-DEPENDENT SEX DETERMINATION
Review round: 1
Reviewer: 1

Output:
Basic reporting: The article is difficult to read and the motivations for the study is unclear. The introduction focuses on previous studies into the mechanism of temperature dependent sex (TSD) in reptiles. Sex determination & TSD occurs in the gonads of the developing embryo and, therefore, previous studies have investigated gene expression in the embryonic gonad (eg. Ge et al 2018, Czerwinski et al 2017 & Yatsu et al 2017). In the present manuscript, the authors measure gene expression in adult gonads of a turtle species. This has little or no relevance to the mechanism of TSD, since TSD occurs in the embryo. The authors instead claim to be investigating mechanisms of 'gonadal maintenance and gametogenesis'. If this is the case, the authors should provide background on this subject in the introduction, rather than discussing TSD, since their data has little relevance to TSD. At present, I am unsure what the authors mean by 'gonadal maintenance and gametogenesis' and why they chose to investigate this subject.
Experimental design: The experimental design is suitable for a comparison of gene expression between adult testis and ovary in this turtle species. However, the experimental design is not suitable fr studying TSD, since this occurs in the embryo (see above comments).
Validity of the findings: The authors have generated a data resource that may be useful for future studies in this species. However, similar data has been previously generated for other turtle species and other reptile species, so the novelty of the dataset is small. Likewise, their analysis has not yielded any original findings - they have simply characterised a number of genes/pathways with previously known roles in sex/gonad identity. In my view, more detailed investigation of a more specific hypothesis is required to be suitable for publication.
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: TRANSCRIPTOME SEQUENCING AND COMPARATIVE ANALYSIS OF ADULT OVARY AND TESTIS IDENTIFY POTENTIAL GONADAL MAINTENANCE-RELATED GENES IN MAUREMYS REEVESII WITH TEMPERATURE-DEPENDENT SEX DETERMINATION
Review round: 1
Reviewer: 2

Output:
Basic reporting: This manuscript offers insights into molecular mechanisms for sex determination in an organism with TSD. However, the background and discussion pertaining to current literature regarding molecular mechanisms of TSD should be expanded on. Specifically, there is very little mention of the importance of the aromatase genes (CYP19a and CYP19b) in regulation of TSD. This mechanism is well studied in reptiles as well as other species, and should be included in the introduction.

The introduction or discussion would also benefit from adding more information on the model organism (i.e. common name, optimal temperature for 50:50 sex ratios, and temperatures at which male/female sex is determined).

There are a few sentences (lines 59-62; lines 74-79) that could be revised or restructured for clarification as they are difficult to understand as written.
Experimental design: The authors present a robust study design, validating findings with multiple methods of quantifying gene expression. However, there are a few key points that need clarification.

More information is need on the rearing conditions of the animals used in this study. Is it possible that their sexual determination could have been influenced by temperature at early life stages? Were the animals sampled independently?

Was expression of genes compared between males and females as suggested by Table 2? More information is needed in the methods on how these comparisons were made.
Validity of the findings: More information is needed pertaining to statistical tests and comparisons made.
Line 136: Specifically what types of tests were used to make comparisons? Were differences detected between sexes or relative to the reference gene? A sample size should also be included here. Were comparisons made using any other data sets in this study?

Calculated efficiency values for primers should be added to Table S1.

Lines 151-152: Was this based on a manufacturer protocol? Why not use the standard 40 cycles?
Additional comments: Citations are needed on a couple sentences in the manuscript:
Lines 152-153: Citation is needed for the paper describing this method
Lines 213-215: Citation is need for this information

Figure 3: It seems that F2 is presented in a continuous color gradient and all other samples are shown in a discrete color gradient? If this is meant to illustrate a particular finding, more information should be given in the figure caption.

Table 2: More information is needed on the tests (sample size, which groups were compared) used to obtain the Q-value in the table caption.

This paper presents interesting insights into the mechanisms of sexual determination. I think this paper would be strengthened by expanding upon the discussion of current literature pertaining to mechanisms of sexual determination in organisms with TSD and including more information on the animals used in this study.


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

Input:
Title: TRANSCRIPTOME SEQUENCING AND COMPARATIVE ANALYSIS OF ADULT OVARY AND TESTIS IDENTIFY POTENTIAL GONADAL MAINTENANCE-RELATED GENES IN MAUREMYS REEVESII WITH TEMPERATURE-DEPENDENT SEX DETERMINATION
Review round: 2
Reviewer: 1

Output:
Basic reporting: The revisions have greatly improved the readability the article, and the motivations of the study (to investigate gene expression patterns involved in gonad maintenance) are more clearly articulated.
Experimental design: My reservations about the experimental design have been resolved by shifting the focus of the article from TSD to gonadal maintenance, since adult tissues were studied. The experimental design is now suitable, in my opinion, for the question being addressed.
Validity of the findings: As above, I did not have reservations about the experiment or its findings, but about their interpretation. By changing the focus from TSD to gonad maintenance, the authors have resolved my concerns.
Additional comments: I think the article has been improved by the revisions and would be happy to see it published.


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: TRANSCRIPTOME SEQUENCING AND COMPARATIVE ANALYSIS OF ADULT OVARY AND TESTIS IDENTIFY POTENTIAL GONADAL MAINTENANCE-RELATED GENES IN MAUREMYS REEVESII WITH TEMPERATURE-DEPENDENT SEX DETERMINATION
Review round: 2
Reviewer: 2

Output:
Basic reporting: This version of the manuscript was improved for clarity throughout.
Experimental design: While most of my concerns were addressed, I still think the methods are lacking on information about the animals (i.e. age, reproductive status).
Validity of the findings: All of my previous concerns have been addressed.
Additional comments: This version of the manuscript is much improved, however more information is needed on the study design particularly about the animals sampled. As is, the study design would be very difficult to reproduce.


