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: RECENTLY-ADOPTED FORAGING STRATEGIES CONSTRAIN EARLY CHICK DEVELOPMENT IN A COASTAL BREEDING GULL
Review round: 1
Reviewer: 1

Output:
Basic reporting: The research questions / hypothesis are currently not well defined so please state these more explicitly.

The tables are currently quite busy and therefore not easy to read - please format these into a standardised formatted scientific table, ensuring columns and rows are well spaced.
Experimental design: The methods would benefit from a little more detail, for example, stating the dates colonies visits were made between, ensuring that it is clear that all free-ranging and aviary chicks are LBBGs, and explaining what the experimental nests are.
Validity of the findings: No comment
Additional comments: This is an interesting and concisely written manuscript exploring marine versus terrestrial diets in Lesser Black-backed Gulls. The experiment with aviary chicks is very useful given the variation in results from free-ranging chicks among colonies in different locations. The introduction provides concise and relevant background to the issues around establishing the consequences of switching from a predominantly marine to terrestrial diet in generalist gulls, and the discussion is concluded well with potential consequences of fishery closures, and how the results may differ if repeated in colonies which are more reliant of terrestrial resources.
1.	My main comment is that some of the results do not seem to be discussed in the Discussion. For example aviary chicks reaching a higher asymptote body mass (and growth rate for body mass?) when raised on terrestrial food. It may also worth mentioning the higher body mass of males than females also found by other studies.
2.	The research questions / hypothesis are currently not well defined so please state these explicitly.
3.	The methods would benefit from a little more detail for example stating the dates colonies visits were made between, ensuring that it is clear that all free-ranging and aviary chicks are LBBGs, and explaining what the experimental nests are.

Line 27: I feel traditionally is a more appropriate word than originally
It would be useful to include a concluding line to the abstract to further highlight the importance of the result that the quantity of anthropogenic food items may lower reproductive success.
Line 54: Suggest replace growing with increasing
Line 68: Could this niche shift away from marine resources also be due to a decline in intertidal forging habitats due to increased coastal development and disturbance?
Line 94 and throughout: italicise latin species names
Line 96: change exhibits to exhibit
Line 102: Please provide the dates data was collected between, and were these visits made from egg laying until fledging? Please clarify.
Please state more explicitly in the methods that free-ranging and aviary chicks were Lesser Black-backed Gulls (i.e. not also Herring gulls).
Line 103: It is not currently clear what the experimental nests are? Please can you clarify. How many eggs did these experimental nests contain after receiving a first- or second-laid egg from other nests?
Line 122: How many eggs were collected in total from Ostend? It would be interesting to know how many of those collected hatched.
Line 150 – 153: refer to your Supplementary figures / tables.
Line 160: add for  - cleaned for 5 minutes
Line 172 and throughout: please italicise δ and subscript the numbers i.e. δ13C
Line 173: Please include what species TEFs corrections were based on from Hobson and Clark (1992). i.e. Ring-billed Gull?
Line 176: is Stable Isotope Analysis a specific analysis within SIAR? Please expand on this.
It would be useful to know the number of male and female field and aviary chicks were sampled.
Line 265: Though aviary chicks reached a greater body mess when fed on a terrestrial diet? This would be worth discussing further.
Line 277: Although in some studies, larger male gulls have been found to exploit landfill resources to a greater extent than females thought to be due to larger ind. Outcompeting smaller individuals – see Monaghan, P. Anim. Behav., 1980, 28, 521-527.
Line 287: Be consistent in Capitlisation of species names – Herring Gull
Line 294: It could be based also on food quality given the range of different terrestrial items the gulls may consume and the differences in quality of these as shown by your Appendix Table 4 shows, which should be acknowledged.
Line 297-299: This is a good point as chicken is likely to contain more nutrients and protein than terrestrial items consumed in farmland such as grain and invertebrates, which is worth expanding on here given that your Lesser Black-backed Gulls consumed earthworms from pellet analysis.

Significant results in figures that are not discussed in discussion. i.e. with head length, body mass and chick condition in aviary chicks?
Figure 1. The palest grey for 20-30 days is not easy to see in plot b.
Figure 4. Does the black regression line just refer to males, and should there also be a line for females?
Table 1. The non-significant interaction between sex and age period was removed is repeated twice.
Table 3. Be consistent in how you abbreviate variables or write them out in full i.e. Prop. Terr. Food


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: RECENTLY-ADOPTED FORAGING STRATEGIES CONSTRAIN EARLY CHICK DEVELOPMENT IN A COASTAL BREEDING GULL
Review round: 1
Reviewer: 2

Output:
Basic reporting: In overall, the manuscript is well written, with clear objectives, nice introduction and discussion. Also, the tables and figures are enough to understand the results.
Experimental design: In my opinion, the experimental design is robust, allowing to answer the aims proposed by the authors.
Validity of the findings: The results are attractive to people working and this thematic.
Additional comments: Some minor comments to be addressed:

Lines 41: change “open-air landfills” to “landfills”. Basically because gulls also can exploit the organic matter present in non-open air landfills.

Line 48: Blonick et al 2003 and Araújo et al. 2011 are two references relating to the presence of individual specialization in diet.

Lines 51-53: I would like that the authors include some reference after this sentence

Lines 57 and 62: Some references that could be useful:
Mendes, R. F., Ramos, J. A., Paiva, V. H., Calado, J. G., Matos, D. M., & Ceia, F. R. (2018). Foraging strategies of a generalist seabird species, the yellow-legged gull, from GPS tracking and stable isotope analyses. Marine Biology, 165(10), 168.
Matos, D. M., Ramos, J. A., Calado, J. G., Ceia, F. R., Hey, J., Paiva, V. H., & Handling editor: Stephen Votier. (2018). How fishing intensity affects the spatial and trophic ecology of two gull species breeding in sympatry. ICES Journal of Marine Science, 75(6), 1949-1964.
Lines 63-65: See these two recent published papers:
van den Bosch, M., Baert, J. M., Müller, W., Lens, L., & Stienen, E. W. (2019). Specialization reduces foraging effort and improves breeding performance in a generalist bird. Behavioral Ecology.
Real, E., Oro, D., Martínez‐Abraín, A., Igual, J. M., Bertolero, A., Bosch, M., & Tavecchia, G. (2017). Predictable anthropogenic food subsidies, density‐dependence and socio‐economic factors influence breeding investment in a generalist seabird. Journal of Avian Biology, 48(11), 1462-1470.

Line 172: please, it is important to indicate the TEFs values and also how the authors estimated the TEFs.

Lines 175-178: MixSIAR model is more recommendable (more credible) than SIAR model. I suggest to estimate again the importance of terrestrial food in the chick’s diet by using MixSIAR modelling.

Line 158: Some reference regarding the feather growth rate is necessary in this sentence

Line 274-276: previously the authors remarked that this species shows low sexual dimorphism. For this, I do not understand why the authors discussed here that males could be better than females in relation to the exploitation of fishery discards.

Figures 2A, 2B, 3A and 3B: change “asymptote for body mass” to “body mass (g)”


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: TOWARDS SEX IDENTIFICATION OF ASIAN PALMYRA PALM (BORASSUS FLABELLIFER L.) BY DNA FINGERPRINTING, SUPPRESSION SUBTRACTIVE HYBRIDIZATION AND DE NOVO TRANSCRIPTOME SEQUENCING
Review round: 1
Reviewer: 1

Output:
Basic reporting: The manuscript “Towards sex identification of Asian Palmyra palm (Borassus flabellifer L.) by DNA fingerprinting, suppression subtractive hybridization and de novo transcriptome sequencing”  attempts to identify sex-linked markers for this palm using PCR-based DNA fingerprinting, suppression subtractive hybridization (SSH) and transcriptome sequencing. Overall, the manuscript conforms to the structure recommended by the journal viz. Introduction, Materials and Methods, Results, Discussion, and Conclusions. The findings of the study are well discussed and supported by existing evidence. The overall English comprehension and grammar of the article is good and attains standards of a scientific article. I really appreciate that authors have well-presented the negative results and discussed the underlying complexity in identifying sex-related markers in Asian Palmyra palm. However, below are a few suggestions that would be useful for the readers and make it scientifically thorough.

1.	In line 123, “using the followed condition should be replaced by “using the conditions as follow:”
2.	Please use either “version” or “ ver” consistently throughout the manuscript to refer to software versions.
3.	In line 193-195, the sentence is ambiguous/incomplete. Please rephrase/update it. Maybe break it in two sentences.
4.	Please replace Go with GO throughout the text, wherever applicable.
5.	In line 423, “understanding” should be replaced by “understand”.
Experimental design: 1.	The two statements from line 178 to 182 are contradictory with respect to quality score cutoff used for filtering. Please be consistent if the author set the quality score cutoff of 20 or 25 to filter low quality reads.
2.	In line 184-186 for similarity search, the percentage of alignment length matched and %identity are also useful parameter to consider along with E-value. Can the author mention in the text avg? alignment length (%) matched and average %identity exclusively in the text along with E-value cutoff?
3.	In line 190-191, please mention specifically in the text which 4 transcriptomes are used for combined assembly using Cufflinks? For e.g. two floral stages of male and female (FY, FO, MY, and MO).
4.	In the “Methods” section, please include the steps/criteria to perform GO enrichment analysis of for the male- and female-specific datasets.
5.	In the “Methods” section, please include the steps/criteria to identify a) transcripts that were shared among the datasets b) specific to one transcriptome).
Validity of the findings: The study is experimentally sound and well presented. However, the findings of the transcriptome analysis would have been more robust with the use of replicates per sample. Moreover, huge variation (47,194,682-90,305,610) in the sequencing depth of the samples makes it more complicated to extract robust conclusions. For further comments, refer section Experimental design.
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: TOWARDS SEX IDENTIFICATION OF ASIAN PALMYRA PALM (BORASSUS FLABELLIFER L.) BY DNA FINGERPRINTING, SUPPRESSION SUBTRACTIVE HYBRIDIZATION AND DE NOVO TRANSCRIPTOME SEQUENCING
Review round: 1
Reviewer: 2

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: I would recommend the following minor revisions:

1) Line 67: Replace “is” by “are” in the first sentence.

