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: GENOME-WIDE IDENTIFICATION AND EXPRESSION PROFILE ANALYSIS OF THE HSP20 GENE FAMILY IN BARLEY (HORDEUM VULGARE L.)
Review round: 2
Reviewer: 1

Output:
Basic reporting: Pass
Experimental design: Pass
Validity of the findings: Pass
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: PREFRONTAL CORTICAL ACTIVATION MEASURED BY FNIRS DURING WALKING: EFFECTS OF AGE, DISEASE AND SECONDARY TASK
Review round: 1
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: The authors discuss the studies based on several comparisons mentioned in the Results section. However, in the Methodology I could not find any hints regarding an exact definition of comparisons. Thus, it is not completely clear what the authors mean when they write for example “11 of the 20 comparisons from 13 studies” (l. 38, l. 189). Two comparisons relate to the comparison of the complex task to the baseline and the simple task for example? Now, it is still ambiguous for me how the authors did the comparisons. I think that the understanding of the Results and the whole manuscript would improve if the authors insert additional information on comparisons in the Methodology section.

Regarding Figure 1:  Four stages were presented in the figure, but stage 1 and stage 2 were only described in the manuscript. Please provide short information to stage 3 with the final inclusion of 35 papers.
Validity of the findings: Regarding Table 1: It shows that the studies are really heterogenous regarding baseline (e.g. sitting/standing/unspecified), speed (e.g. self-selected/controlled), duration and amount of trials, treadmill vs. overground walking, montage, interoptode distance etc. Of course, it is not possible to discuss every difference in detail. Nevertheless, it is important and essential to keep in mind while reading the review – for interpreting and conclude the findings of the review and for planning further fNIRS studies. Therefore, I expect a few more sentences in the discussion and possibly in the conclusion where the heterogeneity of the state of research is mentioned.
Additional comments: General comments:
My overall impression of this systematic review is that it is well-written, good structured, and comprehensible in all parts. The research question and findings are of high relevance as it summarizes a rising number of studies about cortical activation patterns during walking. The methodological procedure is detailed and well-presented. In the discussion section, some comments should be added which are important and essential for interpreting this summary and planning further fNIRS studies. As I have no fundamental concerns regarding methodology procedure, result presentation and interpretation, I have only some additional questions and comments that might help to improve the clarity of the manuscript to get published here or elsewhere. Please see my detailed and specific comments regarding these factors below.

Specific comments:
Regarding Table 1: Be consequent in using terms like oxyHb or oxyHB (e.g. line 1, Results section).

Regarding Table 1: Please add the abbreviations in the subheading.

Regarding Table 2: Please check the information for incorrectness (e.g. Vorghese et al., 2017 used 10s standing as baseline not simple walking). The same comment applies to Table 1.

Regarding Table 3: Is there any reference for the methodological criteria? I wonder at quality criteria such as sample size. I am not really convinced because the sample size and power depend on the experimental design. Power analysis a priori with effect sizes would have been a good approach you have discussed in the study limitations. Please add that your evaluation refers only to the availability of the information, not if the studies have highly qualified experimental designs.

Regarding Table 6: Be consistent with the headings (e.g. young people in Table 2, Healthy young people in Table 6, healthy peers in Table 7).

Regarding Figure 2: The authors present the scores of the methodological quality criteria but what is the message of these data? In case you want to include this figure, I would like to know what the information report. For example, why do only 20% of the papers cover interferences with external light? Additionally, I would prefer to exclude the pie chart. On one side, it does not contain further information or clarify any results. One the other side, there are many figures and tables in the manuscript already.

Regarding Figure 3: Percentages are presented in the figure whereas numbers are presented in the manuscript. Please be consistent in the figures, tables and the manuscript.


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

Input:
Title: PREFRONTAL CORTICAL ACTIVATION MEASURED BY FNIRS DURING WALKING: EFFECTS OF AGE, DISEASE AND SECONDARY TASK
Review round: 1
Reviewer: 2

Output:
Basic reporting: There are some reviews about fNIRS on brain activity during dual-tasks; but the mains idea about this review is original. However, there are many problems that we need to point out and I’m summarizing below the main problems.
1) The main idea to perform a review is summarize the data we have on the literature. I believe that the authors did not summarize in a good way. The tables and figures are huge, with much unnecessary information, information that the authors did not discuss, etc.
2) Another problem is the way you talk about prefrontal activation. To identify an increase on activation, I think that you are talking about the increase in oxy-hemoglobin. You need to clarify it, because the fNIRS can give oxy, deoxy, total hemoglobin, TOI, etc. Nevertheless, I suggest you focus on oxy-hemoglobin changes, focus the paper on this variable.
Experimental design: Abstract
I think this section could be better. The main problem is that results and conclusion do not match. is really good. So, I have some suggestions:
- On row 24, “regarding Prefrontal cortical activation (PFC)…” I suggest “regarding Prefrontal cortical (PFC) activation”.
- Results: this abstract section is really poor. The results presented do not meet with the conclusion of the abstract section. On this results, you are just showing the quality assessment of the papers; while on the conclusions you are talking about the influence of different cognitive tasks on gait, about clinical groups, etc.
- Keywords: I believe that you need to write some keywords at the end of the abstract section.

Introduction
- I suggest a reduction in some sentences. There are many long sentences that should be divided into more sentences. An example is a sentence that starts on line 51 and finishes on line 55.
- On row 59. The fNIRS is used for investigating cortical activation just while participants are moving freely? It is confusing to me. I think that you need to redo this sentence.
- On row 66, you used PFC as Prefrontal Cortex. However, you have been used as Prefrontal cortical activation. You need to adjust it.

Methods:
- On row 99 you said that you used 4 databases. However, on figure 1 you stated 12 were identified through manual search. You wrote it at the end of the methods section. I suggest you write it at the beginning because, on figure 1, you showed that you used the manual search at the beginning of the screaming.
- On the selection criteria section, on the first stage of screaming you have 8 selections criteria. But in figure 1, the excluded motivations are completely different from what you wrote in this section. I suggest you use these 8 selections criteria in figure 1 in order to explain better the exclusion motivation. Similarly it is happening on the second stage of screaming. You need to adjust it as well.
- There are 4 different languages that you selected papers. So, at list two of each author can read Portuguese, Dutch, and French, right?
- How did you define “clinical groups with balance disorders”? I believe that at the methods section you need to give to the readers one definition about this statement.
- On row 127, you wrote: “PFC activity change was the primary outcome measured in this systematic review”. However, the fNIRS can give oxy, deoxy, total hemoglobin, TOI… so, the main outcome should be one of these. In my opinion, it can not be “PFC activity”.
- Table 1: the table is giant, lots of information and so confusing. You have much information that you did not discuss in the paper.
Validity of the findings: Results:
- This section is much longer than necessary. Many times you are writing exactly what you have on tables and figures. You need to decide if you want to show on the text or on the figures or tables. Another point is that most of the tables and figures are not clear, they are big and confusing. You need to summarize these tables and figures in a lower number of tables and figures because many information is not necessary and because you did not discuss this information.
- Table 2: this table is more clear than table 1. However, the numbers that you used to indicate the tasks performed by each study make the table not easily friend when you are looking at. And, you used “clinical group” in all studies. So, if the readers want to identify what pathological group you are talking about, the readers need to search on the paper. It is a confusing way to present the results on a table.
- Figure 2: what methodological criteria did you use to classify the papers on high, medium or low quality. You need to explain it.
- Figure 3: On row 186 you started showing “an increase, decrease or no change in PFC activation”. Are you talking about an increase, decrease or no change on oxy-hemoglobin levels? I believe that you are talking about it, but you need to make it clear. It is crucial. Another important point: These increase or decrease are statistically significant? What is the “p” value? This is another important point because if it is not, doesn’t make sense to report it. This figure has other problem that you have in figure 2: you used: “clinical group” here again.
- Table 6: again; the differences are statistically significant? What is the “p” value? And again, the numbers that you used to indicate the tasks performed by each study make the table not easily friend when you are looking at.
- Table 7: again; the differences are statistically significant? What is the “p” value?

Discussion:
- Several times on this section you just repeated what you have on results or tables or figures, sometimes on results and tables or on results and figures. You need to discuss the results training to understand why you have these results. You need to avoid using “may, might” to justify the results. You need to make your ideas more strong using more papers not included in the 35 selected papers.
- Also, you need to discuss trying to understand if the papers’ results are related to the participants’ age, sample size, etc.
- On row 325, you wrote, “However, increased hemodynamic responses in the PFC appear to be task-specific”. But, how do you classify easy and difficult tasks? For example, why did you consider “crossing obstacles” as an easy task? And, you did not differ cognitive tasks than motor tasks on this statement. To make a conclusion like this one, you need to discuss better the results you have, to show differences between easy and difficult tasks, etc.
- On row 333, you stated, “secondary tasks involving working memory might also involve cortical areas other than the PFC that were not investigated in the published studies”. But if it is involving other cortical areas doesn’t mean that PFC is not involving; and, there are many studies using working memory to analyze differences on PFC activation.
- On row 343; “the PFC was more activated…” compared to what? Single task, young people?
- On row 350, “As with young adults, increased activation may occur in other cortical areas that process visual-spatial stimuli”. You need to use more references to make this statement. Using “might or may” without references supporting the statements make your statement weak.
- On row 366, I saw that you used a study with obese people. Put together in the same “clinical group”, for example, people with stroke and obese people is not a better way to discuss the results.
- On row 373, you wrote a long sentence speculating about hemodynamic responses, using “may” again without any reference.
- On row 380. You need to define what is a difficult task and why it is.