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: ENVIRONMENTAL INACTIVATION AND IRRIGATION-MEDIATED REGROWTH OF ESCHERICHIA COLI O157:H7 ON ROMAINE LETTUCE WHEN INOCULATED IN A FECAL SLURRY MATRIX
Review round: 1
Reviewer: 1

Output:
Basic reporting: The paper is logical in flow and appropriate reference to previous studies thereby providing context for the research.
Experimental design: The methods are appropriate and detail is of nature that will permit replication of the study.
Validity of the findings: The conclusion generated are supported by the data presented.  Authors were cautious to not over generalize the significance of the findings.  Indeed, based on this studies and those in the literature it appears that significant variability can be associated wit behavior of STEC on crops within a region and between geographic regions.  Makes good case for "additional research" must be conducted.
Additional comments: Not very clear why rabbit, poultry (chicken) and pig manures were used. What would be source of these manures...wild (rabbit) and feral (pig) animals?  Application of chicken bedding pellets? Were these types of manure used in other studies (besides Chase et. al., 2017)? Authors may need to provide more information rather than just referring to previous publication.

Why were the inoculated slurries held at 4C? Cold shock the cells? Was this also done in other studies? Why was PBS used for dilution of feces?  Seems water would be more appropriate.

Would the type of feces influence survival as much as other factors evaluated?  I know that only so much can be tested.  Not certain whether information exists form previous studies.


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: ENVIRONMENTAL INACTIVATION AND IRRIGATION-MEDIATED REGROWTH OF ESCHERICHIA COLI O157:H7 ON ROMAINE LETTUCE WHEN INOCULATED IN A FECAL SLURRY MATRIX
Review round: 1
Reviewer: 2

Output:
Basic reporting: This paper is well written and appropriate references have been cited.
Experimental design: Experimental design is  pretty straightforward with no major flaws. The authors referred their previous work but they should briefly describe the methodologies.
Validity of the findings: I don't think this paper is novel but it has useful data.
Additional comments: L25: This study was done in 2012 and I am wondering why it took 6 years to write this paper.
L25: E. coli - name in full.
L117: How many times this strain was grown in the lab?
L127: How did you make sure the feces was fresh?
L127: How many animal fecal samples were collected? Just one for each species?
L133: Did the authors homegenize the fecal slurry?
L150: Please describe the methods briefly here.
L161: 340 - lower detection limit? It doesn't  seem that good considering the low dose of O157:H7
L165: What is recovery rate of this approach?
L166: Provide data as supplemental.
L169: How many isolates were confirmed? 10% is vague.
Table 1: very basic. should be presented in the supplementary materials.


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: IDENTIFICATION OF DIFFERENTIALLY EXPRESSED KEY GENES BETWEEN GLIOBLASTOMA AND LOW-GRADE GLIOMA BY BIOINFORMATICS ANALYSIS
Review round: 1
Reviewer: 1

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

This is an interesting work to investigate the differentially expressed genes in gliomas. Nevertheless, the different types of gliomas, including low grade gliomas and high grade gliomas were not properly introduced. Furthermore, it is not clear how the discussion fits into the overall aim of the study apart from the title. It would seem that the focus on SAA1 and TIMP1 individual genes, without discussing the roles and correlation of the other hub genes with higher degree of connectivity, can be further enhanced in the paper.

It was also noticed at many instances in the paper, the authors did not carefully use the abbreviation for example in the cases of Glioblastoma multiforme, Glioblastoma, GBM, Low Grade Glioma  and LGG.


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: IDENTIFICATION OF DIFFERENTIALLY EXPRESSED KEY GENES BETWEEN GLIOBLASTOMA AND LOW-GRADE GLIOMA BY BIOINFORMATICS ANALYSIS
Review round: 1
Reviewer: 2

Output:
Basic reporting: The manuscript is well written with enough backround information. Manuecript by Xu et al. is based on bioinformatics analysis of differentially expressed genes between glioblastomas and low-grade gliomas. The manuscript is well defined with enough background, I only cannot find the legends to the figures. Hypothesis, figures and tables are well written.
Experimental design: I miss the use of more expression datasets from GEO database, namely authors took only two data sets, GSE52009 and GSE53733. With more datasets they could get more reliable results with higher number of samples.

To state that TIMP-1 and SAA1 could be used as biomarkers, the authors should validate those findings on cell lines or tumor tissues using qPCR/Western blot/IHC or flow cytometry or something similar.
Validity of the findings: In Figure 4, the whole p values are not listed (only 0) and then you cannot know if the result are statistically significat. Please provide p -value, is the p value less then 0.05, 0.01, 0.001?
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: IDENTIFICATION OF DIFFERENTIALLY EXPRESSED KEY GENES BETWEEN GLIOBLASTOMA AND LOW-GRADE GLIOMA BY BIOINFORMATICS ANALYSIS
Review round: 1
Reviewer: 3

Output:
Basic reporting: This manuscript is a professional article. No further comments.
Experimental design: The authors applied bioinformatic tool to analyze the GBM and low grade glioma data from GEO database. Study identified SAA1 and TIMP signaling pathway as plausible biomarker to distinguish low grade and malignant glioma. Authors also propose the contribution of SAA1 and TIMP1 signaling in GBM development.
Validity of the findings: In general, the analysis was good and the descriptions are sufficient to support the results.However, there are some points need to be answer. First, the authors should verify their finding with other data base to see if their finding can be confirm in other database. Second, the role and finding of SAA1 could be very different in different GBM cell lines from previous publications. Such as in Knebel's study, they found dual role of SAA1 to GBM progression; could authors find any correlation or association to explain/distinguish this? Third, the databases are base on RNA expression level in GBM and LLG, which might not be able reflect to the peripheral level. Also, the different region of tumor tissues could expression different SAA1 and TIMP1. Authors could put this into discussion.
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: IDENTIFICATION OF DIFFERENTIALLY EXPRESSED KEY GENES BETWEEN GLIOBLASTOMA AND LOW-GRADE GLIOMA BY BIOINFORMATICS ANALYSIS
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 answer the questions from reviewers.


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: LIGHTWEIGHT DATA MANAGEMENT WITH DTOOL
Review round: 1
Reviewer: 1

Output:
Basic reporting: > _Clear, unambiguous, professional English language used throughout._

The manuscript is written clearly and understandably.