2) Lines 75-76: I would suggest to delete this sentence because it does not make any sense.

3) Line 110: Replace “centrifugation” by “centrifuged” in the first sentence.

4) Lines 175-176: Total RNA was obtained NOT from one male and one female inflorescence samples, but from two male (MY and MO) and two female (FY and FO) inflorescence samples. Please, correct it.

5) Lines 194-195: It is unclear what you meant by “…, unless two transcripts have corresponded of four transcriptomes”. Please, rephrase it.

6) Line 245: Provide also full names for abbreviations FY, FO, MY and MO, such as, FY - female young inflorescences, FO - female old inflorescences, etc.

7) Lines 324-325: Please, list those genes for seven annotated transcripts that were previously identified to be involved in sex determination and flower development, and provide the references for them.

8) Line 395: Replace “potentially” by “possible”, and “young” by “evolutionary young”.

9) Line 399: Replace “… depicted clearly regarding the large difference of the shape and size of the sex chromosomes, …” by ““… depicted clearly large difference of the sex chromosomes in the shape and size, …”

10) Line 429: Replace “study” by “studies”.

11) Line 431: Replace “has been” by “have been”.

11) Line 447: Cite also Ali et al., 2018 together with Al-Mahmoud et al., 2012.

Best regards,


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: TOWARDS SEX IDENTIFICATION OF ASIAN PALMYRA PALM (BORASSUS FLABELLIFER L.) BY DNA FINGERPRINTING, SUPPRESSION SUBTRACTIVE HYBRIDIZATION AND DE NOVO TRANSCRIPTOME SEQUENCING
Review round: 2
Reviewer: 1

Output:
Basic reporting: The authors fully complied with my requests.
Experimental design: sufficient
Validity of the findings: sufficient
Additional comments: No additional comments, but I would recommend additional revisions for Table 5 in the file attached.


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 ATTENTION BIAS TEST TO ASSESS ANXIETY STATES IN LAYING HENS
Review round: 1
Reviewer: 1

Output:
Basic reporting: In terms of sufficient field background provided, it is suggested that the authors include more on peripheral temperature assessment as an indicator of animal affect state in the introduction, such as physiological mechanisms behind temperature increase/decrease given context and perception of animal.

Line 103-105: Perhaps include a semicolon between "chicks" and "interpreted" so make this sentence more clear, or, rewrite entire sentence.

Line 115: Can the authors define "low-ranging birds"? Is that based on distance from pop-hole, or, active movement throughout day, or, duration of day spent outside?

Line 454-461: There appears to be a repetition of the sentences. Can these be distinguished better, or, remove the redundancy?

Line 415: Confusing sentence, could it be rewritten? For example, "Based on previous studies of personality assessment in free-ranging hens, indoor hens display higher levels of fear trait than outdoor free-range hens."

Figure 6: Asterisk is used to indicate significant difference, but in Figure 3, superscript letters are used. Could the authors use a consistent format (unless this is a journal preference).
Experimental design: Line 172: What is the operational definition used to identify "outdoor birds"? Was this a minimum of 4 hours spent on range, or, a minimum of 20% of access time spent on range (for example).
Line 199-200: Did the attention bias testing occur in the same building as the home pens? Could the birds hear the alarm call when not in the test arena? If so, how did the authors account for this confound?
For each experiment, the inter-observer reliability was based on 20% of the data, correct? If this is sufficient cross-coding for reliability assessment, please provide a reference as justification.
Line 309-310: The major confound of birds in the study coming from two separate home pens was not taken into consideration during analysis. Could the authors provide evidence of no impact of home pen on behavior in experiments (or at least attempt to justify this confound)?
Line 326-327: While it is assumed, it is not clearly stated that saline injected birds also waited 20 minutes in the holding pen. In addition, the rationale for using rat model of cMPP metabolism/effect time is not given. It is assumed that no other species (avian, for example) has been examined for correct dosage or time for effect?
Line 390-392: This inclusion of 14 previously handled and isolated saline birds does not appear to be justified other than to increase sample size. These birds experienced the stress of handling and the pain of saline injection, therefore, they could be biased in their interpretation of the alarm call. Also, were these birds evenly split between the two home pens?
Line 418: How feed restricted were the birds? Based on body weight, or reduction in feed quantity?
Validity of the findings: Line 261: It is more appropriate to say that "The majority of birds showed clear vigilant behavior..." instead of "All", since it is stated that 7 birds did not show clear vigilance.
Line 372-373: The authors should remove this statement of non-significant post hoc test (fewer steps by 2 mg/kg dosed birds) as it is misleading.
Line 375-378: No justification is given for why the authors looked to the first 2 minutes of the 10 minute open field test, other than the assumption that the authors are manipulating the data in order to show significance. Is there something about poultry and immediacy effects of novelty or isolation? Why are the first 2 minutes of isolation important?
Line 504: The authors mention that the age of the bird could impact behavior with no evidence to back this claim.
line 538: While this reviewer does not support anecdotal reports in scientific discussions, if this is supported by the editor, than the potential hallucinogenic effects of mCPP should be discussed as well.
Additional comments: This is an exceptionally designed study of using attention bias testing to infer negative affective state in chickens. The authors created a logical story bringing three superb experiments together to tell one concise story. The "proof of concept" objective in the use of anxiogenic drug validated future use of attention bias to assess husbandry and management practices which could impact the welfare state (i.e., result in chronic anxiety) for commercial birds. Hypotheses for each study were clearly written, and the difficulties of assessment (such as, definition of vigilance) was well-written. The limitations of this study are also given in a clear and honest fashion, such as the appetite suppressant effects of mCPP. This is a robust study with clear implications for future comparative cognitive studies of poultry affective states, as well as a tool to assess animal welfare of birds under varying housing/management conditions.


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 ATTENTION BIAS TEST TO ASSESS ANXIETY STATES IN LAYING HENS
Review round: 1
Reviewer: 2

Output:
Basic reporting: I think the manuscript has clear English used throughout. The literature cited is biased towards domestic references, although references within the topic in a slightly more behavioural ecology setting are available and would complement the current references (mainly regarding personality, and behaviour of fowl). I think the structure of the paper is clear. Raw data is not shared what I can see. Results are presented following the structure of the outlined hypotheses.

I think the manuscript “fails” on these aspects (presented in no specific order):
-There are several types of attention biases that can take place, hence the authors need to make this clearer, and also not call their test “THE attention bias test”, but “AN attention bias test”, or more honestly, “responses to playback of alarm call”.

-Regarding the claimed novelty of the use of responses to playback of alarm call. I find this not really appropriate since such a playback call has been used before in behavioural research in domestic fowl (see e.g. Favati et al 2014 Plos One, which seems relevant to this work).

-The authors have currently not described what type of vocalisations they recorded (e.g. in the variables analysed, such as latency to vocalise, number of vocalisations). Chickens can produce very many different calls with different suggested motivation (contact calls, distress calls). The authors have this information in their videos and this could clear out some confusion about the not clear responses of birds with regards to vocalisation. Also, chickens have 2 very distinctive alarm calls for different predators, and this is not clearly explained in the manuscript, nor clearly presented what was used in the study. Further, I suggest to look over and make sure it is clear why 2 alarm calls were played back to each bird, and also then what type of alarm calls. Also, it is not clear to me why a 2nd alarm call was not played if the bird did not resume eating after the first call (L433).

-I think there are too many variables presented. This is of course not a problem per see, since many aspects of behaviour were indeed recorded. My problem is more that several of the measured variables are very likely to be strongly correlated (either positively, like latency to move, latency to first step – and what are the exact biological meanings of differences in these? Or negatively, like vigilance and eating/moving). By reducing the variables that are strongly correlated will both reduce the number of results presented (which the authors currently do not make specific predictions or interpretations of, and should increase the readability of their work) and also claimed “double findings” where in fact only 1 result exists (e.g. feeding vs vigilance if strongly correlated, as they typically are in these kind of tests in chickens). Further, it is not clear to me (from the information the authors provide) how the open field is a social reinstatement test, and which response variable that capture this, in the current study. Overall are each behaviour measured not clearly justified and motivated why chosen, and how it is predicted to be affected by the various treatments.

-The use of different groups of birds make it hard to interpret results across experiments (e.g. vigilance, as defined was not observed in experiment 3). The authors do however try to be clear about this restriction of their work.

-The authors refer to ‘personality’ (which is not defined in the ms as far as I can see) and also papers claimed to look at this in the fowl. I only went through Campbell et al 2016, which is a reference for this, and that paper did not test repeatability of behaviour, hence has not investigated personality. What I suggest that the authors do, is just to rephrase (define personality if using it, not call behaviour personality if not demonstrated repeatability/consistency etc), and also to refer to some of all the literature that has described personality in the fowl (work by Favati et al, Zidar et al).

-Why temperature was measured, is not clearly set up or motivated, in the current methods/manuscript.
Experimental design: The manuscript presents work that fit within the scope of the journal. The research question is relatively well defined. It is not fully clear how the presented research fills a knowledge gap, but I believe the authors will be able to erect this. The investigation is relatively rigorous and with high technical and ethical standards (although weaker with regards to what behaviours were recorded and why). Methods are mainly described with sufficient details.