Conclusions:
- The way you presented the results and discussed the paper did not allow you to make the conclusions you did.
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: PREFRONTAL CORTICAL ACTIVATION MEASURED BY FNIRS DURING WALKING: EFFECTS OF AGE, DISEASE AND SECONDARY TASK
Review round: 1
Reviewer: 3

Output:
Basic reporting: This is an important and methodologically well performed systematic review on an important emerging research topic that will be of interest to the readers of this journal. Overall, this review of the relevant literature is thorough. It evaluates prefrontal cortical activation patterns change when people perform gait tasks of increasing complexity requiring somatosensory, motor or cognitive tasks, it compares young and old and patient groups with healthy controls, and, finally, assesses the quality of existing studies investigating prefrontal cortical activation measured by fNIRS during walking considering the effects of age, disease and secondary task(s).
The Introduction and Discussion sections are well structured, however, could be more informative. What were, for example, the expectations for the cortical activation pattern differences and/or changes and why? It is clear that such an underlying rationale cannot be derived from existing fNIRS works because of a dearth of information; however, brain cortical activation as assessed with other methodologies might be used for this purpose. This could give readers important hints leading to the underlying hypotheses driving this review. What is missing from the paper in the Discussion is a section of the paper where the findings are set in the context of existing works and where possible explanatory hypotheses are given for the observations. This information should be inserted since this will be able giving direction for future studies. A systematic review should not only provide readers a quick way to determine which papers they can trust based on for example a quality assessment, however, they should also offer readers contextual information what those papers are telling us by interpreting the findings and putting these in a context of existing works. They allow us to get a big picture of what is known in a field by doing this.
I do not see obvious issues with methodology or conclusions, but offer a few suggestions in hopes that the authors might find them helpful in revising their manuscript.

The authors should give some information what decreased/increased cortical activation related to walking is thought to represent. From other works we generally assume that decreased activity of a particular brain area may represent decreased use and, therefore, increased efficiency; see for a discussion on this the reviews by Lustig, Shah et al. (2009) and Grady (2012). Some sources use the “compensation hypothesis” as a theoretical explanatory model. In this model additional brain activity in older compared to younger adults was suggested to reflect a compensatory mechanism (compensation hypothesis) (Cabeza, Anderson et al. 2002, Reuter-Lorenz and Cappell 2008) applied to improve performance in a specific task and such over-recruitment might as well be associated with less efficient use of neural resources (Grady 2012). This relation has not only been observed between young and old adults but also between higher fit and lower fit old adults. In the current review the main focus is on the effects of age, disease and secondary task(s); however, why are other possible biasing factors such as level of fitness, physical activity, etc. currently neglected? This should be explained or discussed in the limitations of the study section.
Decreased activity of prefrontal areas are discussed as possibly resulting from a shift from controlled gait to more automatic gait or a shift from the indirect to the direct locomotor pathway, respectively (Hamacher, Herold et al. 2015). This makes it obvious to also focus on this aspect when studying fNIRS while walking and observing the seemingly contradictory findings as presented in this review. While the indirect locomotor pathway regulates gait via prefrontal cortex, premotor area, supplementary motor area, and basal ganglia, the direct pathway comprises primary motor cortex (M1), cerebellum, and spinal cord (la Fougere, Zwergal et al. 2010, Zwergal, Linn et al. 2012). Similar observations of reduced brain activity have also been reported in studies with highly skilled, professional pianists (Jancke, Shah et al. 2000) or soccer players (Naito and Hirose 2014) that both showed lower recruitment of motor areas in which they have richer sensory-motor experiences (finger or foot movements, respectively) compared to amateurs.
From the literature we can also derive behavioral differences between hemispheres regarding activation levels under different task constraints. For example in the paper of Eggenberger and colleagues, included in this review, the comparison of brain activity in the left versus the right PFC showed emerging hemispheric differences at the end of training. Such a finding needs to be discussed in the context of this review by assessing which part(s) of the PFC were actually studied in the included manuscripts (left, right, left and right) and whether the localization of the assessment together with the demographics of the investigated participants (e.g., their fitness level) may or may not account for some of the perhaps unexpected findings. According to the review by Ekkekakis (2009), prefrontal hemispheric asymmetry has not yet been investigated with fNIRS in relation to exercise. However, the observation from Eggenberger et al. seems in line with a cognitive training fMRI study that found increased prefrontal hemispheric asymmetry during dual-task conditions (Erickson, Colcombe et al. 2007). Similarly, Bergerbest, Gabrieli et al. (2009) demonstrated in their fMRI study on implicit memory (repetition priming) an initial bilateral PFC activity in older adults and left lateralized activity in younger adults. These initial activity patterns were followed by repetition related activity reductions, which were smaller for the older compared to the younger adults in the left PFC but larger in the right PFC. These latter findings would correspond with the complementary hypothesis (Colcombe, Kramer et al. 2005), proposing that in older adults bilateral brain activity or a reduction of asymmetric brain activity is not generally related to better performance in a certain task, as it is suggested by the compensation hypothesis (Cabeza, Anderson et al. 2002, Reuter-Lorenz and Cappell 2008).

Other theories that should be reflected on because some of the results of the systematic review are in line with them, are the frontal lobe hypothesis of aging (West 1996) and the cognitive reserve theory which assumes that younger adults increase brain activity by a larger degree to cope with elevated cognitive task difficulty (Stern 2009). Notably, the age-related neural modulation patterns of functional over- and under-recruitment are emerging especially within the transition from middle-aged to old adults (Kennedy, Rodrigue et al. 2015). Moreover, these functional effects of aging are mirroring age-related structural effects proposed by the “last-in-first-out” hypothesis where late maturing brain regions decline first in later life (Raz and Kennedy 2009, Tamnes, Walhovd et al. 2013, Bender, Volkle et al. 2015) and explain gait disturbances.

References used for this peer review
Bender, A. R., M. C. Volkle and N. Raz (2015). "Differential aging of cerebral white matter in middle-aged and older adults: A seven-year follow-up." Neuroimage 125: 74-83.
Bergerbest, D., J. D. Gabrieli, S. Whitfield-Gabrieli, H. Kim, G. T. Stebbins, D. A. Bennett and D. A. Fleischman (2009). "Age-associated reduction of asymmetry in prefrontal function and preservation of conceptual repetition priming." Neuroimage 45(1): 237-246.
Cabeza, R., N. D. Anderson, J. K. Locantore and A. R. McIntosh (2002). "Aging gracefully: compensatory brain activity in high-performing older adults." Neuroimage 17(3): 1394-1402.
Colcombe, S. J., A. F. Kramer, K. I. Erickson and P. Scalf (2005). "The implications of cortical recruitment and brain morphology for individual differences in inhibitory function in aging humans." Psychol Aging 20(3): 363-375.
Ekkekakis, P. (2009). "Illuminating the black box: investigating prefrontal cortical hemodynamics during exercise with near-infrared spectroscopy." J Sport Exerc Psychol 31(4): 505-553.
Erickson, K. I., S. J. Colcombe, R. Wadhwa, L. Bherer, M. S. Peterson, P. E. Scalf, J. S. Kim, M. Alvarado and A. F. Kramer (2007). "Training-induced plasticity in older adults: effects of training on hemispheric asymmetry." Neurobiol Aging 28(2): 272-283.
Grady, C. (2012). "The cognitive neuroscience of ageing." Nat Rev Neurosci 13(7): 491-505.
Hamacher, D., F. Herold, P. Wiegel, D. Hamacher and L. Schega (2015). "Brain activity during walking: A systematic review." Neurosci Biobehav Rev 57: 310-327.
Jancke, L., N. J. Shah and M. Peters (2000). "Cortical activations in primary and secondary motor areas for complex bimanual movements in professional pianists." Brain Res Cogn Brain Res 10(1-2): 177-183.
Kennedy, K. M., K. M. Rodrigue, G. N. Bischof, A. C. Hebrank, P. A. Reuter-Lorenz and D. C. Park (2015). "Age trajectories of functional activation under conditions of low and high processing demands: an adult lifespan fMRI study of the aging brain." Neuroimage 104: 21-34.
la Fougere, C., A. Zwergal, A. Rominger, S. Forster, G. Fesl, M. Dieterich, T. Brandt, M. Strupp, P. Bartenstein and K. Jahn (2010). "Real versus imagined locomotion: a [18F]-FDG PET-fMRI comparison." Neuroimage 50(4): 1589-1598.
Lustig, C., P. Shah, R. Seidler and P. A. Reuter-Lorenz (2009). "Aging, training, and the brain: a review and future directions." Neuropsychol Rev 19(4): 504-522.
Naito, E. and S. Hirose (2014). "Efficient foot motor control by Neymar's brain." Front Hum Neurosci 8: 594.
Raz, N. and K. M. Kennedy (2009). A systems approach to the aging brain: Neuroanatomic changes, their modifiers, and cognitive correlates. Imaging the Aging Brain. W. Jagust and M. D'Esposito. New York, NY, Oxford University Press: 43-70.
Reuter-Lorenz, P. A. and K. A. Cappell (2008). "Neurocognitive aging and the compensation hypothesis." Current Directions in Psychological Science 17(3): 177-182.
Stern, Y. (2009). "Cognitive reserve." Neuropsychologia 47(10): 2015-2028.
Tamnes, C. K., K. B. Walhovd, A. M. Dale, Y. Ostby, H. Grydeland, G. Richardson, L. T. Westlye, J. C. Roddey, D. J. Hagler, Jr., P. Due-Tonnessen, D. Holland, A. M. Fjell and I. Alzheimer's Disease Neuroimaging (2013). "Brain development and aging: overlapping and unique patterns of change." Neuroimage 68: 63-74.
West, R. L. (1996). "An application of prefrontal cortex function theory to cognitive aging." Psychol Bull 120(2): 272-292.
Zwergal, A., J. Linn, G. Xiong, T. Brandt, M. Strupp and K. Jahn (2012). "Aging of human supraspinal locomotor and postural control in fMRI." Neurobiol Aging 33(6): 1073-1084.
Experimental design: This is an important and methodologically well performed systematic review on an important emerging research topic that will be of interest to the readers of this journal. Overall, this review of the relevant literature is thorough.
Validity of the findings: A systematic review should not only provide readers a quick way to determine which papers they can trust based on for example a quality assessment, however, they should also offer readers contextual information what those papers are telling us by interpreting the findings and putting these in a context of existing works. They allow us to get a big picture of what is known in a field by doing this.
I do not see obvious issues with methodology or conclusions, but offer a few suggestions in hopes that the authors might find them helpful in revising their manuscript.
Additional comments: No additional comments