> _Intro & background to show context. Literature well referenced & relevant._

The introduction includes a reasonable description of the problem that the software aims to address.

However, some important and seemingly relevant prior art and corresponding work is not mentioned at all. In particular, one could get the impression that the presented data packaging format and structure is in essence a reinvention of the BagIt standard (<https://en.wikipedia.org/wiki/BagIt>). There may be reasons why this is not quite the case, but the relevancy of BagIt seems strong enough that it should at least be discussed how this differs, and why the authors chose to create their own standards instead of adopting BagIt as an existing standard for package structure and metadata.

> _Structure conforms to PeerJ standards, discipline norm, or improved for clarity._

The manuscript follows its own, alternative, structure. I'm not convinced that the result improves clarity substantially over a more standard structure. Although the chosen structure lends itself well to a descriptive story about how the software can be useful, a more critical reflection on limitations and their underlying design choices is missing. Also, the chosen format doesn't seem to leave no obvious place for putting observational data about data management practices with and without the software (whether as the result of an experiment or anecdotal).

I'd suggest to prefix the subheadings under _Extended use cases_ with "Use case:" or something similar. Or perhaps first give an enumeration of the extended use cases being described? Or both? In the present form, it's easy to lose track for a section that it presents one of the extended use cases.

The fact that there is a section _Extended use cases_ implies that there were also standard (? basic? original?) use cases. But the text does not make it clear what those were, and hence it is unclear how the distinction between extended and other use cases is meaningful. (The "extended" use cases described here all look pretty basic to me for a generic tool aimed at addressing the most common data management challenges.)

> _Figures are relevant, high quality, well labelled & described._

Figures are relevant and good quality, but they are also simply cartoons. This may be exactly right for a software manual, but seems overly simplistic for a scientific article. In other words, by confining themselves to these high-level cartoons, I think the authors are completely missing the opportunity to actually visually present specifics about their software and data package design choices. For example, the structure of the package, including the structure of the metadata directory (`.dtool/`) and its contents within the package are almost entirely missing from the manuscript. (Notice that even the Wikipedia article on BagIt does include this.) Sequence, use case, or activity diagrams (whether UML style or not) are also missing but could be used to present the design of user to software, state to state, and software to storage interactions.

> _Raw data supplied (see PeerJ policy)._

Source code (aside from source code of the presented software) for various scripts and tasks is provided, but printed in the manuscript and in the supplementary file. That requires the reader to copy and paste out of the text, which is needlessly laborious and error prone. Why not provide these as text files, whether under version control (in a Github repo or as Gists), or simply deposited in an archive for research products (such as Zenodo).
Experimental design: > _Original primary research within Scope of the journal._

Yes.

> _Research question well defined, relevant & meaningful. It is stated how the research fills an identified knowledge gap._

Yes.

> _Rigorous investigation performed to a high technical & ethical standard._
> _Methods described with sufficient detail & information to replicate._

There aren't experiments and their results being reported here to which this would apply.
Validity of the findings: The manuscript reports a positive impact of the presented software on data management-related practices at the author's home institution. However, no experiments, metrics, specific observations, or any other data are presented to support this finding. I suspect the authors never collected any before-after data that coul support such findings. However, this is not a minor question. Presumably, creating the software wasn't the objective as an end onto itself, but was expected to be a means towards an end. Hence, simply being silent about the observed impact on data management practices because there are no hard data seems rather unsatisfactory too. If the authors are confident about the positive impact, perhaps they can at least find and describe some concrete cases, even if these had mostly anecdotal character.

The text also in several places speaks about FAIR data, and that the presented software would help with that. I agree that the software should help promote interoperability and reusability, though primarily through the attachment of metadata to the data package. There's also the ability to make publicly available a dataset stored in the cloud, but, given the fact that such storage is ephemeral (it will disappear as soon as one of the bucket owners stops paying the bill, or decides to delete it) isn't that about as good or bad as data stored on some lab's web server? And how does the software help with findability? So it seems how and where this software fits into promoting FAIR data is a little more nuanced as appears from the manuscript, and would benefit from a more critical discussion.

The section about verifying the integrity of a data set needs a more critical treatment of limitations. The verification as currently implemented isn't tamper-proof in any way, and even if this may not be of much significance to individual investigators, I know it is for many institutions, and also for data-producing core facilities. (See under _General comments_.)
Additional comments: Line 41: _need_ is extraneous

Line 83-87: "_there is limited immediate incentive for the people generating the data, most commonly PhD students and post-docs, to care about data management_": This is, I would argue, prone to be misunderstood, because, as the authors point out, "data management" is a broad term. Arguably there's no question that those generating or analyzing the data *have to* care about managing the data somehow. What there is less of an incentive for is to do so in such a way that the result is well-structured, and has sufficiently comprehensive metadata so that the result can be FAIR. And yes, their career relies on generating publications, but publications that receive little attention and reuse because their underlying data are inscrutable are against their self-interest. This is probably what the authors meant anyway. So I suggest the authors be specific here, and not altogether dismiss an incentive for "data management" writ large, and not generically put reusable data in opposition to academic career incentives.

Line 102: _Each technology has its own quirks with which the end user needs to gain familiarity._ I don't necessarily disagree, but on its own this reads overly opaque. Maybe supply an example how these quirks translate into adoption becoming time consuming?

Line 137: _This makes it hard to move this data around without losing metadata._ I think the problem isn't so much that it's hard to move this data around (is it really?) but that it's so *easy* to do so in a way that loses metadata without notice. I.e., isn't the problem one of vulnerability and thus high risk (of metadata loss) than of difficulty?

Line 182 and later: The S3 bucket URIs such as <http://bit.ly/Ecoli-reads> etc will look like a browser-resolvable URL to many, but yield "Access denied". To use these one will have to set up AWS credentials and use dtool (or perhaps some other AWS CLI tool?). This should probably be explained upfront to spare readers from fruitlessly clicking on these URLs (they are in fact hyperlinked -- perhaps they should not be).

Line 188, 216: It appears that `dtool readme show` returns its results in YAML format, whereas `dtool summary` returns JSON format.
Why the choices, and why not use the same format for both commands?