-The largest problem with the experimental design is the use of different groups across testing, which makes interpretation across the 3 experiments, hard.
Validity of the findings: The situation where it is harder to interpret the overall results because experiments were carried out on different groups of birds, is presented by the authors. There are controls within each experiment, and data has ok to low sample size (down to n=9 I think). I am a bit puzzled by the choice of non-parametric analyses of data that so clearly is not normally distributed (e.g. latencies, or categorical data with 3 categories only), but the authors have checked assumptions for the use of parametric statistics, so it should be ok. Conclusion stated are overall unclear due to the design of the study, but linked to the original research question. But from the suggested need to clear up co-varying response variables presented as separate results, I think it should be ok. I think it is clear when the authors are speculating (but from the above regarding personality).

I think the manuscript “fails” on these aspects (presented in no specific order):

-Please provide analysed sample sizes for experiment 1 and 3.

- When recording facial temperature, is body temperature not important to also record and relate to?

- I think the authors need to look over that they do provide descriptive statistics (mean +- SE or equivalent) throughout. E.g. “Result Experiment 3” lacks this (and lack test statistics for several analyses).

-There are more potential responses to a potential threat than only freeze. In general, freeze, fight or flight.
Additional comments: I enjoyed reading your work, and I think the aim to find a relatively simple test to measure fear/anxiety in domestic animals, is a great aim within animal welfare. It is nice to see that the authors aim to validate the test pharmacological.

Of other comments that the authors may want to take part of, I have these:

- I think the style of talking about ‘A’ and not ‘THE’ test is also valid for e.g. Open field test, as there are many variations on this test too. But this is a personal preference of mine, and most open field tests aim to measure relatively similar things/behaviour.

-L150: it is unclear to me what floor reared and cage-reared exactly mean.

-Experimental protocols, around L175: Is the use of the same room later used for playback of alarm call, also used for relatively negative tests (e.g. manual restrain test etc) a problem due to negative associations with the room?

-I believe Zidar & Lovlie 2012, Favati et al 2014 (Plos One), Zidar et al 2018 (BES) describe vigilance in this way in the fowl.

-L240: By excluding birds that did not finish the test within only 2 min, I think you remove relevant behavioural variation.

-Statistical analyses throughout. The authors keep repeating that they “censored the results” for birds that got max values. It is said in the exact same phrasing, and never explained. Please consider explaining why, and what consequences this has.

- For the description of the statistical analyses of the first experiment, all fixed effects are not defined (i.e. groups used = , time = ).

- There is quite a lot of overlap regarding information on housing and animal housing across the experiments, that I think the authors can reduce.

-The heading “open field testing” is one paragraph too early for experiment 2, in my mind.

- L418-19: Look over these sentences, as the feed restriction and not food deprived information is not clear to me.

- Is not recorded number over time, rate and not proportion?

-Are there problems with differences observed in already the first open field of hens exposed to 2mg/kg vs. controls, considering they both should be habituated to the arena?

-Again semantics, there is no “alarm call threat” (L502) but a playback of an alarm call, which we think should function as a threat. Further, we do not know that hens show an increased perception of the alarm call, but we assume things around the playback (L506). Please re-phrase.

-Zidar et al 2017 (Behav Proc) show that early life experiences affect vigilance in the fowl.

-L520-536: It seems to me that the temperature response to playback is similarly hard to interpret as other tests then, if both excitement and anxiety/fear can result in a drop in temperature (similar to how increase in cort can be both due to stressed and active individuals).

-I do not follow why the authors think that the use of a different reward during playback would be helping in better understand what the response of a bird means (L591-). Please develop.


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 ATTENTION BIAS TEST TO ASSESS ANXIETY STATES IN LAYING HENS
Review round: 2
Reviewer: 1

Output:
Basic reporting: No comment.
Experimental design: No comment.
Validity of the findings: No comment.
Additional comments: The authors' response to the reviewer's recommendations were sufficient and changes made to the manuscript are acceptable. No additional changes are recommended by this reviewer.


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 ATTENTION BIAS TEST TO ASSESS ANXIETY STATES IN LAYING HENS
Review round: 2
Reviewer: 2

Output:
Basic reporting: This is the 2nd time I review this ms, and I am pleased with the revision the authors have produced. I only have minor comments on the current version (see below).
Experimental design: No further comments.
Validity of the findings: No further comments.
Additional comments: My additional minor comments on your work are:
L136-: A reference more relevant to your work, is comparison of personality and coping style in fowl (Zidar et al 2017 Animal Behaviour), more than comparison with social status?
L285-: All the 3 result sections. I think it is more transparant to present Mean and SE also for non-significant result, than to only due so for signitficant ones.
L361 & 365: I would change the word significant to 'observable' here.
L365: I would clarify what the random aspect refers to.
L386: Consider re-phrasing 'frozen' since the bird did not litteraly freeze.
L408-: This section lacks test-statistcs L417, 422, 426, 428, although interactions (so both for consistency and clarity I would present test-statistics for also these).


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: BIOINFORMATICS ANALYSIS OF MICROARRAY DATA TO IDENTIFY THE CANDIDATE BIOMARKERS OF LUNG ADENOCARCINOMA
Review round: 1
Reviewer: 1

Output:
Basic reporting: This paper is nice, and has  real-world applications; I see many positive aspects in this work and would like to see it published. In fact, this paper will be of value and interest to as a significant portion of potential readers of the journal.
Experimental design: The experimental design and results seem to be much appreciable, while the authors might discuss and list the contributions of this paper.
Validity of the findings: This paper identified several differentially expressed genes by integrating four GEO 230 datasets and extracted 11 hub genes from PPI network.
Additional comments: This paper identified several differentially expressed genes by integrating four GEO 230 datasets and extracted 11 hub genes from PPI network. This paper is nice, and has mathematical real-world applications; I see many positive aspects in this work and would like to see it published. In fact, this paper will be of value and interest to as a significant portion of potential readers of the journal. In my opinion, this paper can be further improved in the following aspects:

1. The experimental results seem to be much appreciable, while the authors might discuss and list the contributions of this paper.

2. More detailed review of the literature is expected in separate section. Specially, it is required that the previous solutions to this problem be addressed. Then, the advantages (and disadvantages?) of the proposed work and should be discussed.

3. Some parts of mathematic derivations are not given in details. I suggest the authors a carefully checking and give all the necessary manipulations for the derived formulas.

4. The authors should clarify if the work requires data processing or not. More detail information should be added to make clear explanations.

5. Some latest references about Microarray and related methods should be added to give readers an up-to-date picture. In this sense, the following papers can be referred:
[a1]A novel ensemble machine learning for robust microarray data classification. Computers in Biology and Medicine. 2006 Jun 1;36(6):553-73.
[a2]Wavelet denoising algorithm based on NDOA compressed sensing for fluorescence image of Microarray, IEEE Access, vol. 7, no. 1, pp. 13338-13346, 2019.
[a3] Multilevel segmentation optimized by physical information for gridding of Microarray images, IEEE Access, vol. 7, no. 1, pp. 32146-32153, 2019.

6. Some future directions can be discussed in the conclusion part.

7. The authors still need a careful check of English, formulas and format/style.

I recommend the publication of this paper after the authors address the above concerns.


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: BIOINFORMATICS ANALYSIS OF MICROARRAY DATA TO IDENTIFY THE CANDIDATE BIOMARKERS OF LUNG ADENOCARCINOMA
Review round: 1
Reviewer: 2

Output:
Basic reporting: Clear and unambiguous, professional English used throughout (X).
Experimental design: No comment.
Validity of the findings: Conclusions are well stated, linked to original research question & limited to supporting results (X).
Additional comments: In this study, through combining integrative bioinformatics analyses, Guo et al identified 11 hub genes, among which 10 genes were shown to be related to the occurrence and development of LUAD. Finally, the authors concluded that 6 hub genes might be regarded as potential biomarkers. The study is interesting, however, in my opinion, the manuscript is not of PeerJ standard in its current version.

Other remarks:
1.	This is an integrated bioinformatics study based on the published data without any experimental validations, thus, the Title seems to be very ambiguous.
2.	The Discussion is too long. There are lots of re-description of the results, such as the Third and Fourth paragraphs.
3.	Line 213-214, the authors claim that the relation of LUAD and these identified 6 hub genes has not been reported. This description is inaccurate (Kontic et al., 2012; Stavrinou et al., 2015; Zhai and Li, 2014; Zhong et al., 2015).
4.	The figures and tables do not meet the standards for publication, especially that the font and its size should be uniform and clear. The authors should carefully revise all these figures and tables.
5.	If possible, the authors should use qRT-PCR or western blot to validate the expression of these selected hub genes, and it will be preferable to employ the gain- and loss of function studies in vitro.

References:
Kontic, M., Stojsic, J., Jovanovic, D., Bunjevacki, V., Ognjanovic, S., Kuriger, J., Puumala, S., and Nelson, H.H. (2012). Aberrant promoter methylation of CDH13 and MGMT genes is associated with clinicopathologic characteristics of primary non-small-cell lung carcinoma. Clinical lung cancer 13, 297-303.
Stavrinou, P., Mavrogiorgou, M.C., Polyzoidis, K., Kreft-Kerekes, V., Timmer, M., Marselos, M., and Pappas, P. (2015). Expression Profile of Genes Related to Drug Metabolism in Human Brain Tumors. PloS one 10, e0143285.
Zhai, X., and Li, S.J. (2014). Methylation of RASSF1A and CDH13 genes in individualized chemotherapy for patients with non-small cell lung cancer. Asian Pacific journal of cancer prevention : APJCP 15, 4925-4928.
Zhong, Y.H., Peng, H., Cheng, H.Z., and Wang, P. (2015). Quantitative assessment of the diagnostic role of CDH13 promoter methylation in lung cancer. Asian Pacific journal of cancer prevention : APJCP 16, 1139-1143.