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

Input:
Title: PREFRONTAL CORTICAL ACTIVATION MEASURED BY FNIRS DURING WALKING: EFFECTS OF AGE, DISEASE AND SECONDARY TASK
Review round: 2
Reviewer: 1

Output:
Basic reporting: No comment
Experimental design: No comment
Validity of the findings: No comment
Additional comments: The authors did a nice job of revising their manuscript.
In Table 4: Write “Nieuwhof” not “Nieuwholf”


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: LEPTIN-A MEDIATES TRANSCRIPTION OF GENES THAT PARTICIPATE IN CENTRAL ENDOCRINE AND PHOSPHATIDYLINOSITOL SIGNALING PATHWAYS IN 72-HOUR EMBRYONIC ZEBRAFISH (DANIO RERIO)
Review round: 1
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: Please also see comments point c but in my opinion, the core findings of this study could be replicated in a biological replicate and a qPCR setting to increase validity.
Additional comments: This paper on leptin A mediated effects in Zebrafish development is a very timely and important study on the effect of leptin in a non-mammalian vertebrate as the role of leptin is not entirely clear in the non-mammalian species thus far looked at.

Specific points

1) line 119 Daneau -> Danieau

2) lines 114-124 - 2nL of 0.4mM morpholino was injected or a 1:1 solution of 0.4mM morpholino with rx leptin - also 2nL (i.e. half the concentration) or 4nL (i.e. a larger injection)?

3) As far as figure references in text are concerned I would find it helpful to reference 2a and 2b seperately as opposed to all references leading to figure 2 as a whole.

4) lines 187-198 - The heatmap contains a network of genes that follow similar trends - what exactly do you mean? I think this paragraph could benefit from some further explanation at least for non-microarray afficionados like me. From what I understand the most interesting samples are samples which show a higher intensity in morphants compared to WT (upregulation) which is reversed by the rescue (i.e. RS intensity comparable to WT) which looks like the first cluster Figure 2a and conversely samples that have a lower intensity in MO vs WT which is "reversed" in RS, i.e. again comparable intensity between RS and WT (cluster 2). Is this what you mean by "follow similar trends"? Also, what do you define as "reciprocal trends" and specifically which genes are you talking about regarding the "delta/notch" pathway and the "phototransduction pathway"?

5) lines 199-206 supplemental figure 2 (line 195) is referenced before supplemental figure 1 (line 201) - could be reversed or this paragraph could go before the heatmaps?

6) Am I missing something or is Figure 2b not referenced in the text? I think this is a highly interesting figure. How did you classify "leptin signaling genes"? What is the overlap of leptin responsive genes in zebrafish compared to other organisms? I think a very interesting analysis would be to compare overlap between a) rx zf leptin on zf sample (this study) with an in silico analysis of previously published mamalian microarrays/RNAseq of leptin responsive cells (the recent leptin responsive neuron paper from Martin Myers comes to mind) and b) with the recent microarray published by Bob Denver on xenopus leptin treatment. Are there common signalling modalities?

7) Why use one way of expressing the data in Figure 3 (including # of DEGs, p-value and gene ratio) and another to express the data for figure 4 (excluding gene ratio)? I believe that it would be easier for the reader to express the data in a consistent manner.

8) line 276/277 - please see the 2017 paper by Bob Denver on leptin treatment of Xenopus as well as a 2018 paper by Borski on tilapia.

9) lines 304-324 - You are listing a bunch of leptin target which are differentially regulated in lepa rescue but not knockdown and conclude that therefore leptin a mediates the JAK/STAT... cascades. I am not sure I follow the argument. Leptin morphants would have reduced leptin signalling which should be rescued on co-administration of rx leptin. On the example of SOCS 2, 5b and 9 - figure 2 shows that there is no or little difference between WT and MO but a reduction during rescue. However, exposure of the animal to the LepR agonist should induce SOCS (admittedly 3a/b). Why then would there be a reduction in SOCS upon co-injection of leptin and leptin morpholino and no change upon morpholino injection? While I have not carefully looked at the other targets mentioned I am concerned about the change specific to the rescue.

10) lines 356-368 - You mention side-effects of morpholino treatment, shared obsevations in leptin a morphants and conclude the argument with expression changes. What do the expression changes (ncRNA metabolism, RNA processing, mRNA decay) mean? Are these expresion changes specific to morpholino treatments? If so, is mRNA decay for example upregulated in various morphants and upregulated in these morphants as well? An expansion of this discussion would be helpful as the connection of the mentioned expression changes to the morphological fetures discussed is unclear.

General comment

I think this is a very good paper and am very interested in the results. However, personally I think the results could benefit from a clearer presentation. The authors present data on leptin knockdown compared to it's rescue (incomplete or with potential induction as the leptin concentration is not titrated) compared to WT. However, what I am missing is

a) a clear analysis as to the direction of changes. There are points where the authors discuss these such as phototransduction which appears to be differentially regulated in the same direction (up or down?) in both morphants and rescued animals compared to control. But what about genes that to me would constitute the most interesting - down or upregulated in morphants which is partially or fully mitigated in the rescue (i.e. 3 fold up in morphant compared to WT; no change, less of a change or even a reduction in the rescue compared to WT and consequent significant difference between rescue and morphant or not)? I do not see much discussion on these - why? Microarrays are semi-quantitative.

b) (also covered in point 6) What about a compare and contrast to other similar experiments? In the discussion there is a comparison to the dataset by Borski. What about Denver's dataset and possibly some mammalian datasets? While I understand that the specific treatments are different, Denver's dataset is also during development for example. Overlap of DEGs and lack thereof could be telling. The core leptin regulated immediate early genes likely overlap?

c) The core genes found in this study and also mentioned in the abstract appear to me to be hidden in the lists and GO/KEGG terms. This could be clearer, maybe with a figure of expression changes in the different treatments dedicated to these? This is the case in the expression heatmap of figure 2, but since these are taken as the key finding in this study I would find it helpful if they were highlighted in some way. Further, the core genes could warrant to be checked by qPCR as the microarray results should be replicatable in a qPCR setting.


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: LEPTIN-A MEDIATES TRANSCRIPTION OF GENES THAT PARTICIPATE IN CENTRAL ENDOCRINE AND PHOSPHATIDYLINOSITOL SIGNALING PATHWAYS IN 72-HOUR EMBRYONIC ZEBRAFISH (DANIO RERIO)
Review round: 1
Reviewer: 2

Output:
Basic reporting: This was a well written manuscript by a group that has deep (if not seminal) roots in teleost leptin research. As such, they offer novel insight into how leptin affects a teleost transcriptome. Granted that the current experiment pertains specifically to whole embryonic danio (as acknowledged), the experiment sets the stage to further explore lepa function is adult fish organs. Their findings provide specific and categorical connections between lepa and gene activity, and will likely help guide future research in the elusive topic of fish leptin(s) by.
Experimental design: This is original, primary work to my knowledge, addressing a relevant research topic that has received increasing attention over the last decade. As in past work, this group provides a rare comparative perspective that puts fish leptin function in a context with that of other vertebrates.  The general design seems sound in their directness (transcriptome of KO and Rescue groups compared to a Control group), although the techniques used are beyond this reviewer’s personal experience. That said, the methods are established and the author provides references. Specific concerns or questions are listed below.
Validity of the findings: The group openly addresses limitations of their methods or conclusions; however, this reviewer defers to assessment of the stats to someone with experience in this technique.
Additional comments: Intro:
Background info about leptin function in vertebrates generally, and fish specifically, is provided. Brief description of the experimental design good, with groups clearly defined, (KO, rescue and wild type), and their comparative value within the experiment. Hypothesis and goal clearly stated. A reader unfamiliar with this type of KO might appreciate a few details about how the technique works; this information is included more so in the Discussion, but might be better moved to the Intro. Similar brief explanation of GOs and KEGG Pathways in the Intro might help the reader follow the data.

MM:
Treatments are described in adequate detail.