Line 288, "Verifying the integrity of old data". I think the far more interesting case of detecting dataset corruption, accidental or from tampering, is verifying that the file *contents* are exactly what they are expected to be. A little experiment shows that dtool doesn't detect file content corruption. Also, if one deletes a file and then also deletes the manifest entry, `dtool verify` reports that all is good. So this is just simply MANIFEST checking, with no checking of file content integrity (aside from file size checking), and no detection of tampering. Perhaps the authors consider these checks out of scope for dtool, but such intentional limitations should then be clearly stated so that the "verify" capability is not misunderstood. Cryptographic signature checking is not hard to implement and widely employed by, for example, package managers of all flavors, and hence may easily be expected to be taking place if it's not made clear that only the data manifest is being checked.

Line 328-332: The presented output from the script seems to be missing the `PROCESSING ITEM` line that the script should output, suggesting that what was run to generate the output isn't what's printed in the manuscript. Same for lines 336-339.

Line 395: This is the first time the `dtool` command `uuid` appears, which seems unfortunate and a missed opportunity?

Line 446-448: Why isn't this just another `dtool` command, such as `dtool publish`? It seems like an unnecessary surprise that doing this requires remembering a separate command line tool?


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: LIGHTWEIGHT DATA MANAGEMENT WITH DTOOL
Review round: 1
Reviewer: 2

Output:
Basic reporting: This paper is well written. First I have to say that I love this idea! I work in biology and bioinformatics and have been advocating for keeping data and metadata together for many years. This is a key, and under appreciated issue! I was really excited reading the paper about the potential for the tool and would be excited to try it out with my data. Before I would commit to employing it more broadly though, I would in particular want more information on the implementation of the software and to understand how it has been used and received at the institute where the authors work, and outside if others have used it. More specific comments below.

Overall it seems like this was a tool that was developed for the needs of the John Innes Centre, and this paper is the opportunity to present it to a broader audience and hope that others also want to use it and find it useful. That's great! However to make that transition, it would be helpful and important to have more of a general context for the paper throughout. In particular, that starts with the introduction. The introduction section lacks references that put the tool and paper in the context of the literature. Overall the intro doesn’t demonstrate a strong understanding or appreciation for the space. That’s potentially OK, but when presenting some new tool as better than current approaches it’s important to put it into context and also appreciate other contributions. A few particular examples are cited below.

Introduction:
- More references are needed in the introduction for such statements as "has resulted in data management becoming one of the big challenges faced by the biological sciences”. There are many reports and papers documenting this as an issue. It's a good opportunity to highlight them here, and that it is broadly recognized as an issue. Also important for recognizing others who have explored this topic.
- The statement "Open access is increasingly viewed as a public good”. I don’t think it’s ‘open access’ that’s viewed as a public good, but data, which means that data should be open access.
-Are there no more recent reference than 2015 on funding agencies requiring data sharing plans?
- I don’t think ‘laboratory notebooks’ are one extreme. They are historically and currently important.
- A few examples are cited. Why are these particular ones selected?
- In the 'Data management problem' section, overall challenges in data management are presented as ‘just so’ stories and not in relationship to research that has highlighted the challenges.
- Rather than separating ‘Data Management challenges’ and ‘Our motivation’ the authors either need to expand and add citations to the ‘Data Management’ section, or could combine into one ‘Challenges of data management at an independent research institute’. Then they could focus on their issues in particular, and also help ground the tool in the type of organizations it was developed for. The ‘Our motivation’ section was much more compelling and provided a clearer picture of why the tool would be important.

The section describing the use of the tool was exciting and made me want to try it out! Good examples and nice to include the code that would do those tasks. However, I didn't have much of a sense of how it was implemented 'under the hood' from the paper. More on that in further sections.

Similarly, while it looked exciting and useful, it would have been good to hear how it's been received where it's been used. This likely has been used at John Innes Centre. How has it been received? What have been the issues? How many people are using it?  Has it been used at other places? More information on how it has been used and some ‘lessons learned’ would be very valuable. The authors have data on reliability and usage! That would be great to include and help people evaluate if they want to use it.
Experimental design: Because this is a software paper, there isn't exactly an 'experimental design' section. So, here I'll discuss 'software design'.

The authors provide links to the software and documentation. Both are excellent. I was able to install the tool and use it with the example described. Code is clear and well documented.

This paper could do with essentially a 'Methods' section that describes how the tool was implemented. What's the conceptual framework for the approach? Why was it chosen? How was it implemented? Does the implementation rely on other theoretical (e.g. graph theory, hashing) or practical implementations? This is really important for evaluating its speed and reliability and since this is a paper, is a great opportunity to discuss this component. The paper is focused on how to use the software, rather than about the software. Both are important, but if there is not a more theoretical discussion of the software, this is more of a 'how to' paper than an academic publication. I definitely recognize the challenges with software of walking this line of what to include in a paper, but I think this is important in this journal.
Validity of the findings: With a software paper, again a different model. So here I'll comment on 'usage of the tool'. As mentioned in the other paper, it's difficult to evaluate the effectiveness of the tool without information on how it has been used and even potentially 'lessons learned'. Biologists do really want to see evidence of it being used in a 'real world' scenario before they are eager to try a new approach or tool. Since the authors likely have some of this information from using it in their context, it would be very helpful to include.

This is a totally different workflow for biologists, so for adoption there is a strong social/cultural issue. For making that sort of change, this kind of information is crucial.

As this is open source and they are putting it out for usage and contributions, it would also be interesting to hear if they had plans for how they are going to sustain and support this open source project. This is not often included in papers, so the authors wouldn't' be expected to include, but it is an important element of open source and should be more discussed.
Additional comments: Thank you for this nice tool! I recognize the challenges of wanting to publish software in a journal. From my perspective, this paper needs a bit more general background, and discussion about the tool and its use for it to be included in this particular journal. That's a great goal though and would really help with adoption, so I would encourage the authors to keep working on this paper.