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: BIOINFORMATICS ANALYSIS OF MICROARRAY DATA TO IDENTIFY THE CANDIDATE BIOMARKERS OF LUNG ADENOCARCINOMA
Review round: 2
Reviewer: 1

Output:
Basic reporting: Results shown that the proposed method is successful and efficient. Moreover, this paper is very well written and organized, and the contributions are well conveyed.
Experimental design: Results shown that the proposed method is successful and efficient.
Validity of the findings: This paper is very well written and organized, and the contributions are well conveyed.
Additional comments: The new version has been more clearly written and the results have been more rigorously proved. I think that the paper is suitable for publication.


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

Input:
Title: BIOINFORMATICS ANALYSIS OF MICROARRAY DATA TO IDENTIFY THE CANDIDATE BIOMARKERS OF LUNG ADENOCARCINOMA
Review round: 2
Reviewer: 2

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


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

Input:
Title: EXPLORING REGULATORY NETWORKS IN PLANTS: TRANSCRIPTION FACTORS OF STARCH METABOLISM
Review round: 1
Reviewer: 1

Output:
Basic reporting: This review was intended to focus on the transcriptional regulation of starch metabolism. To my knowledge, this topic has not been reviewed recently, and I believe a review in this area is necessary and timely. However, this particular review has many flaws (see below) which will need to be corrected before it is considered for publication.

Overall, the article is poorly structured, lacks focus, and is unnecessarily long – making it unsuitable for the general reader. For example, the authors propose that there are "master" transcriptional switches that control starch metabolism which have not been found yet. This is an idea that is not widely accepted, and the authors could have spent more of the text discussing their perspective on why they believe such specific "switches" for starch exist and how to find them, rather than spend entire sections trying to make unhelpful generalisations about plant vs animal transcription factor networks. The comparison to regulatory networks in animal cells can be dramatically shortened (the text between lines 230-232 summarises everything said between Lines 144-224, and is sufficient).

The paragraphs detailing the influence of hormones and the discovery of TFs that regulate starch metabolism (Lines 271-344), read like lists of one-sentence summaries of each cited paper rather than building a specific argument or perspective.  Moreover, each paper describes work conducted in a different species, and either in leaves or storage organs/seeds. This makes it very confusing for a general audience. Perhaps a better strategy would be to build an argument - eg: first comparing differences in starch metabolism between photosynthetic and heterotrophic tissues -> then detailing which TFs have been identified in each different tissue -> and then discussing how and why their transcriptional regulation is likely to follow different cues (eg: linked to photosynthesis or circadian clock, or by availability of incoming sucrose)? Currently, the article seems to give the reader the impression that the regulatory network is the same in all tissues (See the point about Figure 1 below), which directly contradicts the authors own conclusion that cell-specific information is necessary in the future (Lines 410-421).  I would have expected any review on transcriptional regulation of starch to make clear distinctions between leaf starch, and storage starches in endosperms or tubers.
Experimental design: no comment
Validity of the findings: See above - In my opinion, a central argument or perspective is lacking, or is not delivered clearly to the reader.
Additional comments: Other major points:

Line 18:  I have concerns about including a sentence like “We first need to find the master switches of primary metabolism” in the abstract. This implies that “master switches” exist, but there is no evidence to date that they do. Thus, if the authors believe such switches exist, it would be better to expand on that concept in the text rather than state this as a blunt fact in the abstract. A more acceptable alternative would be “We first need to identify key factors controlling these regulatory loops”

Line 31: I think there is something wrong with the wording here – are the authors referring to the cells or the organisms? – but in either case they are wrong. Plant cells are not sessile due to the fact that they respond to many external signals. They are sessile because they cannot move because they are bound together by cell walls.  What does “create their own internal environment” mean for animal cells?? That sentence implies plant cells cannot make their own internal environments, but they do in regards to pH and ion concentrations. How totipotency is relevant in this review is also unclear. I would strongly advise the authors not to start a review with such a flawed sentence.

Line 113: “participate in the starch network” is very vague. To make it more suitable to the general reader, make it clear that in the cereal endosperm, the cytosolic AGPase IS the major ADP-Glc generating activity.

Line 117:  “assembly” should be “assemble”, but also, not all starch biosynthetic enzymes assemble into HMW complexes. This generalisation should be removed. Only one example of complex formation in maize endosperm is provided, neglecting the important work by Tetlow, Emes and colleagues on complexes in other cereals.

Line 140: Since the referred papers were published, there has been vast progress in understanding new regulatory proteins involved in starch synthesis in leaves, particularly PTST proteins that form complexes with the starch synthases.

Figure 1: Firstly, this figure is far too confusing to any reader. The overlap of arrows in the central part is very difficult to read. Secondly, the figure is fundamentally flawed in that it seems to be a hybrid of the pathway of starch synthesis in heterotrophic organs with the pathway of starch synthesis and degradation pathways in leaves. Most seriously, many of the TFs indicated in this figure was shown to regulate those genes in heterotrophic tissue OR leaves, but not both! Thus it is very misleading to depict all of these things happening within a single cell. This figure needs to be split into at least two separate figures – one for the leaves and one for the heterotrophic tissues. Also: Proteins should be capitalised. AMY3 makes both linear and branched glucans. The position of SEX4 and LSF2 above the arrow implies that they directly generate linear glucans, which they don't.

Table 1: “Beta-amylase “is misspelt in the table. Check accession number for SS1 in Arabidopsis. Also, an Arabidopsis accession number is given for GBSS1, but actually, it is known that the Arabidopsis GBSS is more related to GBSS2 (Cheng et al. (2012) PLoS ONE 7(1): e30088)

Minor points:

Line 38: Zhang et al. (2012) – I can’t find this in the reference list.

Line 40: Sentences should not start with a “But”.  Rather: “However, their regulation at the transcriptional…”

Line 46: “concludes” might be better than “ends”

Line 71: Is it necessary to mention “R1”? Why not just say “Additional enzymes included GWD, PWD,….” etc?

Line 108: Unclear what is meant by “strategic position”? Maybe expand and explain it is the first committed step?

Line 121: This is the first mention of PPDK. An explanation should be included in the text to make it suitable for the general reader.

Ref list: Tetlow and Emes 2011a and 2011b appear to be the same reference???

Line 164: Genes are not built from letters. They are built from base pairs.

Line 165 – omit “somehow democratic”


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: EXPLORING REGULATORY NETWORKS IN PLANTS: TRANSCRIPTION FACTORS OF STARCH METABOLISM
Review round: 1
Reviewer: 2

Output:
Basic reporting: No comment
Experimental design: No comment
Validity of the findings: No comment
Additional comments: The review article provides with up to date literature on the regulatory network of starch metabolism. The later is used to illustrate an original point of view of the hierarchy of plant regulatory networks in comparison with animals. The authors’ conception diverge from an anthropocentric perspective and thus contributes to the understanding of plant biology. The future directions proposed in this review (i.e. single cell transcriptomics and metabolomics, swaping of promoter elements) are particularly relevant.
The following minor points should be addressed prior to publication:
- Line 62: Please do not use the word “reversible” here. Most of the stated enzymes (e.g. starch synthases, starch branching enzymes,…) do not catalyze reversible reactions.
- Figure 1: Please replace the two-directional arrow between ADP-Glc and starch by a one-directional arrow.
- Line 117: replace “assembly” by “assemble”
- Line 161-162: “Therefore, in gene networks connections are one-directional arrows that have a certain hierarchy” should be corrected as follow: “Therefore, in gene networks, connections are one-directional arrows that have a certain hierarchy”
- Line 207-208: “In comparison, plant cell ….” should be corrected as follow: “In comparison, plant cells ….”
- Line 209: “similar as in bacterial cells” should be corrected as follow: “similar to bacterial cells”
- Line 218, 219 and 220: “TF” should be corrected as follow: “Tfs”
- Line 230: “This contrast” should be corrected as follow: “This contrasts”
- Line 243: “Also some SS isoforms are affected by photoperiods” should be corrected as follow: “Also, some SS isoforms are affected by photoperiods”
- Line 244: “…, but much less is known….” should be corrected as follow: “…, much less is known….”
- Line 252: “In lentil leaves some...” should be corrected as follow: “In lentil leaves, some...”
- Line 276: “A highly...” should be corrected as follow: “The highly...”
- Line 350: “under a changing environments” should be corrected as follow: “under a changing environment”
- Line 411: “But it” should be corrected as follow: “But if”


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: POLYP EXPANSION OF PASSIVE SUSPENSION FEEDERS: A RED CORAL CASE STUDY
Review round: 1
Reviewer: 1

Output:
Basic reporting: The topic of the paper is highly interesting for biologists and ecologist studying suspension feeders. Several different approaches are applied to highlight the effect of flow speed and seston concentration on the espantion cycle of  Corallium rubrum (an high valuable octocoral) polyps. Also if authos collected a wide bulk of data, however the manuscript is confused in some parts and Results must be reconsidered and more clearly explained and Discussion must be reorganised and condensed.
Experimental design: Also if a lage amount of data have been collected  the general experimental design should be more clearly explained and  synthesized.
Validity of the findings: Some main findings are valid and important, however they must be more clearly reported,  explained and discussed. Tables should be reduced and  must be more synthetically sketched.

Some of the points of the manuscript that must be readdressed are reported in the following Comments for the author.
Additional comments: I suggest to the authors to expand the Results section and to describe in a more detailed way  the main results that must be more clearly highlighted.
The number of tables should be reduced and they must be condensed.

Some main points that should be readdressed:

Abstract

line 26 "polyp activity correlated positively with particle concentration, which was related to current speed.." -  polyp activity which was significantly (?), positivelty correlated to particle concentration, which, in turn, was singnficanly correlated to current speed ? Is this the meaning of this sentence?

lines 28-31 "....which showed differences in polyp activity.......but especially in response of nutritional stimuli and the presence of zooplankton. Zooplankton  and water moviment..." - Differences means oscillations? presence means density?  Zooplankton density? Whater speed? this sentence must be write again.