Line 121. Microinjection (of MO): is there a justification/reference for the MO and lepa concentrations? I see a volume for the MO, but not the leptin treatment (1:1 = double volume of MO alone?). Is it possible to verify the extent to which lepa is KO’d with this method? (i.e. with gene KO, it can be confirmed by PCR. Perhaps southern blot for lepa expression?) Sham injection to controls? (Please consider if any of these need to be addressed)

125. Does 72 hpf coincide with a particular developmental stage? As I read it, each treatment n=4 x5 pooled embryos = 20 embryos (the numbers a little confusing). If so, this seems adequate.

153. Comparisons are nicely presented. DEG criteria is explicit and contains reference.

Results:
3.1 I suggest that Fig 1 be labelled A and B1-B3, or in such a way as to make this section easier to follow in parallel with the data presented. It is not clear which figure the # of DEGs refers to, and more detailed figure labeling and specific references would help clear this up, especially since the #s mentioned are not apparent in the graphs/diagram. This should be easy to clarify.

176. Clarify “top-5 ranked genes.” E.g. upregulated genes? DEGs? Total expression? Otherwise, the various up/downregulated genes/classes of note are well described.

187-198. This section refers to the two heatmaps, and Figure 2 is mentioned 4 times in one paragraph. This section would again benefit from an A/B distinction in Figure 2.

204 Good data check of date vs group.

208. Again, a brief description of GO would be nice, as well as what ‘enriched biologically processing terms’ means, since these are key to the analysis. The reader pieces it together eventually, but no reason not to provide a quick explanation. The terms listed seem somewhat randomly chosen from the list on the figure – maybe these are just the more interesting functions.

224. Need a figure reference somewhere in this paragraph. Presentation is otherwise consistent with that of previous data.

239. Suggestion to use either ‘wild type’ or ‘control’ throughout text to make it easier to skim for information.

250. Again, a brief description of KEGG pathway would be nice.

Discussion:
275. First study of its kind in non-mammals = great.

278-290. Nice description of MO that would be beneficial earlier in the paper (either move or include in intro).

297. Confused: Why would lepa morpholinos rescue lepa KO? Otherwise nice suggestion of developmentally distinct roles of leptin.

304-310. This section seems to address if the rescue group should look like the WT group (i.e. both expressing lepa), or else why they might in fact be different in some aspects, which could be very interesting. Should this be more explicitly discussed?

317. Kinase written 3 times

325. A welcome, but conservative/general, conclusion after much data is referenced. Consider if any further conclusion is warranted?

356-86. Be consistent between lepa and lepA. This is paragraph (along with the last paragraph in the Discussion) is a good acknowledgement of MO limitations, but that the group is confidence that the experiment functioned as expected. Could 365-68 be considered a validation of lepa KO using the MO technique (as mentioned earlier)?

Note: Fig 1 does not appear to be mentioned in the Discussion.

Conclusion:
We see the first explicit mention of metabolic and appetite (genes named, but not “appetite” specifically) regulation. These concepts should probably be mentioned in the Discussion prior to showing up in the conclusion since they are classically associated with leptin.


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: LEPTIN-A MEDIATES TRANSCRIPTION OF GENES THAT PARTICIPATE IN CENTRAL ENDOCRINE AND PHOSPHATIDYLINOSITOL SIGNALING PATHWAYS IN 72-HOUR EMBRYONIC ZEBRAFISH (DANIO RERIO)
Review round: 1
Reviewer: 3

Output:
Basic reporting: Tuttle et al. present work detailing the effects of lepa on embryonic zebrafish gene expression using a well designed combination of lepa knockdown and lepa rescue models and a microarray approach to transcriptomics. The language is largely clear and the literature review is thorough. The rationale for and structure of the article are both nicely laid out.

There are a few minor comments regarding the basic reporting.
Lines 28-32: I believe the authors are stating that leptin signaling in humans is known to regulated a number of factors whose homologues are regulated by lepa rescue in the present studies. The wording is a bit unclear though. Could you confirm this is the intended meaning of your sentence and possibly rephrase this to be more clear.
Line 57: For clarity should read "with ob/ob rodents, but not db/db".
Line 70-73: Restructure for clarity. There appears to be a comma splice here and the sentence is difficult to follow as currently punctuated.
Lines 98-99: The wording of the comparisons made is unclear. Use the phrasing from the methods as that was readily understandable.
Line 277: It seems, by the authors statements, that Douros et al. have previously performed transcriptomic analysis of altered leptin signaling in a non-mammal. This sentence should be rephrased to give the sense that this work adds to the comparative, species specific literature.
Line 346: Change vs. to verses and remove the parenthesis surrounding "even in the same species".

Additionally there are major concerns that, in my opinion, would have to be addressed before this manuscript is published.
Experimental design: Line 126: How many total embryos were used for each group? By my math the WT groups had 40 total embryos pooled to form 8 biological replicates?
Validity of the findings: Line 278: It is unclear from the data that lepa knockdown was actually achieved. Because the antisense morpholino blocks translation but not transcription the microarray data is useless to confirm the knockdown. Therefore, it is necessary to some validation that this knockdown AND rescue functionally ablate and enhance leptin protein levels in this context. A western blot, immunoassay, or mass spec approach is required.

Line 300-303: The statement that your findings highlight a role for leptin-a in phosphatidylinositol signaling pathways is not wholly warranted as there is no data suggesting that leptin actually regulates this pathway. Rather the data wholly support a role for leptin to regulate the gene transcription of proteins involved in this pathway. The distinction is critical as this manuscript offers no functional data regarding the physiologic outcomes of leptin signaling in this particular model.
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: LEPTIN-A MEDIATES TRANSCRIPTION OF GENES THAT PARTICIPATE IN CENTRAL ENDOCRINE AND PHOSPHATIDYLINOSITOL SIGNALING PATHWAYS IN 72-HOUR EMBRYONIC ZEBRAFISH (DANIO RERIO)
Review round: 2
Reviewer: 1

Output:
Basic reporting: Since this re-submission addresses my concerns I limit myself to the "general comments to the author".
Experimental design: Since this re-submission addresses my concerns I limit myself to the "general comments to the author".
Validity of the findings: Since this re-submission addresses my concerns I limit myself to the "general comments to the author".
Additional comments: The authors have presented a revised manuscript that addresses the main points I had reserves about in the first version. The current manuscript is a good read for the leptin community and will hopefully engender research into some of the pathways identified as leptin targets.

One minor point - the qPCR and the microarray - were these the same RNA samples or was the qPCR carried out on a freshly prepared set of RNA? The authors address this in line 424 and it seems to be implicit in their argument that the qPCR data also constitutes a biological replicate. However this is not clear from the manuscript. If these were technical replicates (as it seems to me many other authors use), then I would be worried.


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: LEPTIN-A MEDIATES TRANSCRIPTION OF GENES THAT PARTICIPATE IN CENTRAL ENDOCRINE AND PHOSPHATIDYLINOSITOL SIGNALING PATHWAYS IN 72-HOUR EMBRYONIC ZEBRAFISH (DANIO RERIO)
Review round: 2
Reviewer: 2

Output:
Basic reporting: See general comments.
Experimental design: See general comments.
Validity of the findings: See general comments.
Additional comments: I followed the author's rebuttal and am generally satisfied with the way my comments were addressed. As long as the editor is ok with the merit of the Liu 2012 paper that is heavily referenced for precedence in the current paper, then some of the major concerns about validation seem to be addressed. Otherwise, the manuscript reads more clearly now, in part by paraphrasing some of the material covered in Liu, as suggested (rather than requiring a parallel read of that paper), as well as by clarifying/explaining some of the technical aspects and pathways, etc., that are discussed in the current paper The figures have been completely revamped, I see, although I am not very familiar with the types of analyses in Fig 2 &3, so cannot comment on them; however, the other tables and figures look intuitive. In a nutshell, the paper now more clearly illustrates differentially expressed genes, and hence has potential to inform readers of possible pathways affected by leptin, which has potential to drive new directions of leptin research.


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: LEPTIN-A MEDIATES TRANSCRIPTION OF GENES THAT PARTICIPATE IN CENTRAL ENDOCRINE AND PHOSPHATIDYLINOSITOL SIGNALING PATHWAYS IN 72-HOUR EMBRYONIC ZEBRAFISH (DANIO RERIO)
Review round: 2
Reviewer: 3

Output:
Basic reporting: No change from previous review.
Experimental design: No change from previous review.
Validity of the findings: No change from previous review.
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: PHYLOGENOMIC ANALYSIS AND REVISED CLASSIFICATION OF ATYPOID MYGALOMORPH SPIDERS (ARANEAE, MYGALOMORPHAE), WITH NOTES ON ARACHNID ULTRACONSERVED ELEMENT LOCI
Review round: 1
Reviewer: 1

Output:
Basic reporting: Since I’m not an English native speaker, I did not make any changes regarding the grammar. The manuscript is well written, clear and technically correct.

The introduction presented a background of previous phylogenetic evidences on the study clade of Mygalomorphae and also discussed about the use of ultraconserved elements in phylogenetic analyses. Although the background is complete, the introduction could include some information about biogeographic history of the clade, or comments about the genera distributions.