If they wanted to publish just the software, there are options like the Journal of Open Source Software. Publishing there would not preclude publishing here, but would get a publication and DOI for the software itself and a paper like this could focus on the background, methods and usage.


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: LIGHTWEIGHT DATA MANAGEMENT WITH DTOOL
Review round: 2
Reviewer: 1

Output:
Basic reporting: The text is substantially improved in structure and introduction of relevant prior art. The authors' responses to my earlier comments are largely satisfactory, and the introduction of the two new figures definitely add useful information to the manuscript.

That being said, two issues remain.

One, the text across the Methodology and Results section still feels in parts undecided on what in this piece of scholarship constitutes methodology, what constitutes a result, and what constitutes interpretation. For example, the Methodology section seems to consider the product that is the report's primary subject, the dtool software a methodology, in the same vein as explaining design decisions. On the other hand, major use-cases are mostly considered Results, even though presumably these drove, in the form of a-priori requirements, the need for and the design of the software from the beginning. And the description of the use-cases in the Results sometimes veers into interpreting the facts, for example where it speaks to the ease of using the tool, or that it gives a "clear overview". I.e., as described it remains unclear for this example whether ease of use and clarity of certain output is something the authors (as the developers) believe to have achieved, or something that users have reported or confirmed to them, or both. I think the text would benefit from a more thorough follow-through on the new structure. As another example, aside from an enumeration of driving requirements (which presently are sort-of implied by the use-cases in the Results), other elements of the development methodology, are glossed over as well, such as the use of Python as implementation language (was there a design reason for this choice, or is it simply what seemed up to the task and what the developers were sufficiently fluent in?), how and with which objectives (e.g., percent code coverage aimed for?) testing is implemented, and how documentation is authored, and then rendered/published.

Two, the introduction and discussion of the BagIt standard goes a long way to addressing my earlier comment about it. But it also still leaves some questions open, perhaps in part because I wasn't clear enough in my earlier comments. Specifically, the authors' point seems to be mostly that dtool is a both a tool for end-users and a programmatic interface for programmers, whereas BagIt is a standard structure for a dataset and its metadata. That is obviously both true and fair enough. However, dtool under the hood does employ a structure for a dataset and its metadata, only it is an ad-hoc created one rather than BagIt. If, as the authors state, the dataset structure was designed to survive the tool used to create it, i.e., to stand on its own, then if the authors aren't using an existing standard for this they are de-facto creating a new (a  d-hoc) standard, whether they meant to or not. I think this merits a more thorough treatment in the text. The authors don't really provide a good argument as to why BagIt didn't and/or doesn't meet their needs for the underlying dataset structure. ("implementing a consistent convention for storage of this metadata on top of BagIt" seems, if possible and not overly complicated, still preferable to creating an entirely new standard ad-hoc?) Perhaps what happened is that in the design of the underlying dataset structure existing standards such as BagIt were not considered for adoption. If so, what could still be done now is to acknowledge that, and to outline steps that would need to be taken to adopt BagIt or another existing standard in a future version (such as layering some kind of metadata storage convention on top so that dtool's requirements are met). Alternatively, if BagIt (or other standards) was considered for adoption but had to be rejected, this would deserve a more substantive case than provided in the revision.

However, overall while addressing both of these issues would improve the scholarly clarity and value of the manuscript, I don't feel these rise to the level of preventing publication if left as is.
Experimental design: N.A.
Validity of the findings: Some of the features of the reported software continue to be described in the Methods and Results sections interpretative ("easy", "clear", "powerful", etc) rather than strictly fact-based. See comment under (1).

The case for de-facto rejecting BagIt as the underlying dataset structure as currently described is weak and, IMHO, unconvincing. See comment under (1).
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: LIGHTWEIGHT DATA MANAGEMENT WITH DTOOL
Review round: 2
Reviewer: 2

Output:
Basic reporting: This article is much improved. My concerns around references in the introduction, organization of the paper and discussion of the tools use have been addressed. In particular, I appreciate the Use Cases and the new figures. I still would like to have seen more discussion of how dtool is being used at JIC. I think there's a lot of valuable information there on social and/or technical barriers to use, not only of the tool, but of the concept of data management. Given the close association of the tool developers with its users, this is a good opportunity. As the authors continue to develop and deploy dtool, I hope that they will do some 'user testing' and incorporate it into their development and documentation. I look forward to their next paper that reports on further uptake and barriers encountered. :) The authors mention the development of a GUI to help with uptake. I'm not convinced that's the best use of resources, so some analysis before embarking on significant GUI development would be valuable.
Experimental design: Methodology section now address methods of development and design choices.
Validity of the findings: The use cases and Uptake at JIC sections now address how the tool is used. As mentioned above, I would have liked to see more about how it is being used at JIC, however deployment was not set up to collect that data. In the 'Uptake at JIC' section, perhaps the authors could add a sentence to that effect and that they plan to continue to evaluate as they deploy in more labs. Something along the lines of "We did not initially plan deployment to systematically collect information on usage, strengths and barriers to use. As we continue to use and deploy dtool at JIC, we will more systematically collect information and work with users to continue to improve dtool."
Additional comments: Thank you for so thoughtfully addressing our comments. The paper is much improved.


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

Input:
Title: AN ASSESSMENT FOR HEALTH EDUCATION AND HEALTH PROMOTION IN CHRONIC DISEASE DEMONSTRATION DISTRICTS: A COMPARATIVE STUDY FROM HUNAN PROVINCE, CHINA
Review round: 1
Reviewer: 1