Material and methods

lines 123- 175 - This part may be condensed.

line 176-177 "..2) the polyp activity index, obtained from subsequent image differentiation. - This sentence is not clear, please describe this index in a more detailed way (formula?).

Introduction

line 54 "..is to change their feeding activity.." - to tune their feedng activity?

Results.

Results must be more exhaustively and clearly written and the number of subdivisions must be reduced (Laboratory experiments is made of 3 lines only..)

242-244 " No significant relationships were directly observed between the tested environmental variables and red coral polyp expansion" -  This is main point of the Results that must be explained and  deeply discussed in the Discussion. "Tested variables" = recorded variables.

Line 251  MDS plots of seston cmposition........show separation of sycles and sampling times" - Indicating or suggesting what?

Line 264 "..moviments increase until the colony suddently closes” – This sentence must be written in a more clear way.

Line 269 “Pairwise analyses clarified the significance of individual comparisons “ – As the Interactions in this ANOVA are significant, no further analysis can be done.

Discussion

278 “The present study provides new insights into…”  - This study try to provied some new insights into…

292 “ Our in situ results suggest complex hydrodynamics conditions act in a complementary way….”
 -  Go deeper, discuss in a more eshaustive way.

295 “polyp efficiency” – polyp activity?

323-326 –This sentence is not clear and must be written in a more plan way.

360 “…presence of naural prey..” – Density?

375 “The relationship between food pulses and feeding activity has also been studied in other ….”
– A relationship…….was also found in…

382 “ Many authors have shown the relationship between..” – Positive relationship between…

405 “ ....bring energy pulses to the bentic community which may change our view of the energy budgets of benthic communities”. – bring energy pulses to the benthic communities. (end here this sentence).

Some minor points

 Abstract
line 17 "has been linked" - Has been cosidered to be affected by....
line 22  " testing various ecological varables"  - please change in:  examining...

Material and method
line 96 C.rubrum polyp expansion was monitored at high frequency.." - by a video camera....
line 101 "Several environmantal and biological parameters..." -       The following parameters..

189 and 192 - "The differences in.. " - Variability  in...
198 "seston data" - seston density? composition?
377 “ We hypothesize…” – We consider that…

Results
246 "Polyp expansion.." - Polyp expanson rate? Percentage of expanded polyps?

Discussion
282 “even on the small scale. “ – End the line here and start the new sentence on a new line.


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: POLYP EXPANSION OF PASSIVE SUSPENSION FEEDERS: A RED CORAL CASE STUDY
Review round: 1
Reviewer: 2

Output:
Basic reporting: The manuscript prepared by Rossi and colleagues is written in professional English and easy to understand.

General:

1. What were the temperature and currents speed used in the experiment? And what are the different nutritional stimuli? I believe this is very important information, so I do not understand why this is not mentioned in the text.

2. The wording is precise though some times very specific so readers from a slightly different field might be confused. To make it easier, the terms 'seston' and/or 'flume' could be explained in the introduction.

3. I appreciate the amount of statistical detail given in the results section, however, the numbers break up the flow and make it rather difficult to follow the chain of thoughts. May be the authors would like to only highlight the key numbers of the statistics.

4. The manuscript contains a lot of figures. Figure 2 is very difficult to read in its current preparation, may be a landscape type of outline would be easier or only focus on the graphs that are really important for the understanding of the manuscript. Figures 3 and 4 are very similar though focusing on different aspects of the experiment (seston and zooplankton, respectively). Can they however be combined? For figures 5 and 6 I am missing information in the figure caption on the temperature and current conditions as well as the nutritional stimuli.

5. The tables are very repetitive and should contain more detail as mentioned for the figures. In table 4 TexCu reads as one word, Te x Cu would increase the readibility.
I believe that the caption for figure 7 is the one for figure 5.

6. The discussion is very long and some of the information is a bit confusing. I recommend to condense this section and focus on the main aspects. Further I would appreciate more clearly separated paragraphs this would increase the readibility.
A good idea might be to first discuss the two experiments separately and then have a concluding section.

Minor:
Line 166: I believe there is a typo 'coenenchyme' instead of 'coenenchima'.

Line 288: Does 'and by seawater movement' refer to the current study/manuscript?

Line 295: 'polyp efficiency' is explained later in the text as particle caputre but may be already explain it here.

Line 358: Is Anthony (1999) the only paper? Otherwise the sentence could be rephrased as followed 'The response to a chemical signal indicating increased zooplankton concentrations has not previously been experimentally tested in octocorals, but in scleractinian corals particle concentration also elicited a response (Anthony, 1999).

Line 577 and 578: May be 'significance' is a better term instead of 'significativity'
Experimental design: The experimental design as comparison of field survey and experimental observations is a great approach to validate field observations under laboratory controlled conditions.
However the description appears to be somewhat confusing to me. Therefore, I recommend to revise this section and may be add some more details.
Further, I do miss some more detailled informations about the design:

1. Can you give more information about the sampling locations, e.g. were the colonies for the experimental observation collected at the same location as the field survey was conducted? Further was the temperature also recorded as part of the environmental parameters?

2. How far apart were the groups of colonies? What type of light was used for the night observations? Could that have influenced the opening/closing of the polyps?

4. What type of axe (line 114) was used for the colony collection? This is more a 'out of curiosity question' as I haven't used an axe for coral sampling before.

5. What light conditions were used in the culture tanks and the flume?

6. Would it be possible to describe the plankton addition in more detail? I do not quite understand the different volumes used for homogenisation (line 146) and what was added.

7. The statistical analysis section is very well described!

Line 106: '..., determined  by analysing two samples...' Does this mean two samples at each group of colonies of two samples during each dive?

Lines 134-136: What does it mean 'acclimated for 30min' and 'experiments conducted from 24 to 72 hours'?
Validity of the findings: As mentioned above, the results and discussion should be revised. The authors present interesting results which could be highlighted more. The conclusions are valid with the findings however should be more precise and condense.
Some of the information given in the discussion could already be highlighted in the introduction which would specialise the paper more.
Additional comments: The manuscript 'Poylp expansion of passive suspension feeders: a red coral case study' is well written and will contribute to its respective area of research. Prior to publication, I recommend careful revision of the manuscript with some improvments to increase the readibility and to make it more attractive for researchs with other backgrounds.


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: POLYP EXPANSION OF PASSIVE SUSPENSION FEEDERS: A RED CORAL CASE STUDY
Review round: 2
Reviewer: 1

Output:
Basic reporting: The manuscript has been improved and is basically ready for publication. I only suggest some minor change. Moreover I suggest to the authors that Discussion should be condensed.
Minor changes are reported in the following.
Experimental design: This was examined in my previous revision.
Validity of the findings: This was reported in my previous revision.
Additional comments: The manuscript has been improved and is basically ready for publication. I only suggest some minor changes. Moreover I suggest to the authors to condense a little the Discussion section.

Some minor changes:

Abstract
line 25  - please, delete comma.

Material and Metods
line 113 - his sentence is not clear, do you mean that a similar,previosly described area is 40 km apart from the sampling site? If so you can delete the sentence or explain that a similar area (40 km part) was described by.....

Still water - Do you mean marine natural water or marine filtered or stelirized water or what?

161 maximum poyp expansion - Is it the maximal percentage of polyps expanded in a colony or what? Please, expain in a detalied way.

172 Polyp activity index - Please, explain more clearly this index and add a formula. You have to explain widely these indexes so that they can be appied by other researchers in the future ofcomparison....

Results

236 PEMANOVA - So what is the meaning of this result? Can you assess that an higher variation occurs between cycles or between times? Should you go deeper in this result?

239 Significant differences among temporal scales? Is there some differences between scales?

243 - OK
244 on higher water moviment?
249 - OK
254-255 ...so what? this sentence must be completed...
256   significant Te x Cu term - significant Te x Cu interaction
258-258 increase of temperature support a rapid expansion of C. r expansion - to much expansions, the first one should be: increase..
254 simultaneous effects - interactions?

Discussion
line 278 confirming its capacity to detect....- to be sensitive to food availability
280 the obtained outcomes contribute....-try to contribute..
290 Water moviment is essential to understand..- the knowledge of water moviment effects is essential to understand...
296 has been seen in studies..- was reported in ....
404 but the presence of chemical signals have...- however, chemical signals have....
409 ta may be in consonance..- that may be in phase with tydal rhythm and be also affected by storms and upwellings


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: POLYP EXPANSION OF PASSIVE SUSPENSION FEEDERS: A RED CORAL CASE STUDY
Review round: 2
Reviewer: 2

Output:
Basic reporting: The submitted version by Rossi and coauthors has been extensively revised and the reviewers comments have been answered in great detail.

However, I found a few typos and have some suggestions for rewording to increase the clarity of the manuscript:

L30: '...stimulus consisting in zooplankton'. My suggestion would be '...of zooplankton'
L53: Please replace 'suspension-feeders' by 'suspension feeders'
L77: I would suggest '...using the red coral...'

L139: Replace pending with until '... and stored at -20°C until the experimental setup'
L145: '... uniform mass of the zooplankton'
L145-146: 'The homogenate was filtered through a 10µm mesh to remove the solid part and ...' might be eaiser to undestand
L150: Please use ground instead of grinded
L169: Do you mean calculation when saying computation?
L181: '... the software also allowed to measure...'
L187: '... was assessed...'
L212: '.... we performed another experiment...'
L242: '... pairwise analyses revealed...'

L260: May be rephrase to if it's correct 'This rhythm was most dominant when the colony's polyps are fully extended as could be observed in the records of individual polyp activity'
L272: '...the analyses showed...'

L322: '...with the polyp activity being lower...'
L324: '...current speed...'
L325: Above which temperature did the oxygen consumption decrease, 20°C?
L378-382: Can you please break this phrase into two. This would greatly increase the understanding of this sentence.
Experimental design: The experimental design and descripton of methods was revised according to reviewers comments.
Validity of the findings: I appreciate the revisions on the discussion part of this manuscript. The new version is more readable and easier to follow.