Figures are appropriate and the supplementary material provided is very complete and informative.
Experimental design: The work is well-designed and the chance to analyze specimens of particular genera that were not previously studied adds a relevant feature. Methods are described with a good level of detail, just some details on general comments about this section are presented below.
Validity of the findings: The data of the manuscript which the conclusions are based are available through various repositories. The conclusions are related with the main questions of the work, and are discussed including updated references. The findings are of great interest, since some taxonomic changes at family levels are proposed in a relevant clade of spiders. This work will promote new studies to develop that could understand the evolution of some clades.
Additional comments: Introduction

Lines 65–66. Maybe you can add some references to this statement, by adding some of the works related with theraposid phylogeny (e.g. Hamilton et al. 2011, Guadanucci 2014, Ortiz et al. 2018).
Lines 73–74. Explain with more detail this statement.
Lines 80 – 82. Add some references about the morphological characterizations of Atypoids, for example, Gertsch & Platnick (1979).
Line 100. Please update the reference to “World Spider Catalog (2019)”.
Lines 130–132. This sentence is long and little confusing, try to rewrite it.

Methods

Line 137. Update the reference to 2019.
Line 139. You stated “…(species now included in Antrodiaetus)…”. I assume that this is not a result from the present work, thus include a reference.
Line 140. Which means by “traditional”? please explain better.
Line 145. Samples of Mecicobothrium, Calommata and Atypus were represented by single specimens. Could you state to which species corresponded the materials?
Line 213. The unpartitioned analysis were conducted with the GTR + gamma model. This was achieve by using the ModelFinder, as stated below?

Discussion

Lines 387–388. Add the reference of Goloboff (1993).

Figures

It would be great if authors could include a figure of a member of Mecicobothrium in Figure 1.

Figure 2. I suggest to present the different phylogenetic hypotheses from previous works in a chronological order, thus it is easy to follows.


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: PHYLOGENOMIC ANALYSIS AND REVISED CLASSIFICATION OF ATYPOID MYGALOMORPH SPIDERS (ARANEAE, MYGALOMORPHAE), WITH NOTES ON ARACHNID ULTRACONSERVED ELEMENT LOCI
Review round: 1
Reviewer: 2

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: MS# 34648 Review of Phylogenetic analysis and revised classification of atypoid mygalomorph spiders (Araneae, Mygalomorphae), with notes on arachnid ultraconserved element loci

In this manuscript, Marshal Hedin and colleagues employ UCE loci for inferring the phylogeny of atypoid mygalomorph spiders, revise the group’s taxonomy, comment on the evolution of these spiders (including divergence dating and silk use), and discuss the utility of UCE loci in arachnid phylogenomics.  The paper is clear and well researched and is an important contribution to our understanding of the systematics and evolution of the Atypoidea, a group with a contentious phylogenetic and taxonomic history.

The discussion of character evolution and divergence times are supported by rigorous analyses and do not require additional commentary.  My remarks primarily deal with the taxonomic section:

•	Mecicobothriidae is polyphyletic and requires taxonomic revision.  The authors propose to solve this by making Mecicobothriidae a monotypic family; creating two new families to accommodate Hexurella and Megahexura; and transferring Hexura to Antrodiaetidae.  While I do not disagree with any of the proposed taxonomic changes, I do wonder how the authors would have treated Hexura had it been placed sister to the other antrodiaetids rather than nested within them.  Would the authors have named a new family (“Hexuridae”) or would they have still considered it an antrodiaetid based on shared palpal bulb morphology?  I bring this point up because the classic antrodiaetids (Aliatypus + Antrodiaetus/Atypoides) can be considered adaptively/ecologically divergent (trapdoors versus collars/turrets) and I suppose it can be argued that the two lineages perhaps warrant family status on their own accord (e.g., “Aliatypidae”).  Similarly, could Megahexura – although ancient and quite distinct – not now be considered an antrodiaetid given its phylogenetic placement?  The authors should be more explicit on what they consider an atypoid spider family and discuss the factors they consider most important for family-level recognition (e.g., morphological apomorphies, adaptive divergence, age, nomenclatural stability, etc.) so that the taxonomic conclusions are repeatable.

•	The three antrodiaetid species formerly placed in Atypoides (= Antrodiaetus) form a monophyletic group sister to the “original” Antrodiaetus – and as a result, the authors resurrect Atypoides (contra Hendrixson & Bond, 2007).  This change is justified based on the inclusion of Antrodiaetus roretzi (a species spanning the root node of the genus and who shares a plesiomorphic palpal bulb morphology with Atypoides).  Given this change, the authors should also provide a revised diagnosis for Antrodiaetus and summarize the diagnostic morphological characters for the genus in Figure 6.  The authors may consider including a reference to Hendrixson & Bond (2009) who also found strong support for Atypoides (in at least some analyses) but did not call for any taxonomic changes.

•	For the sake of consistency and efficacy, the authors should consider making reference to specific figures of diagnostic features in the diagnoses for each revised taxon (as is done for Megahexuridae, e.g., Line 508).
Additional comments: No additional comments


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

Input:
Title: TRANSCRIPTOME ANALYSIS OF CHELIDONIUM MAJUS ELAIOSOMES AND SEEDS PROVIDE INSIGHTS INTO FATTY ACID BIOSYNTHESIS
Review round: 1
Reviewer: 1

Output:
Basic reporting: The manuscript "Transcriptome analysis of Chelidonium majus elaiosomes and seeds provide insights into fatty acid biosynthesis" presents reliable and interesting data on the fatty acid biosynthesis in Chelidonium majus elaiosomes and seeds using comparative transcriptomic approach. The English is professional and well used throughout the text, however the authors did not avoid some repetitions, as e.g. in line 344 the word "further" used twice "...further studies are required to further verify....".
The authors deal with the interesting issue of myrmecochory and provide sufficient background to understand the results.
Experimental design: The manuscript is within the scope of PeerJ. Research question is well-defined and fills an important gap of knowledge on the fatty acid biosynthesis in C. majus elaiosomes and seeds.
However, there is inconsistency in the description of developmental stages of C. majus between sections "Plant Material and Treatments" and "RNA isolation and Sequencing" - in line 140 it is stated that Ch04 stage is called "mature seed stage" and in line 160 the same stage is named "early mature seed stage". Please make it consistent and explain what is the background behind your division on stages (maybe the small table could help to organise it in a more comprehensible way).
Additional comments:
- the legend of Figure 1 should contain symbols of the distinguished developmental phases (Ch01, Ch02, Ch03, Ch04), which should be added to this figure legend in the relevant places,
- line 176, "fragments per kilobase of the exon per million mapped reads" should be abbreviated as "FPKM", not as "FRKM".
Validity of the findings: The data presented are reliable and the authors prove this fact in several places of the manuscript, however in some points of the manuscript inconsistencies could be found:
- in line 198 it is stated that "The results are shown in Table 1"; however when looking at Table 1 mentioned data in preceding lines (196-198) are not present therein, it contains other values, so it must be reviewed and potentially expaned to contain relevant data,
- lines 201, 208 - is the number of 63,064 unigenes found in the study the total number for all studied samples?
- lines 220-221 - the authors state that their data is reliable, and it is true, but this fact is just the simple consequence of presented data and does not have to be mentioned several times? Please consider to transfer these data (presented on Fig. 3A) to supplementary material.
- the data presented on Figures 8-10 are very complex and their description between the lines 294-356 seems to be too descriptive. Please consider preparing additional table(s) to better present the main differences between dev. stages and selected genes and enzymes, what could better support final conlusions. Figures 8 and 9 could be then considered to be included as supplementary information.

Additional points:
- line 193, "de novo" should be written in italics throughot the manuscript; the word "Assembly" should be written here beginning with small letter "assembly",
- supplementary information (lines 425-427) should contain titles of each tables, and supplementary tables themselves should contain the title and the legend on one of the panels inside the file to better present the results and make them more understandable.
Additional comments: No additional comments


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

Input:
Title: TRANSCRIPTOME ANALYSIS OF CHELIDONIUM MAJUS ELAIOSOMES AND SEEDS PROVIDE INSIGHTS INTO FATTY ACID BIOSYNTHESIS
Review round: 1
Reviewer: 2

Output:
Basic reporting: 1. Unigene is a Trinity specific term. The authors should define and describe it first. The general audiences wouldn't know what is a unigene.
2. Please check the grammar and typos throughout the text, especially the figure and table captions!
Experimental design: The experimental design is sound, carefully designed time series expression data with biological replicates, and qRT-PCR. However, not enough data is shared to ensure the result can be replicated. For example, the script for finding DEG genes; the script/tool for functional-enrichment analysis.  For those are not code, e.g., the unigenes from Trinity, the authors can also provide the program output files as supplementary files. The bottom line is to share as much as possible to ensure replicative research.
Validity of the findings: The text says saturated fatty acids and unsaturated fatty acids but figure 8 caption mentioned only unsaturated fatty acids

"The above results indicated that the biosynthesis of saturated fatty acids and unsaturated fatty acids in C. majus elaisomes and seeds primarily occurred during the flowering stage to the young seed stage (Ch02 to Ch03), consistent with the remarkable cell growth of the elaiosome observed during this period (Yang et al., 2015)."

I think this is an overstatement. The above results indicated there are more relevant DEG genes in Ch02 vs Ch03 compared to Ch01 vs Ch02 and Ch03 vs Ch04. However, whether that indicates the biosynthesis occurred primarily during Ch02 to Ch03 requires more explanations or evidence.

"Therefore, we inferred that the high palmitic acid content in C. majus elaiosomes is similar to that in the ants that disperse the C. majus seeds. However, further studies are required to further verify the findings in C. majus and the ants."