Output:
Basic reporting: •	English language:  The English language should be improved to make sure that the international audience can understand the text.  Think about having a native English speaking colleague review the manuscript.  Some examples where the language could be improved include lines 28, 29, 42-43, 44, 71-72,…
•	There is clearly a need for the creation of quality indicators to measure health education and promotion efforts.   Potentially think about working on flow of introduction to help the author better grasp the need.
•	Professional article structure, figs, tables:  I am not familiar with RSR, so I am only commenting on Delphi-related tables and figures.  Potentially think about expanding Figure 1 to visual show what occurred during Round 1 and Round 2 of Delphi method.  You have the potential to show a lot of information in an aesthetically pleasing manner that will be more easily understood versus the text.  See Li e al. (2014) “Framework of behavioral indicators for outcome evaluation of TB health promotion:  a Delphi study of TB suspect and TB patients” for their Figure 1.  Great article to use as a resource.
•	Raw data shared and accessible.
•	The article is a bit busy with three primary purposes.
Experimental design: •	Primary research within Aims & Scope of journal:  This article is focused on Health Sciences – mainly monitoring population health through a novel method.
•	Research question well defined, relevant & meaningful:  the research question could be improved and written more clearly.  Abstract:  “This study was designed to construct such quality indicators, have a grasp of the work status of health education and promotion in the districts, and explore the main factors involved to promote the work.”  This is a bit busy, potentially think about splitting up.  “This study was designed to establish a framework of indicators for outcome evaluation of health education and promotion efforts in Chinese districts.”  And then continuing with your secondary purposes.
•	Rigorous investigation performed to a high technical & ethical standard:  Lines 131-132 – typically informed consent is a bit more formal than “it was regarded as informed consent.”  (I am not an expert on RSR and therefore am not commenting.)
•	Methods described with sufficient detail & information to replicate:  improvement needed.  More information about the Selection of Delphi experts, Instrument for Delphi surveys, consider putting the consensus level in bullet form for easy reading, Figure 1 could be improved (see Basic Reporting section), consider splitting Table 2 into two separate tables for ease of reading with 1st round results in one table, and 2nd round results in the second table.
•	Creating a table devoted to the framework of indicators would be helpful to readers to cleaning depict your results.
Validity of the findings: •	Again, I’m not speaking on RSR only Delphi method.  Reporting the median and standard deviation is not what I am accustomed to, typically mean and standard deviation or median and mode.  Would consider changing standard deviation to mode in Table 2.
•	I suggest having a native English speaker assist in editing the discussion and conclusions to help make the sections more easily understandable and readable for your audience.
Additional comments: I think the creation of the quality indicators by the modified-Delphi process should be the main focus of the paper.  More information could be provided on the process while lessening the focus on the descriptives of the counties and RSR.


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: AN ASSESSMENT FOR HEALTH EDUCATION AND HEALTH PROMOTION IN CHRONIC DISEASE DEMONSTRATION DISTRICTS: A COMPARATIVE STUDY FROM HUNAN PROVINCE, CHINA
Review round: 1
Reviewer: 2

Output:
Basic reporting: English is hard to follow with adjectives used to define results without any quantitative parameter.
Experimental design: Research question well defined, relevant & meaningful. Rigorous investigation is not performed.
Validity of the findings: Data is not controlled.
Additional comments: The number of participants in this study is too small. It is not possible to reach any conclusion with this number. There are other problems. The manuscript is hard to follow, and its fundamental hypothesis is not underlined.  Abstract is written very poorly and does not provide clear understanding of results. The authors did not define DELPHI process in Introduction or results. The authors use adjective to define their results however how that conclusion is reached is not addressed.
1.	The study participants are senior doctors with a mean of 21 year experience. What is the control of this group, a comparison of individuals with similar work experience who are not doctors. The other group of individuals who are doctors but have significantly less experience is required to compare with first group.
2.	Line 23 “This study was designed to construct such quality indicators, have a grasp of the work status of health education and promotion in the districts, and explore the main factors involved to promote the work. “ The authors did not define quality indicators.
3.	Line 82 “A modified Delphi method (conducted between late 2013 and 2014) was firstly used to build quality indicators. “ What is quality indicators?
4.	Line 204 “Overall, NCD demonstration districts performed better in health education and health promotion  than that of non-NCD demonstration districts. “ Define better ?
5.	The English language needs to be improved. Many sentences are too long and are vague.


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: AN ASSESSMENT FOR HEALTH EDUCATION AND HEALTH PROMOTION IN CHRONIC DISEASE DEMONSTRATION DISTRICTS: A COMPARATIVE STUDY FROM HUNAN PROVINCE, CHINA
Review round: 2
Reviewer: 1

Output:
Basic reporting: The English language is improved.
Experimental design: The experimental design is sound.
Validity of the findings: The study has interesting findings although validity remains unknown due to low number of samples.
Additional comments: I do not have additional comments. I will like to congratulate authors for their work.


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: ULTRA-PERFORMANCE LIQUID CHROMATOGRAPHY-MASS SPECTROMETRY FOR PRECISE FATTY ACID PROFILING OF OILSEED CROPS
Review round: 1
Reviewer: 1

Output:
Basic reporting: The manuscript for "Ultra-Performance Liquid Chromatography Mass-Spectrometry for precise fatty acid profiling of oilseed crops" is well written and data has presented in an interesting manner. Please correct a few spelling mistakes in the manuscript as follows:

Line 127 Change eppendorf to Eppendorf
Line 142  Correct mixining to mixing
Line 143: Correct chlorophorm to chloroform
Line 183 Correct standarts to standards
Line 187 Change peak Table to peak table
Line 340, change sunflower to sunflowers
Experimental design: Well design experiment with the appropriate number of replicates for the analysis. However, some useful information is missing in the manuscript and it will be useful to have that information added. Please include the validity of the two LC-MS methodologies with linearity, repeatability and reproducibility of the data. In addition, include the lower level of detection (LOD) as well as lower level of quantification (LOQ) using standards for each fatty acid standard reported.
Validity of the findings: This is a novel approach of detecting fatty acids using LCMS and it will be useful to the research community in fatty acid analysis in plant and biomedical research.
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: ULTRA-PERFORMANCE LIQUID CHROMATOGRAPHY-MASS SPECTROMETRY FOR PRECISE FATTY ACID PROFILING OF OILSEED CROPS
Review round: 1
Reviewer: 2

Output:
Basic reporting: The writing is mostly adequate, but some checking for subject-verb agreement would help, especially in the methods section.

Sufficient references and context are provided.

It’s good that all the original data is provided in the supplemental tables.  However, it could be organized in fewer tables. For example, all the original data could be combined with Table 1, by labeling each column that follow the sample information with a method/metabolite and including the data for that method/metabolite for each sample.