I do have one more general question: How can you differentiate between opening for food and opening for gas exchange? (L342-344)
Additional comments: Rossi and coauthors made great improvements on the manuscript. I appreciate the great detail with which reviwers' suggestions and comments were answered.


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

Input:
Title: A NEW COMPREHENSIVE EYE-TRACKING TEST BATTERY CONCURRENTLY EVALUATING THE PUPIL LABS GLASSES AND THE EYELINK 1000
Review round: 1
Reviewer: 1

Output:
Basic reporting: See general comments
Experimental design: See general comments
Validity of the findings: See general comments
Additional comments: In this paper, Ehinger et al. introduce a test battery in order to compare different eye trackers with one another. They then demonstrate the applicability of this battery in a comparison between the EyeLink 1000 and the Pupil Labs Glasses. Overall I am positive regarding this manuscript. There aren’t many eye-tracker tests in the literature, and any efforts to improve this are welcome. However, I also have some critical comments, that I believe should be addressed in order to make this manuscript publishable. While there are many technical details throughout the manuscript (which I generally applaud), it is often unclear what problem these techniques solve (which should be stated up front). In other cases, there is information that doesn’t add anything, and quite a few repetitions occur. Addressing these issues may shorten the methods/results section, which is quite lengthy at present. Second, I find the framing of this test battery lacking in terms of its motivation and its comparison to previous work. I outline all my comments below. Given that it is a long paper, and I have many comments, I will note them as they first appear in the paper. As such, the comments are not ordered by importance. I will explicitly mention when points are essential to me.

Introduction p.1 line 44: “a single index will not be sufficient to characterize the suitability of an eye tracker for a specific application, but a more comprehensive test is needed.” I disagree. A single index can be sufficient, it just depends on the application. And each application may require a different measure to address suitability. Please nuance: a comprehensive test can inform suitability for more applications.

Introduction p.2. The paragraph from line 48-66 is unnecessary. Instead, I urge the authors to explain the different metrics they investigate, and highlight with example from the eye-tracking literature where they are important. For example, high accuracy may be important for reading research, high precision for microsaccade research, robustness to head movement for infant research, etc.

ESSENTIAL: No clear definitions of fixations, saccades, smooth pursuit, etc. are given. Upon introducing the different eye-movement terms such as fixation, saccade or smooth pursuit, the authors should refer to:
Hessels, R. S., Niehorster, D. C., Nyström, M., Andersson, R., & Hooge, I. T. C. (2018). Is the eye-movement field confused about fixations and saccades? A survey among 124 researchers. Royal Society Open Science, 5(180502), 1–23.
In this paper, semantic disagreement among eye-movement researchers is characterized and the different ways of defining eye movements are discussed. I believe it is essential that the authors give clear definitions as well, as it directly impacts on their work. For example, in the Introduction p.2, lines 67-70, the authors write “It is clear that such a range of paradigms … that types in a controlled manner.”
I agree with the second sentence in this short paragraph. However, I disagree with the first. What is considered by researchers as a specific “eye movement” may differ depending on the research setting as is shown by Hessels et al. (2018).
As a second example, a few lines later (73), the authors write “even when participants think they fixate …”. Here what is considered a fixation is of vital importance. There are many examples in the literature of where fixation is considered to happen when participants follow the instruction to fixate, as such drift etc. are part of the fixation as “fixational eye movements”. This should be clarified.

Introduction p.2 lines 86-88. These sentences are unclear. In my experience, the pupil labs (~1000 euro) and the EyeLink 1000 (~30.000+ euro) can vary much more than a factor 15, or is that not what is meant? I also don’t which “features” the authors refer to in the last sentence.

IMPORTANT: Introduction p.2 lines 89-90. Please acknowledge the work that IS available on the performance of eye trackers from independent researchers. These are, at least:
Blignaut, P., & Wium, D. (2013). Eye-tracking data quality as affected by ethnicity and experimental design. Behavior Research Methods, 46(1), 67–80. http://doi.org/10.3758/s13428-013-0343-0

Hessels, R. S., Cornelissen, T. H. W., Kemner, C., & Hooge, I. T. C. (2015). Qualitative tests of remote eyetracker recovery and performance during head rotation. Behavior Research Methods, 47(3), 848–859. http://doi.org/10.3758/s13428-014-0507-6

Niehorster, D. C., Cornelissen, T. H. W., Holmqvist, K., Hooge, I. T. C., & Hessels, R. S. (2018). What to expect from your remote eye-tracker when participants are unrestrained. Behavior Research Methods, 50(1), 213–227. http://doi.org/10.3758/s13428-017-0863-0
The latter two also make the explicit point that accuracy and precision values are not sufficient to characterize eye trackers for e.g. infant research in their case and compare robustness to movement of many eye trackers. Niehorster et al., 2018 also test the EyeLink 1000 in explicitly during yaw and roll movements, and the findings of the manuscript should therefore be directly compared to the findings in Niehorster at al.

Introduction p.2 line 69. “Calibration decay” and Introduction p.2. line 97. “Decay of accuracy”.
Please define decay of accuracy here. There is also empirical work on the decrease of accuracy over time that is missing here:
Nyström, M., Andersson, R., Holmqvist, K., & van de Weijer, J. (2013). The influence of calibration method and eye physiology on eyetracking data quality. Behavior Research Methods, 45(1), 272–288. http://doi.org/10.3758/s13428-012-0247-4

Methods p.3 line 114. “Section 1”, should be supplementary materials I presume?

Methods p.4 line 143. Round trip delays are noted to be <1 ms. I assume that the authors do not claim that a gaze coordinate is calculated from the video images within the system below 1 ms? Please clarify what this sentence adds, and what problem is solved.

Methods p.4 lined 152-153: change author names to initials, write out < and > signs, e.g. “with more than 5 years experience”

Methods p.4 line 162 “in their own time” -> “at their own pace”

ESSENTIAL: Methods p.4 line 164. “each task either measures attributes of the eye-tracking devices (e.g. accuracy) …”.
This is wrong. Accuracy is an interaction between at least the eye tracker, the participant and the operator, and the environment (see e.g. Nyström et al., 2013 mentioned above).

Methods p.4 line 168-169. Sentence on task sequence is redundant, please remove.

Methods p.5 line 180-181. “the eye movement classification algorithms for event detection”. How do these differ? See also Hessels et al., 2018 for a discussion on the problems inherent in the term “detection”, and the preference for using “classification”. Please reconsider all mentions of “classification” and “detection”.

Methods p.5 line 185. “Maximal manufacturer filter settings”. Make this explicit. What filters and what settings?

IMPORTANT: Methods p.6 lines 195-200. The gaze coordinates of the Pupil Labs are mapped to a world-centered coordinate system. In combination with the fact that the participant does not move around, this means that you consider the Pupil Labs as a remote-like eye tracker here. Please make this explicit (saying that it is a lab-setup is not explicit enough). In this manner, it doesn’t differ much from e.g. the EyeLink 2 head-mounted eye-tracker, which also reports gaze in screen coordinates. Nor does it mean that your findings will generalize to situations including locomotion.

Methods p.6 section Blink detection. From this section it seems that the authors are not only comparing eye trackers, but also blink detection methods. Why do the authors not conduct the blink detection for the EyeLink data themselves as well?

Methods p.7-8 lines 254-279. This section is unclear. Moreover, it is unclear what problems are being solved. Please revise: start with the problem, why it is important for the measures, and how it is solved. Optionally, this could be moved to an appendix.

Methods p.8 line 282. “Actual target point”, or “Instructed target …”

Methods p.8 line 293. Please add a link to the reference for Holmqvist 2017. It is not included in the reference list.

Methods p.8 line 297. Spatial precision is often noted as the variable error in the gaze coordinate signal. This seems a better description than “consistency of samples”. What is meant by consistency?

Methods p.9 line 306. “Task 2.3.9” doesn’t exist, it is a section.

Methods p.9 line 318. “The task sequence…”. This is the third iteration, please remove.

Methods p.9 line 337. “Fixation were manually accepted”. Calibration points were manually accepted I assume? Fixations doesn’t seem like the right word, unless the Pupil Labs software returns a set of “fixations” from the eye-tracking data that the user can choose from.

IMPORTANT: In many cases, dashes (-) are missing in the writing. This is not grammatically correct, and impedes the reading of the manuscript. E.g. “13 point verification” -> “13-point verification” or “validation accuracy limit” -> “validation-accuracy limit”. This occurs in many locations. Please revise the manuscript critically for such instances.

Methods p.10. I like the randomization process!

IMPORTANT: Methods p.10 line 393. “It [smooth pursuit] is especially common while we move relative to a fixated object”. This is in stark contrast with the views held by many researchers, who hold that an object needs to move with respect to the world and continuously looked at before it is considered smooth pursuit. Movement of oneself with respect to the world is not considered smooth pursuit by many researchers. This is worked out in Hessels et al., 2018. This again emphasizes the importance of giving definitions, or mentioning where researchers are divided on the definitions.

Methods p.10 line 394. “Smooth pursuit detection is still in its infancy”. Please cite the works that HAVE done this, at least:
Larsson, L., Nyström, M., Andersson, R., & Stridh, M. (2015). Detection of fixations and smooth pursuit movements in high-speed eye-tracking data. Biomedical Signal Processing and Control, 18, 145–152. http://doi.org/10.1016/j.bspc.2014.12.008

Methods p.11 section “Measures” starting from line 410. It is unclear why this method is used. Please first describe the problem, and then how it is solved.

Methods p.11 line 431. Remove “a thesis evaluating … EyeLink 1000”

Methods p.11 line 445-446, please cite the relevant study (it is mentioned before).