Does the Hughes et al. (1994) include C. majus and the ants? If yes, just cite their study. If not this statement is speculation without any support from the text.
Additional comments: This study provides valuable research and data to study the expression patterns of genes associated with fatty acid biosynthesis in elaisomes and seeds of C. majus. And I tend to believe it is the first study to use times series RNA-Seq to study this problem since no relative results are compared against. And I think the author should make it clear in the text. The results are significant and consistent with prior knowledge. However, I do believe to make this work more impactful, the authors should also provide
1. The Raw RNA-Seq data (can upload to SRA), and qRT-PCR result.
2. The intermediate files, such as Trinity results and analyses scripts.


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

Input:
Title: TRANSCRIPTOME ANALYSIS OF CHELIDONIUM MAJUS ELAIOSOMES AND SEEDS PROVIDE INSIGHTS INTO FATTY ACID BIOSYNTHESIS
Review round: 2
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: Hereby I would like to congratulate the authors and repeat the opinion from my previous review, that the manuscript "Transcriptome analysis of Chelidonium majus elaiosomes and seeds provide insights into fatty acid biosynthesis" presents reliable and interesting data and fills an important gap of knowledge on the fatty acid biosynthesis in C. majus elaiosomes and seeds. The authors have answered on all my questions and corrected the manuscript accordingly. Therefore I recommend to publish the manuscript in PeerJ as is.


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

Input:
Title: TRANSCRIPTOME ANALYSIS OF CHELIDONIUM MAJUS ELAIOSOMES AND SEEDS PROVIDE INSIGHTS INTO FATTY ACID BIOSYNTHESIS
Review round: 2
Reviewer: 2

Output:
Basic reporting: The new manuscript has addressed the previous comments.
Experimental design: The new manuscript has addressed the previous comments.
Validity of the findings: The new manuscript has addressed the previous comments.
Additional comments: The new manuscript has addressed the previous comments. This study is in scope with PeerJ publication interest. The result and its RNA-seq data fill in some gaps of knowledge about fatty acid biosynthesis in C. majus elaiosomes and seeds. I recommend accepting this manuscript.


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

Input:
Title: CHARACTERIZATION OF THE MITOCHONDRIAL GENOME OF ANALCELLICAMPA XANTHOSOMA GEN. ET SP. NOV. (HYMENOPTERA: TENTHREDINIDAE)
Review round: 1
Reviewer: 1

Output:
Basic reporting: An extensive language revision of the manuscript is necessary, both in terms of grammar and style of the text (e.g. "clads" [clades] (line 80), "Analcellicampa gen. nov. and Monocellicampa Wei is undoubtedly a member of Hoplocampini" (lines 90-91) [should be given in plural], "according to the manufacture’s protocols" [manufacturer’s protocol] (lines 123-124), "Host plat" [plant] and "Larvae are borer" [borers] (line 223), "epicnemium vestigiel" [vestigial] (line 231), "larvae boring leaf petiole or known" [unknown] (line 324), "which were usually used" (line 387), "condon usage" [codon usage] (line 409), "because of the present" [presence] (line 413), etc.). I therefore strongly recommend the language of the paper to be checked by a native English speaker. Moreover, all genus and species names must be given in italics (please see the first paragraph of the Conclusions section, lines 474-480, as well as the reference list), and some hyphens apparently coming from previous versions of the text (see e.g. lines 389, 400, 405 and 460) should also be removed.

Not all files can be downloaded from the location provided by the authors. I personally could not download figures 2, 6 and 7.

Some sources cited in the reference list, e.g. those by Ashmead, P. Cameron, Dierckxsens et al., Konow, Malaise, Nawrocki, Rohwer and Middleton, Ross, must be carefully checked for both correctness and completeness.

In addition, most files in the Raw data section cannot be opened with usual text processors or similar software.
Experimental design: No comment
Validity of the findings: No comment
Additional comments: The authors list the names of four outgroups used in the analysis (lines 452-453), but I would replace the latter one, Adephaga, to Coleoptera, since the other three represent various insect orders.

Overall, I believe that inaccurate submission of the manuscript (please see above} effectively prevents its further consideration by the Editorial Board of PeerJ.


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: CHARACTERIZATION OF THE MITOCHONDRIAL GENOME OF ANALCELLICAMPA XANTHOSOMA GEN. ET SP. NOV. (HYMENOPTERA: TENTHREDINIDAE)
Review round: 1
Reviewer: 2

Output:
Basic reporting: This manuscript presents the mitochondrial genome of a new species from a new described genus of Analcellicampa with a phylogenetic analysis of Symphyta based on the previously known mitogenome sequences. The authors report general features of this genome and emphasise that this mitochondrial genome shares similar characteristics with the ancestral mitochondrial genome. The phylogenetic tree recovered Analcellicampa and Monocellicampa as sister group within the family of Tenthredinidae. However, the manuscript must be reviewed by a native speaker (or a professional corrector) in order to improve the flow and clarity of text. I have tried to correct as many English mistakes as possible but unfortunately there are far too many and therefore it needs to be checked very thoroughly. Apart from the problem with the English, I have highlighted also other problems on the manuscript that should be solved or explained better. Moreover, it is essential to draw an aim paragraph in the last part of the introduction. Authors should also revise the references both in text and references list adding some missing references. There are also questions regarding the presentation of the results that the authors will have to address.
Experimental design: I think "unlinked branch lengths” should be used in the analysis with PartitionFinder. Moreover, this “linked branch lengths” preference may have led to inaccurate results of phylogenetic analyses. When considering the trees, I clearly realised this case from misplacement of pamphiloid species. They are not clustered together as a monophyletic group.
Validity of the findings: Although the findings have been discussed and supported by relevant literature sufficiently, the main problem is linguistic throughout the MS. It is not clearly presented and needs to be improved.
Additional comments: Here, the manuscript presents the characterisation of the mitochondrial genome of Analcellicampa xanthosoma for the first time. Then the authors aimed to compare the various genomic features against other reported symphytan species. The manuscript presents a newly built phylogenetic tree using the known symphytan mitochondrial genomes to verify the phylogenetic position of the species. All changes are marked in the MS and some major/minor changes or comments are also listed below:
•	Lines 85-88: “It seems that the branch of (Susana + Hoplocampa + Cladius) of Nematinae s. lat. is probably a monophyletic group, though the systematic position and the in groups of the branch are uncertain: the position of Monocellicampa, Craterocercus and Moricella is a question.” What means of “in groups of the branch”?
•	Lines 135-136: This sentence should be moved to the beginning of the paragraph.
•	Line 149-154: “The secondary structures of the rrnS and rrnL were partitioned into 4 areas and 6 areas entirely. The structures were artificially transformed to the relative secondary structure of Italian honey bee. The structural adjustment of remainders was predicted by specific structure models, which were built in SSU-ALIGN (Nawrocki, 2009) with multiple sequence alignments from MARNA (Siebert & Backofen, 2005), in view of the primary sequence and the secondary structure of above-mentioned partial species as well.” It is unclear and it would be better to rewrite considering the related literature.
•	Lines 434-435: “The rrnL and the rrnS that accounted for about 9% and 5% were essential for the translation of messenger RNAs into mitochondrial proteins.” What means of this sentence? I think this is not necessary.
•	Lines 445-446: “The number of the loop was determined by its length.” What do you mean?
•	The authors should rearrange the order of Tables, which I highlighted at the MS.
•	The figures 9 and 10 should be coloured and presented using only species name (removing accession numbers) demonstrating their family and superfamily names.


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: CHARACTERIZATION OF THE MITOCHONDRIAL GENOME OF ANALCELLICAMPA XANTHOSOMA GEN. ET SP. NOV. (HYMENOPTERA: TENTHREDINIDAE)
Review round: 2
Reviewer: 1

Output:
Basic reporting: Dear Colleagues,
I have carefully read the revised version of the manuscript by Niu et al. entitled "Characterization of the mitochondrial genome of Analcellicampa xanthosoma gen. et sp. nov. (Hymenoptera: Tenthredinidae)". As I wrote you earlier, the paper apparently contains important information on the new sawfly genus and species together with its mitochondrial genome, and therefore I believe that it could be published in PeerJ. In addition, substantial changes have been done to the recent version of the manuscript to meet some of the reviewers’ suggestions. However, certain issues related to the paper still remain unresolved.
Specifically, an extensive language revision of some parts of the manuscript, especially of the abstract and introduction, is necessary. During the previous round of the reviewing procedure, I strongly recommended a revision of that kind by a native English speaker. Nevertheless, it seems that the paper still contains many language errors, although the authors state in their rebuttal letter that they "have carefully proof-reading the manuscript to minimize the errors" (by the way, the latter phrase is grammatically incorrect as well). Moreover, section "Etymology" in the description of the genus Analcellicampa is obviously duplicated (see lines 231-232 and 233-235). Furthermore, ALL genus and species names given in the reference list must be given in italics. In addition, a few sources cited in the list must be carefully checked for both correctness and completeness (e.g. authors’ surnames in line 528 should be spelled out).
Experimental design: No comment
Validity of the findings: No comment
Additional comments: Overall, I believe that the manuscript can be published in PeerJ only after a careful major revision of all sections of the text and figures.