Also I could not locate legends for the supplemental tables and figures.

Lines 192-193: “Since the method used do not allow to distinguish between isomeric FAs”--  Suggested rewording: “Since the method does not distinguish isomeric FAs”

Tables: “Procentage fraction” should be “percent”. Significant digits should reflect the precision of the measurements (i.e., excess digits appear to be present in GC data). “NA” should be defined.

Figures 3-5: Legends should describe the samples (lines).

Line 349: “which allows detecting 2.5 times higher amounts of FA” -- Consider: “which detects 2.5 times more FA species”

Line 142: mixining should be mixing

Line 143: chlorophorm should be chloroform

Line 183: standarts should be standards

Line 248: sperman rho should be Spearman.

Lines 183 and 215: GS should be GC?  If so, you don’t need to add “chromatograph”.

Supplemental Table 1: Sheet label is not in English.
Experimental design: The article is within the scope of the journal. The research question asks how LC-MS with TOF detection in negative mode compares with GC as a method for fatty acid analysis. It demonstrates that LC-MS can detect more fatty acid species, but that the quantification is poor.  It was a little disappointing that there wasn’t any attempt at absolute quantification (using internal standards) for either method and that there was apparently no attempt to describe the linearity of the LC-MS detected response as a function of the amount analyzed. Additionally, the fact that there are two methods, one with one chromatography solvent system and concentrated samples and the other with a different solvent system and dilute samples makes it difficult to assess the effect of the solvent system and the sample concentration independently. Was there any advantage/disadvantage of one solvent system over the other?

Line 133-134, describing the LC method: “For FA quantification, three isotopically labeled internal FA standards were added to the extraction mixture (3 μg of each FA per sample).”  These standards do not appear to be used in any calculations.  Only calculating percentages is described (lines 206-208).

If the authors wanted to make a great LC-MS method for fatty acids, it would be possible to determine the response factor for each fatty acid by comparison to the GC data, ideally over a range of concentrations through which both methods are linear.
Validity of the findings: The authors should consider in their calculations that GC-FID response is approximately proportional to the mass of the fatty acid methyl esters, and, thus, to calculate mole percent, the molecular weights of the methyl esters should be included in the calculations.  On the other hand, MS response is theoretically proportional to moles (if all species ionize equally, though this doesn’t seem to be the case) and mole percent can be calculated directly from intensities.  Putting values from GC and LC-MS both in mole percent would be appropriate.

Another thing that the authors should/could consider is that, because the m/z of the fatty acids vary quite a lot, the fraction of the intensity in the major isotopologue also varies.  For example, for 14:0, the lowest m/z peak at 227 for [M-H]- accounts for ~ 85% of the total intensity, whereas for 28:0, the lowest m/z peak at 423 accounts for only ~73% of the total intensity, so it would be appropriate to divide the intensity associated with the main isotope by the intensity of that peak over of the total intensity of all the isotopes (i.e., 0.85 or 0.73).  This would alter the percentages a bit.

What is the rationale for using Spearman’s correlation coefficient vs. Pearson’s for method comparison?  It seems like you want to determine if there is a linear relationship which might be best accomplished with Pearson’s correlation.

In Supplemental table 3, the authors calculate the coefficient of variation across the samples. It would be nice to see the coefficient of variation calculated on the same sample run at intervals throughout the analysis.  That would give information about the repeatability of the analysis.
Additional comments: Figure 1: The panels for A and B seem to be reversed in relation to the description.  This figure would be better if the peaks were labeled with the fatty acids that they represent.  “Dynamical” should be deleted or replaced with “dynamic”.

Line 68: “significant differences in the directions of the selection process in these two crops”: No mention is then made of anything about sunflower selection.

Lines 199-201: The mention of “net-like” patterns here does not seem to make this any clearer than just indicating the effect of chain length and double bond number on the retention time.

Line 157: It would be helpful to indicate that the MS scan is with the TOF, rather than the quadrupole.

Line 322: “while GC-FID requires at least 4-5g”  This seems like way more than most people use, especially for oilseeds.


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: ULTRA-PERFORMANCE LIQUID CHROMATOGRAPHY-MASS SPECTROMETRY FOR PRECISE FATTY ACID PROFILING OF OILSEED CROPS
Review round: 1
Reviewer: 3

Output:
Basic reporting: This paper describes a method of fatty acid quantification by UPLC/MS on rapeseed and sunflower seeds. This method was previously successfully used to quantify fatty acid in different type of organisms. The authors compare the FA profile of 50 lines of each plant species analyzed by the UPLC/MS method or by GC-FID. They conclude that UPLC/MS is more sensitive and detect more FA molecules.
Experimental design: Major remark:
The methods of lipid extraction from the seed material are totally different in the two methods: in the GC-FID method, extraction is done only with hexane and will be therefore specific for highly apolar lipids such as triacylglycerols, whereas in the UPLC/MS method, the extraction is done with the MTBE method that will extract triacylglycerols but also polar lipids such as phospholipids. Therefore, it is not surprising to find more diverse FA molecules with the UPLC/MS method.
There is no comparison of recovery or quantification, only fatty acid distribution… When the number of detected fatty acid changes, the percentage also change… Maybe a comparison by quantity of fatty acid per seed will help to really compare the two methods.
Minor remark:
Is the method of fatty acid hydrolysis specific enough? Normally the use of methanol and KOH produces also transesterification and therefore FAMEs.
Where does the odd chain fatty acid come from?
A lot of details are missing for the GC-FID analysis procedure such as the column length that can explain why the very long chain fatty acids are not detected.
Validity of the findings: No comment
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: ULTRA-PERFORMANCE LIQUID CHROMATOGRAPHY-MASS SPECTROMETRY FOR PRECISE FATTY ACID PROFILING OF OILSEED CROPS
Review round: 2
Reviewer: 1

Output:
Basic reporting: Well presented manuscript and sufficient literature have provided.No further comments.
Experimental design: No comment.
Validity of the findings: No comment
Additional comments: Well presented methodology with a novel approach using LCMS for FA analysis.