Methods p.11 line 448. “the Engbert algorithm” -> “the Engbert & Mergenthaler (2006) algorithm”.

Methods p.11 line 453-454. Is there a specific reason? If not, the sentence doesn’t add much.

Methods p.12 line 474 “8 different”, shouldn’t that be 7?

IMPORTANT: In the Results section, every subsection starts with a paragraph repeating the Methods section. This unnecessarily lengthens the manuscript. Please remove, shorten or revise.

Results p.16 line 531. What does the “(4)” refer to?

Results p.16 line 569. How can it be that the difference is -0.001s while the means are 0.004 apart? Maybe I missed something.

Results p.19 line 606. “Significant shortcomings”. Who/what is the standard here? Keeping the sentence neutral (i.e. not using “shortcoming”) is already informative enough.

Results p.19 line 611. “The eye never stays still”, please define the reference frame. With respect to what?

IMPORTANT: Results p.20 line 623. “Pupil Labs has problems identifying microsaccades”. This is wrong. The pupil labs doesn’t do this, the researchers do this using some algorithm. “It is not possible or problematic to classify microsaccades in pupil labs data” would be an accurate description. There are many such instances throughout the manuscript. Please revise such that the Pupil Labs and the EyeLink 1000 are not the acting agent. See e.g. also line 704.

Results p.21 line 643. “Blinks were detected correctly”. Please specify the benchmark here. Is that the EyeLink 1000? Or the authors?

Results p.21 lines 645-647 are unnecessary.

Results p.21 Figure 12. The legend for plot D is not complete. I do not understand what the two different lines refer to.

Results p.21 lines 654-657 are unclear.

Results p.21 line 658. “For this reason”, please explicitly mention the reason, as it is the start of a new paragraph.

Results p.22 line 662-663. “In order to investigate eye movements in more natural paradigms”. What is meant by natural? There are many lab-based studies that have to deal with participant movement, e.g. research with infants or certain patient groups, see Hessels et al., 2015, Niehorster et al., 2018 as mentioned above.

Results p.22 line 665, aren’t there 5 different target positions?

Results p.22 Figure 13. Define systematic drift here, and how it different from a constant offset.

Results p.23 line 688. What are “Biases”? How do they differ from constant offsets or systematic drifts. Please carefully define these, or revise the terminology. To me, it is an offset, not a bias.

Discussion p.24 line 717. “Accuracy is the dominant metric …”. Says who? and why?

Discussion throughout: please replace the Barsingerhorn thesis citation with the actual papers, e.g. at line 724 is should be:
Barsingerhorn, A. D., Boonstra, F. N., & Goossens, J. (2018). Development and validation of a high-speed stereoscopic eyetracker. Behavior Research Methods. http://doi.org/10.3758/s13428-018-1026-7

Discussion p.24 line 732-733. The following paper gives a much better simulation and provides code for running your own simulation for AOI size:
Orquin, J. L., & Holmqvist, K. (2017). Threats to the validity of eye-movement research in psychology, Behavior Research Methods. http://doi.org/10.3758/s13428-017-0998-z

IMPORTANT: Discussion p.25 line 757. “Our main motivation for this study was to…” This does not seem to match the content of the manuscript. The authors do not determine whether different experimental paradigms have different requirements, they assume this. That is what the development of their test battery is predicated on. Please rephrase this carefully, and make sure that the introduction and discussion match in their description of the goal of the manuscript: introducing the test battery and giving a proof of concept by comparing two eye trackers.

Discussion p.25 line 769. The word “indebted” in the sentence seems grammatically incorrect.

Discussion p.25 line 771, please cite these recent exception, e.g. Larsson et al., 2015.

Discussion p.25 line 771-772, this sentence is unclear. I do not see how the catch-up saccades are linked to the first part of the sentence. Please revise.

Discussion throughout. The authors talk about “mobile settings”. In many fields of eye-tracking research, an eye tracker is considered “mobile” if it can easily be moved around labs. Please define this carefully when first mentioning mobile settings, and explain that “mobile settings” refers to situations with wearable mobile eye trackers (glasses) where the participants moves with respect to the world.

Discussion p.25 line 793-796, relate these findings to Niehorster et al., 2018 for the EyeLink 1000.

IMPORTANT: Discussion p.26 line 798. “This study allows researchers to plan and select the eye-tracking equipment according to the design of their studies”. This is incorrect. Only if the choice is between an EyeLink 1000 and a Pupil Labs in “remote” mode would this be possible based on the present manuscript. This manuscript present researchers with a test battery in order to test their eye trackers on a range of measures. Please revise.

Discussion p.26 line 809. “Due to uncontrolled pupil dilation changes”. What is meant by this exactly?

Discussion p.26 line 821. As stated before, there are more smooth-pursuit classification algorithms than only the Pekkanen & Lappi algorithm.

Discussion p.26 line 828. The authors here describe the use of added depth calibration or recording. Please note that there is quite some discussion on whether this is reliably possible at all with video-based pupil-cr eye trackers, see e.g.:
Hooge, I. T. C., Hessels, R. S., & Nyström, M. (2019). Do pupil-based binocular video eye trackers reliably measure vergence? Vision Research, 156, 1–9. http://doi.org/10.1016/j.visres.2019.01.004

Discussion p.26 line 848-849 / p.27 line 852. Please write out the two problems. Not “Blink detection: …”. E.g. “First, blink detection was a problem …”

Discussion p.27 line 854. “poor fusion of the eyes”. By whom? By the participant or the eye tracker. This part can easily be misunderstood by readers, please revise.

Discussion p.27 line 855. I did not see any standard deviation precision measures, only RMS.

Discussion p.27 line 865. Please add that the Pupil Labs in “remote” mode is what is meant here. When participants walk around, it may be very different.

Discussion p.27 line 875. How do you know the performance will certainly be worse? Please nuance. “It may be worse” or “Is likely to be worse”.

Discussion p.27 line 878. “Determining the detection algorithms…” This sentence does not make sense to me.

Discussion p.27 line 880-881. The statement about SMI and EyeLink being the best eye trackers needs to be backed up by facts or stated as an opinion. In many situations (e.g. when head movement can occur), they are NOT the best, and this is based on facts. E.g. Hessels et al., 2015 / Niehorster et al., 2018.

Discussion p.27 line 884. Please clarify how the algorithm output affects precision. It is usually considered to be exactly the opposite. See e.g.
Holmqvist, K., Nyström, M., & Mulvey, F. (2012). Eye tracker data quality: What it is and how to measure it. Proceedings of the Symposium on Eye Tracking Research and Applications - ETRA '12, 45. http://doi.org/10.1145/2168556.2168563


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

Input:
Title: A NEW COMPREHENSIVE EYE-TRACKING TEST BATTERY CONCURRENTLY EVALUATING THE PUPIL LABS GLASSES AND THE EYELINK 1000
Review round: 1
Reviewer: 2

Output:
Basic reporting: Important citations are missing.
Experimental design: Nothing to object against
Validity of the findings: No comment
Additional comments: Review of A new comprehensive Eye-Tracking Test Battery 1 concurrently evaluating the Pupil Labs Glasses and the EyeLink 1000 (Benedikt Ehinger, Katharina Groß, Inga Ibs, Peter König )

This is an interesting paper that has collected a number of paradigms into a test battery, and then evaluate the battery on co-recorded EyeLink and Pupil lab eye-trackers. I think the test battery is an interesting proposal, that gives this paper its major merit. I recommend publication, but would like to see a revision based on the comments I provide below.

Few studies have been made that co-record participants with two eye-trackers in order to test data quality. Surprisingly, this manuscript neither cites Jan Drewes co-measurements of the EyeLink with coils, or the co-recording comparison of the SMI RED with the EyeTribe by Agnes Scholz and colleagues. Co-recording with two VOGs is particularly tricky, as the infrared illumination of the two systems onto the same eye may interfere with the measurement of gaze in both of them.

p2, l76-78: I strongly disagree with the implied statement that the EyeLink is a gold standard eye-tracker. The only currently existing gold standards in eye-tracking are scleral search coils and the DPI. See Collewijn 1998 for the original term. The EyeLink is good for a video-based eye-tracker, but only marginally better than the SMI HiSpeed or the Tobii Spectrum. All these three VOGs have decent precision and accuracy, but they have a large number of artefacts that have been reported in literature: The accuracy errors due to varying pupil dilation, the PSOs, the erroneous measurement of saccade amplitudes below 1.5°, and of course artefacts due to movement of the participant. I do not mind using the EyeLink as baseline in a comparison, but it must be done with full acknowledgment of its weaknesses.

The proposed battery of tests is the first I have seen. I think the considerations here constitute the real merits of this paper. I have several comments, though:

Figure 4: Doubling the sampling frequency by interlacing left and right signals is also done in the LC Technology eye-tracker and in the Tobii Glasses 2. The Tobii glasses have the same problem as shown in C.

Task 6 the Pupil dilation task: When you were making this manipulation, why did you not check whether the accuracy of the gaze position is affected by the altered pupil dilation, like Jan Drewes and others have done? I think it should be part of this task.

Why do you not make a PSD-analysis (Power Spectral Density) analysis of the fixation task? A peak at 90Hz could indicate that the system is good enough to measure tremor. Ko, Poletti and Rucci 2016 made that comparison on the coil vs DPI.

Task 4: I showed in my talk at the Scandinavian Workshop on Applied Eye-Tracking 2018 that the EyeLink (and 10 other VOGs) mis-measure the amplitudes of small saccades (including micro-saccades). The paper is in review. I find your co-measurement very interesting, and I predict that if you make a item-by-item comparison between EyeLink vs Pupil Lab microsaccade amplitudes, you will find a large variation, as the two mis-measure different microsaccades in different directions (which I take your figure 10 C and D to confirm?).

lines 531-534: Yes, the relationship between sampling frequency and RMS is complex. However, RMS can be compared between systems of different sampling frequencies. What we used to think was an effect of sampling frequency was in fact an interaction between downsampling and the colour of noise (which changes with filtering). This was my talk at ECEM 2017. The paper is in review. My point is that you can exclude sampling frequency as an explanation. The binocular interlacing must be really bad for RMS and is likely the main explanation.