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: CHARACTERIZATION OF THE MITOCHONDRIAL GENOME OF ANALCELLICAMPA XANTHOSOMA GEN. ET SP. NOV. (HYMENOPTERA: TENTHREDINIDAE)
Review round: 2
Reviewer: 2

Output:
Basic reporting: I am happy to see the revised version of the manuscript about the mitogenome features and taxonomic position of A. xanthosoma. I am also glad to see that newly performed phylogenetic analyses were included. I feel that the text improved but I have highlighted some English mistakes and some modifications on the manuscript attached. Also, the manuscript must still be reviewed by a native speaker (or a professional corrector) in order to improve the flow and clarity of text and to avoid unnecessary repetitions. Moreover, as I stated before, it is essential to draw an aim paragraph in the last part of the introduction. There are also questions regarding the presentation of the results. Figure 6 seems to be problematic. I couldn’t see some tRNAs in the figure. Figures 9 and 10 are on phylogenetic trees obtained from ML and BI methods. Both trees have same topology and therefore could be presented as a single tree highlighting the supports values of each methods. Posterior probabilities should be shown as decimal. Also, Symphyta is a paraphyletic group and Orussoidea is thought to be a sister group with Apocrita. So I think it’s better to rotate the clade including "Apocrita + Orussoidea + Cephoidea + Pamphilioidea" in the phylogenetic tree to see the relationship between Orussoidea and Apocrita.
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: CHARACTERIZATION OF THE MITOCHONDRIAL GENOME OF ANALCELLICAMPA XANTHOSOMA GEN. ET SP. NOV. (HYMENOPTERA: TENTHREDINIDAE)
Review round: 3
Reviewer: 1

Output:
Basic reporting: In my previous review, I already noted that this manuscript was written in unacceptable English. I have just begun to read the present version of the text, and I again see that the language errors remain uncorrected. I believe this prevents me from further discussion of the manuscript. I am sure that the authors are unable to prepare an acceptable version of the text. I also do not think that constant submission of poorly prepared versions of the manuscript is the best strategy to get it published in any journal including PeerJ.
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: CHARACTERIZATION OF THE MITOCHONDRIAL GENOME OF ANALCELLICAMPA XANTHOSOMA GEN. ET SP. NOV. (HYMENOPTERA: TENTHREDINIDAE)
Review round: 3
Reviewer: 2

Output:
Basic reporting: I am happy to see again the revised version of the manuscript about the mitogenome features and taxonomic position of A. xanthosoma. I feel that the text is mostly improved but I have highlighted some English mistakes and some modifications on the manuscript attached. Also, the section on the mitochondrial genome characterization and comparison of the manuscript still contains unnecessary repetitions (for example, presentation of tRNAs, rRNAs). It probably leads to need for the improvement of the flow and clarity of the relevant text. In my opinion, the manuscript should be reconsidered by an expert in this area, particularly in the field of insect mitochondrial genomics. Furthermore, I could not see the accession number of this newly sequenced mitogenome throughout the manuscript and it should be provided. There are also question regarding the presentation of the phylogenetic tree. As I stated before, Symphyta is a paraphyletic group and Orussoidea is thought to be a sister group with Apocrita. So I think it’s better to rotate the clade including Apocrita + Orussoidea + Cephoidea + Pamphilioidea in your phylogenetic tree to see the relationship between Orussoidea and Apocrita. The family and superfamily names could not been read in this new version of figure (Fig. 9) and should be improved.
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: CHARACTERIZATION OF THE MITOCHONDRIAL GENOME OF ANALCELLICAMPA XANTHOSOMA GEN. ET SP. NOV. (HYMENOPTERA: TENTHREDINIDAE)
Review round: 4
Reviewer: 1

Output:
Basic reporting: I am happy to see again the revised version of the manuscript about the mitogenome features and taxonomic position of A. xanthosoma. I feel that the text is mostly improved. I have highlighted some minor changes on the manuscript attached.
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: A 5-YEAR STUDY (2014–2018) OF THE RELATIONSHIP BETWEEN COASTAL PHYTOPLANKTON ABUNDANCE AND INTERTIDAL BARNACLE SIZE ALONG THE ATLANTIC CANADIAN COAST
Review round: 1
Reviewer: 1

Output:
Basic reporting: No comment.
Experimental design: No comment.
Validity of the findings: No comment.
Additional comments: 1. General comments
In this paper, the authors report on a 5-year study (2014-2018) on the relationship between coastal phytoplankton biomass and intertidal barnacle size along the Atlantic Canadian coast. ” To do so, they used satellite data to document chl a concentrations and they measured the size of intertidal barnacles over this period at eight locations along 415 km of the Atlantic coast of Nova Scotia. For almost every year, the authors found a positive relationship between the summer barnacle size and chl a concentrations for the months before summer. This study adds on current research in the field benthic-pelagic coupling along marine intertidal coasts. The scientific value of the paper could be considered as high since it helps explaining the inter-individual variation in barnacle size along the western boundary of the Atlantic. The topic meets the criteria laid down for publication in PeerJ. The manuscript is generally well written and could be accepted after some revision (see specific comments).

2. Specific comments
Abstract: Although the content of the abstract is informative, it should present the working hypothesis that appears in the Introduction.

Introduction: There is little published information available on benthic-pelagic coupling along the western boundary of the Atlantic. As a result, the results of the present study could be valuable. This is somewhat well explained. The objectives are relevant and strengthen the content of the paper.
Lines 47-50: A reference is needed here. Also, the fact that the temperature is not taken into account in the present study should be justified.
Line 58: I would suggest to use the term biomass rather than abundance to relate phytoplankton chl a concentrations to barnacle size.

The Material and methods are adequate for the problem tackled and the analyses seem to be justified for the data set. Overall, this section is well-written.
Results: Overall, the presentation of results is fine. Some clarifications, however, are needed on two aspects.
Lines 122-128: How do the authors explain the lack of significant correlation between chl a concentrations and barnacle size in 2017? Is there an effect of the water temperature?

Presentation of discussion and conclusion: The discussion is well-written and the answers to the objectives presented in the introduction are reached and convincing. However, I wonder of it is necessary to have a long paragraph discussing about the causes of the variation of chl a concentrations along the coast of Nova Scotia. The end of the discussion is interesting but speculative.
Lines 144-146: It is written that Chl a concentrations explain half or more of the alongshore variation in barnacle size. Do the authors have any idea of other potential factors explaining this variation?


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 5-YEAR STUDY (2014–2018) OF THE RELATIONSHIP BETWEEN COASTAL PHYTOPLANKTON ABUNDANCE AND INTERTIDAL BARNACLE SIZE ALONG THE ATLANTIC CANADIAN COAST
Review round: 1
Reviewer: 2

Output:
Basic reporting: I have perception that document is result like cut and paste derived secondarily from past publication not a new independent "stand alone" work, not establishing new ground integration  satellite chlorophyll-a data with in-situ sampling has a sense of being "tacked on" to establish basis for paper. However, I like idea and has great potential if in the analysis use additional available satellite data   (thermal> gulf stream?, visual photos > discern wave action, rip tide, calm zones of inaccessible areas to compare to areas he has access to. See if something pops out of the noise), NOAA currents, wind charts, bathymetric data.  Needs to study and utilize existing public databases to solidify work The author needs to expand his vision on pertinent investigative avenues and oceanographic viewpoint. Oceanographic features important but not considered.

A weakness of the work, that has to be considered,  lies in the use of chlorophyll data derived from satellite sensors is problematic, its use for coastal waters can be skewed, in addition to assuming that this data is an indicator of phytoplankton abundance.

Though out document makes a lot of general statements, not specific point, then cites literature where information contained. Should be giving specific information from literature pertinent to argument and integrate into current work with citation. Example: Line 157-159 obvious, because barnacles are on the rocks of Nova Scotia and they are filter feeders. How is a spatial connection established that “can occur”, adding to the relationships previously reported for eastern ocean boundaries (Menge et al., 1997, 2003). Reader made do a lot of work to see if what said is relevant. Should give relevant specific information, supporting work and citation
Experimental design: Original primary research within Aims and Scope of the journal. Research question is well defined

The methods were not written in enough detail. some specific details are listed:

Was there a specific reason for the selection of 100 sq. cm. area? In your methodology isn’t clear did you use same exact quadrant each year cleaning off last year’s growth or establish new quadrant in same area?

Not clearly stated that you are establishing your transects along coast and have more than one quadrant at each site until look at table 2, available quadrants in parenthesis, at each station

Line 17: importance of site exposure to wave action

Line 93: Your usage of “random” selection. In one photo A I see 10 candidates and in the other photo B, 7 candidates. What is your criteria for making “random”, clarify please.

It is suggested to include more details of the chlorophyll data, such as the coordinates of the area considered for the extraction of the data and their temporal resolution.
Validity of the findings: Assuming equivalence between coastal phytoplankton abundance and chlorophyll-a concentration is not correct.

Phytoplankton abundance and/or biomass does not covary with chlorophyll over the entire seasonal cycle. Chlorophyll cell concentration changes as response in nutrients and light conditions. Thus Chlorophyll is a poor indicator of the phytoplankton abundance or biomass variations. I suggest the author review the publication of Bellacicco et al 2016 and references there in (http://dx.doi.org/10.1016/j.rse.2016.08.004)

Lines 97-102 Chlorophyll-a concentration from coastal waters color satellite is affected by other water constituents, such as non-phytoplankton particles and colored dissolved organic matter. In what way are you considering this? I suggest reading Yang et al., 2018. Remote Sens. 2018, 10, 1335; doi:10.3390/rs10091335

Article based on satellite data, but very little available data utilized.