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

Input:
Title: ULTRA-PERFORMANCE LIQUID CHROMATOGRAPHY-MASS SPECTROMETRY FOR PRECISE FATTY ACID PROFILING OF OILSEED CROPS
Review round: 2
Reviewer: 2

Output:
Basic reporting: It seems to read better than previously, but there are still some minor issues:

287-288: Sperman should be Spearman (multiple places)
214: rt should be retention time
157: Daltonik should be Daltonic (unless this is a European spelling)
131: Concentrator should not be capitalized.

Supplemental Tables seem to be called 2 and 3 with no 1. I still could not see the legends for supplemental figures and tables.
Experimental design: No comment
Validity of the findings: Table 1A: It seems like it would be hard to detect 3 or 5 ng/ml when these values are only 3-5% more than the y-intercept (and it’s out of the linear range).  Similarly, how can you quantify things below the linear range?  I understand that you defined the LOD and LOQ based on the signal/noise ratio, but shouldn't at least the LOQ be in the linear range?
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: ULTRA-PERFORMANCE LIQUID CHROMATOGRAPHY-MASS SPECTROMETRY FOR PRECISE FATTY ACID PROFILING OF OILSEED CROPS
Review round: 2
Reviewer: 3

Output:
Basic reporting: The authors corrected well their manuscript, describing through the text that the two methods differ not only by the detection method (GC-FID vs LC-MS) but also by the extraction. Therefore the manuscript is accurate.
Experimental design: No comment
Validity of the findings: No comment
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: ASSOCIATION OF LATE-ONSET POSTPARTUM DEPRESSION OF MOTHERS WITH EXPRESSIVE LANGUAGE DEVELOPMENT DURING INFANCY AND EARLY CHILDHOOD: THE HBC STUDY
Review round: 1
Reviewer: 1

Output:
Basic reporting: There needs to be a more comprehensive review of the literature, and in particular consideration given to the effects of severity and duration of maternal postnatal depression.
Experimental design: The severity and duration of disorder are not taken into account and are likely confounded with the characterisation of depression as early or late. Analysis also needs to  take account of socio-demographic factors that are typically important moderators of depression effects.
Validity of the findings: likely invalid due to lack of accounting for severity and duration of depression
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: ASSOCIATION OF LATE-ONSET POSTPARTUM DEPRESSION OF MOTHERS WITH EXPRESSIVE LANGUAGE DEVELOPMENT DURING INFANCY AND EARLY CHILDHOOD: THE HBC STUDY
Review round: 1
Reviewer: 2

Output:
Basic reporting: no comment
Experimental design: The authors should describe the Mullen Scales of Early Learning in detail.
Validity of the findings: no comment
Additional comments: Aoyagi et al. investigated the effects of maternal postpartum depression (PPD) on language development in offspring.  The authors found that late-onset (between weeks 5 and 12 postpartum) PPD had a significant effect on expressive language development, and that the effect was not evident in early-onset PPD (within 4 weeks after childbirth).  The findings are clinically important, and the manuscript is generally very well-written.  However, there are points that need to be addressed.

- The description on the measurement of outcome variable
The authors employed the Mullen Scales of Early Learning (MSEL) to assess expressive language development.  An evaluation using MSEL is conducted by assessor(s) but not by a caregiver, which may be one of the strengths of this study.  This can be mentioned.
Were the assessors masked to information about previously evaluated data and maternal PPD?  These can affect the results, and thus, should be clarified.

- “, including postpartum depression,” in Line 56 should be deleted.
- References should be added to “other studies” in Line 60.


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: ASSOCIATION OF LATE-ONSET POSTPARTUM DEPRESSION OF MOTHERS WITH EXPRESSIVE LANGUAGE DEVELOPMENT DURING INFANCY AND EARLY CHILDHOOD: THE HBC STUDY
Review round: 1
Reviewer: 3

Output:
Basic reporting: It's clear written, with sufficient background provided
Table and figure were well designed
The content include results relevant to hypothesis
Experimental design: The study was conducted based on a relative large sample size, prospective birth cohort. The methods were clearly described and considering the majority of confounding factors (or at least discussed as limitation)
Using growth curve for time evaluation of expressive language score over different time points is a relative novel idea and easy to understand.
Validity of the findings: The data is statistically sound and controlled
The discussion including the shortage and strength of this study,  conclusion are well stated
Only some description of data  need to state more clearly.
Additional comments: This study aims to investigate the association between maternal postpartum depression (PPD) and infant’s development of expressive language, focus on the onset of PPD. The manuscript is well written and organized. Some comments are listed:
1.	Line 103. The original expressive language scores were converted to Z score. What’s the reference population, this study cohort or general population?
2.	Line 138. Does the change in growth refer to change in (expressive language) score?
3.	The value in Table 2 refer to mean scores or regression coefficient? Please state more clearly.
4.	Please merge line 208 “On the other hand…” with next paragraph (line 213 to 217).


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: ASSOCIATION OF LATE-ONSET POSTPARTUM DEPRESSION OF MOTHERS WITH EXPRESSIVE LANGUAGE DEVELOPMENT DURING INFANCY AND EARLY CHILDHOOD: THE HBC STUDY
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 all of the 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: ASSOCIATION OF LATE-ONSET POSTPARTUM DEPRESSION OF MOTHERS WITH EXPRESSIVE LANGUAGE DEVELOPMENT DURING INFANCY AND EARLY CHILDHOOD: THE HBC STUDY
Review round: 2
Reviewer: 2

Output:
Basic reporting: The whole manuscript were well organized and written in fluent English.
The figures were clearly presented
The results and discussion were relevant to the hypotheses
Experimental design: A longitudinal cohort design with mother infant pair and repeated measure is the strength of this study.
The measurement of exposure and outcome were clearly described
Validity of the findings: Conclusion are well stated
Additional comments: The authors have answered my questions point to point with clear expression.
There are only two minor comments: the introduction part is a little bit to lengthy at current version. Second, the authors add more description about MSEL "as one of the child composite scale". Maybe more information such as how many items? in what dimension of language, or give few examples, will be helpful.