Note that the EyeLink is filtered by default (unless you turned off filters). It can be considered unfair to compare precision between a filtering and a non-filtering eye-tracker. Filters reduce RMS, but have no large effect on STD, which often confuses comparisons between eye-trackers, which find, just like you, that the two measures seem to tell a different story.

Task 8/9: I am quite confused that you compare the effect of head movements on eye movement data in a head mounted eye-tracker vs a remote system. From the view-point of the eye-camera in each system, the same head yaw or roll movement looks very different. It is not really a fair comparison, as you are not testing the same thing in both eye-trackers.

Line 721-729: A major difference between your study and Holmqvist 2017 appears to be that while you made conditions ideal, Holmqvist 2017 recorded with many sub-optimal participants wearing all kinds of glasses, lenses and make-up. Noone was rejected.  This is again relevant on line 869.

Line 732: Wrong citation. You should cite Orquin and Holmqvist 2018

Line 783-788: It is very optimistic to think that Pupil Labs can be used to study microsaccades. I think your Figure 10 C and D hint that for measurement of small saccades, both eye-trackers have issues (which my SWAET talk from 2018 also shows).

Line 794: Why do you think that head roll is less common in mobile settings? I would think the opposite is true.

Line 809: Incomplete sentence about the pupil

Line 816: I think your test battery is a good one. But have you really proven reliability? And I do not think the battery is exhaustive. I know of several tests that you have not included (some referred to above, such as the effect of pupil dilation on gaze accuracy).


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

Input:
Title: A NEW COMPREHENSIVE EYE-TRACKING TEST BATTERY CONCURRENTLY EVALUATING THE PUPIL LABS GLASSES AND THE EYELINK 1000
Review round: 2
Reviewer: 1

Output:
Basic reporting: See general comments
Experimental design: See general comments
Validity of the findings: See general comments
Additional comments: The authors have addressed all of my concerns. I have no further objections. I'd be happy to see this paper in print.

One thing that might be amended before publication is that the abstract and Figure 3 still refer to "detection" whereas the rest of the manuscript now states "classification", but this  a minor detail and I trust the authors to catch this in e.g. the proofs.


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

Input:
Title: A NEW COMPREHENSIVE EYE-TRACKING TEST BATTERY CONCURRENTLY EVALUATING THE PUPIL LABS GLASSES AND THE EYELINK 1000
Review round: 2
Reviewer: 2

Output:
Basic reporting: This updated version of the manuscript leaves very little to desire, and I am happy to recommend publication if the authors make a few minor additions and corrections.
Experimental design: I am happy with this version.
Validity of the findings: I am happy with this version.
Additional comments: My minor suggestions:

1) On page 19, figure 9, there are histograms that compare saccade amplitudes and fixation durations between the two eye-trackers (9F and 9G). This is a very coarse method for comparison of events. Recent literature, for instance

Zemblys, R., Niehorster, D. C., & Holmqvist, K. (2018). gazeNet: End-to-end eye-movement event detection with deep neural networks. Behavior research methods, 1-25.

discuss several recent methods for event-on-event comparisons between algorithms. I do not expect you to carry out the analysis in Zemblys et al, but it would be valuable to point out that these methods exist.

2) On page 28, line 828, there is a missing citation, to what I think might be a paper I have in review - entitled "Small eye-movements cannot be reliably measured by current video-based P-CR eye-trackers". I would recommend you to keep the sentence questioning the assumption that the EyeLink can measure microsaccades adequately, which my paper show that it cannot. You may not want to cite a paper in review, though.


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: BIOSHAKE: A HASKELL EDSL FOR BIOINFORMATICS WORKFLOWS
Review round: 1
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: Attached annotated PDF contains general comments and addresses grammatical issues.

I really like the idea of leveraging a strong type system to validate workflows during construction, nicely done!

I didn't see it mentioned anywhere, but does Bioshake support any sort of dynamic workflow structure?  E.g. if you wanted to run one tool per chromosome, but the number of chromosomes changes depending on the input, can those jobs be generated dynamically or does the entire graph have to be compiled statically prior to execution?

You mention, "It is feasible to compile a specification to a different backend instead of Shake, such as Toil [...]." Do you have plans to do this or is this an area of active work? This would get around the issue of Bioshake lacking any type of cloud support.

I think the text needs to be tightened up a bit before submission. Both to address small grammatical issues as well as to avoid repetition between sections. I like the bullet points in the background for succinctly describing the advantages of your tool, but that information is essentially repeated later on without much more additional information presented to the reader.


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: BIOSHAKE: A HASKELL EDSL FOR BIOINFORMATICS WORKFLOWS
Review round: 1
Reviewer: 2

Output:
Basic reporting: Please make the following changes:

Line 14:"Bioshake is an embedded domain specific language embedded in Haskell" -> "Bioshake is a domain specific language embedded in Haskell"

Line 78: Dependencies in shake are specified in an extrinsic manner "called internal/external" -> "called implicit"
Experimental design: I also request you make the following changes/enhancements:
- You need to briefly define the term "template Haskell". Not many readers outside the Haskell community would know what that means.
- You should address the most popular DSL frameworks in your Table 1, which according to this recent poll by Albert Vilella https://docs.google.com/spreadsheets/d/1plkAsT_S3CzSeb7ivxyjRnHyrK3JclUCXeUMf_azraY/ are NextFlow and Snakemake by a huge margin. Ruffus is now seldom used and should be removed.
- The bioshake github repository uses git-lfs to store the sequence files. If you try to simply clone the repo without having git-lfs installed, the pipeline breaks mysteriously. 99.999% of git users do not have git-lfs installed. Once that is fixed, the example runs without errors but Platypus produces a VCF that has a header but no entries. Please warn the user they will need git-lfs and come up with some example files that actually produce a variant call.
Validity of the findings: Bioshake does implement some significant advances over the state-of-the-art. Bioshake introduces a strong typing mechanism, inference, and support for the EDAM ontology. It is also the first Haskell-based DSL. Haskell is a fast and perhaps underrated functional language in the bioinformatics community. The advanced type system offered by Haskell is leveraged in Bioshake to implement compile-time validation of workflows, which is novel for frameworks.

While these are impressive accomplishments, I don't want the manuscript to oversell these features so much that they confuse the reader. Please add the following disclaimers:
- Put a disclaimer that configuration-based workflow languages CWL, do have some primitive mechanisms for matching inputs and outputs between tasks, although these are accomplished using file suffix patterns and not a formal language type convention as implemented in Bioshake. You can then explain how your system allows for hierarchy and faceting (such as "sorted").
- Put a brief disclaimer that Bioshake does not actually check the file output contents for conformance to these types. It doesn't actually parse SAM files to make sure they are sorted. (This might seem obvious to you but I think you might give this impression to some readers.)
- Mention that CWL support EDAM entities in tool definitions, but this is mostly for semantic annotation, not type-checking
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: BIOSHAKE: A HASKELL EDSL FOR BIOINFORMATICS WORKFLOWS
Review round: 1
Reviewer: 3

Output:
Basic reporting: The author should be consistent with the capitalisation of "Shake" throughout the manuscript.

The difference between "compile time" and "execution time" may not be obvious to some of the target audience.  I suggest it is made clear early in the paper that "compile time" refers to the development of the pipeline and that a pipeline that passes these checks will be guaranteed free of many classes of errors that only appear the user runtime of other workflow frameworks.

The formatting of the titles for "Example 1" and "Example 2" are difficult to find on scanning the manuscript, I suggest these be in bold.

line 83: s/make/made/

line 94: replace "recurse" with "refer".  I think "recurse" necessarily implies self-referential which I don't think is the case here.

line 105: s/build/built/

line 119: s/ensure/ensures/

example after line 195, I suggest the variable "t" be replaced with a more meaning variable such as "nthreads"

line 141: I am not clear what is meant here by "only compiled if they do not already exist".  Is this referring to during the construction of workflow?

The paper includes basic examples from the point of view of a developer of a new workflow.  There should also be an example of how such a workflow would be used by a  end-user, particularly with multiple input files and requested threads.

The code near line 141 is difficult to understand - for example including an operator "&%>" that is not defined anywhere.  I'd suggest removing this, or replacing with pseudo-code.
Experimental design: Table 1 has a highlevel comparison to other similar tools/frameworks.  I recommend the author include frameworks that are more common to the intended bioinformatics audience, such as snakemake & nextflow.

I do not understand the difference between "$(is ...)" and "$(isP ...") around line 168.  Could the author clarify in the text the differences
Validity of the findings: This paper introduces Bioshake which is a useful contribution to workflow construction that allows development of workflows with less errors.
Additional comments: I commend the author on a new an interesting approach to workflow development for bioinformatics.

I think the paper would benefit from a more in-depth example of a workflow.  And it would be nice to have more than one example on the github site at : https://github.com/PapenfussLab/bioshake/tree/master/examples

I was unable to test bioshake on my laptop as the documented build instructions require Nix which I cannot install as it requires admin privileges.   I also failed to build using stackage, and have raised a corresponding issue on the github site.


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: BIOSHAKE: A HASKELL EDSL FOR BIOINFORMATICS WORKFLOWS
Review round: 2
Reviewer: 1

Output:
Basic reporting: The author's revisions addressed all of my concerns regarding comparable tools and standards. The author also made substantial improvements to the inline examples in the manuscript and source code which I feel adequately address the comments from the other reviewers.
Experimental design: This is a well-designed and documented tool.
Validity of the findings: no comment
Additional comments: Great contribution!