David (1938) recommends the use of Confidence Intervals for Pearson correlations only if n ≥ 25. Why didn’t author put all your raw data (total barnacle size data) instead of average in analysis? This would make his analysis more robust in supporting his results.

Line 127-128: Why exclude data? The reason is not clear.

David, F.N. (1938). Tables of the ordinates and probability integral of the distribution of the correlation coefficient in small samples. Cambridge: Cambridge University Press.

Bonett and Wright (2000), Sample size requirements for estimating Pearson, Kendall and Spearman correlations, Psychometrika, 65(1)
Additional comments: Why are filters feeders important along rocky shores? Important or ubiquitous?
Most likely in water larvae released to coincide w/ spring algal bloom. How does barnacle know algal bloom occurring?  Type of Ecological signal? Phytoplankton concentration, Temp., etc. Article’s theme benthic-pelagic coupling ecological relation should be some type of environmental/ ecological signal. If relationship hasn’t been found should be stated reference cited (Scrosati & Ellrich, 2016)
Says currents could hinder recruitment by moving away from intertidal zones. No bathymetric, current data or wind chart data?  Station # 8 close to Bay of Fundy tidal bore influence no mention of mechanical resuspension of sediments and nutrients and dispersive zone of influence. Turbidity
No mention of effect of Gulf Stream pinched off gyres or tributaries etc. satellite photographs thermal imagining
Line 22: chl-a explains nearly half of growth what’s other part
Line 51-57 relevance to article’s topic plankton abundance vs. growth
Change “were done on eastern ocean boundaries” by “have been conducted”
Could full moon be part of release signal, tides?
Line 139-141: “This outcome is remarkable because the five examined correlations were based on data for just eight locations (or seven in 2017), a limited number that, by curbing statistical power, often prevents field studies from detecting patterns in ecology.” Why not restate idea? Awkward phraseology
Line 162-164: upwelling move nutrients away from shore?? Explain add citation
Are there another local nutrients sources?
Currents transporting barnacle larvae along shore?
Could there be physical topographical/ bathymetric features that make a favorable microclimatic area?
Line 141-142 Safety concerns or lack of access renders the majority of coastline unknown. He has barnacle size data and chlorophyll data at eight sites, has satellite data for entire coastline.


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 5-YEAR STUDY (2014–2018) OF THE RELATIONSHIP BETWEEN COASTAL PHYTOPLANKTON ABUNDANCE AND INTERTIDAL BARNACLE SIZE ALONG THE ATLANTIC CANADIAN COAST
Review round: 2
Reviewer: 1

Output:
Basic reporting: The submission defined the research question. The authors' clarifications are satisfactory to establish a sequential chain of a research for the relationship between coastal chlorophyll a and intertidal barnacle size.
Experimental design: No comment
Validity of the findings: No comment
Additional comments: After a careful revision of the manuscript "5-year study (2014-2018) on the relationship between coastal phytoplankton biomass and intertidal barnacle size along the Atlantic Canadian coast",  the authors' clarifications and supplemental information improved the document to a satisfactory level. It is generally well written and could be accepted for publication in PeerJ.


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

Input:
Title: THE RESPONSE OF CUCUMBER PLANTS (CUCUMIS SATIVUS L.) TO THE APPLICATION OF PCB-CONTAMINATED SEWAGE SLUDGE AND URBAN SEDIMENT
Review round: 1
Reviewer: 1

Output:
Basic reporting: The Ms by Wyrwicka and Urbaniak is written in a clear way, easy to read and with appropriate reference to data in figures and tables. Overall, the structure of the work sounds valid. The state of the art is correctly depicted and the scientific context (Phytoremediation) appropriately described. Literature is updated  and properly reported and discussed.
Experimental design: The topic of the Ms fits well with the scope of the journal. The research questions are properly defined and addressed. The investigation is sound with a good technical approach and the methods appropriately described and applied.
Validity of the findings: The novelty of the work and how it can contribute to the advance of the knowledge on the topic are sufficiently discussed. The data set is properly focused to sustain the essential hypothesis on which the work is based. The statistical approach needs a revision following the comments reported in the general comments section. Conclusion should be more consistent with  the statistically correct data interpretation in order avoiding unidentified speculation.
Additional comments: As reported in the previous sections, the work by Wyrwicka and Urbaniak is certainly of interest as highlighting an interesting approach and bringing useful information on the topic of the phytoremediation of PCB present in the sludge or urban sediments.
My main concern is referred to the statistical  treatment of data that in my opinion should be accurately revised. Namely, following the statistical approach performed by authors (see specific comments below reported), it seems that most of the data referred to plants grown in amended soil did not show a statistically supported difference from control. Therefore, a lot of statements made by authors to present and discuss data are not accurate and should be modified accordingly. Specific comments are reported below. For your convenience, I numbered the page starting from the abstract one.
Abstract
P1 L12 Not clear if the treatment is only referred to sludge or also the sediments or both together. Please clarify.
P2 L1 This statement is not clear. Did authors refer to heavy metals possibly present in the sludge? I suggest to reformulate the sentence avoiding to mention other compounds that should be quantified.
Introduction
P3 L9 As the reference “Urbaniak et al., 2017” is at first appearance I suggest to denominate it as “a” rather than “b”.
P4 L8 I wonder whether no food plants could be profitably exploited for such scope instead of plants that can enter the food chain.
P5 L12-13 I think that the aim of the work can be better summarized.
M&M
P5 L24 A brief description of the main soil parameters could be reported.
P5 L27 I suppose that such doses are referred to wet matter while authors used dried material. Did authors take into account this issue?
P6 L12 Add “the apex of” before “the control” if this detail  is correct.
P7 L28-32 Not clear about six replicates if n= 6-8. Please correct or specify. About the statistical approach, I wonder whether authors did not use ANOVA followed by a statistical test to separate means. Did data fail to pass normality test?
P8 L1-2 Assuming that statistically different data were displayed by the “p” factor as in Figs. 2, 4 and 5, the statement referred to data on Fig. 1 is not appropriate as no differences among such data are reported. Such aspect sounds a bit strange as different doses of sludge/sediments were applied to soil. Moreover, PCB concentration in control soils seems not corresponding to a background value. I suggest authors to revise this part, possibly adopting a different statistical approach in order to better highlighting the reduction of PCB concentration after plant growth.
P9 L2 Also in this case, no statistical outcome allowing for such statement was produced.
P9 L14 I suggest to change the term “gradual” as referred to a trend that was not highlighted.
Discussion
P10 L7-9 See comments on P8L1-2 and revise this part accordingly.
P10 L12-14 Really, to better describe the microbial contribution  on  PCB concentration decrease, pots with sludge/sediments but without plants could have been tested.
P11 L7-16 Some statements reported in this part are not properly supported by the statistical outcomes reported in figs. and table (e.g. no greater total chlorophyll content was evidenced). I suggest to revise these sentences.
P11 L33-34 TBARS content was not affected by the exposure to the sludge/sediment added soil. This sentence is not consisting with the statement in P13L15.
P12 L31-34 These statements sound not accurate if referred to the statistical assessment of discussed data.
P13 L2 Contrarily, it seems that CAT activity is increasing  in plants treated with sludge.
P13 L17 Again, this sentence should be revised in accordance to the statistical evaluation.
Conclusion
I suggest to reformulate the first part of the section to make it better coherent with the discussed data.
Reference
The reference “Lakhdar et al.” and  “Srivastava et al.” are not reported in the list. Check the date of the reference “Zhang et al.” and order by alphabet.


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

Input:
Title: THE RESPONSE OF CUCUMBER PLANTS (CUCUMIS SATIVUS L.) TO THE APPLICATION OF PCB-CONTAMINATED SEWAGE SLUDGE AND URBAN SEDIMENT
Review round: 1
Reviewer: 2

Output:
Basic reporting: The paper is timely, dealing with a worldwide problem of environmental pollution and the usability of sewage sludge and sediments. The text is clear and understandable. There are references covering the field, the introduction is explaining all the aspects of the performed research. While the use of sediments and sludges is not new, the novelty is in evaluation of benefits of activity as fertilizers together with some antistress effect on Cucurbita plants
thus supporting their phytoremediation potential.
The structure of the paper is OK, as well as figures and the table, the raw data are available. There are very few typing errors, see comments for authors, where are suggestions for minor corrections.
Experimental design: The paper presents original primary research which is in agreement with the scope of the journal. It is clearly stated what is the research question, it is meaningful and helps ti identify the dangers and benefits of the use of sludge and sediments
The methods and materials are appropriate and were used correctly. The experimental setup is well described and clear.
What I am missing is more description of how the PCBs were analysed - at least a rough description, the citation of an other paper is OK, but I might appreciate at least a basic info on analytical method also in the text, on used standards, which PCBs were analysed, a sum, indicator congeners only or individual peaks evaluated? It would be enough to add a single paragraph with proper citation.
Validity of the findings: The paper brings new information concerning the application of sewage sludge or sediments, describing the danger of introducing toxicity of soil, but evaluating the advantageous effect of supporting phytoremediation potential by introducing nutrients into the soil. Authors discuss also the delay of senescence of cucumber plants caused by sludge addition. The conclusions and results are matching the experiments, which are statistically confirmed.
Additional comments: The paper brings new information important for evaluation of potential phytoremediation technology. I have only a few comments. As already stated above, one most important question concerns missing more description of how the PCBs were analysed - at least a rough description.
Other minor problem:
on page 7, line 18, probably you meant Miracloth, and not Micracloth.
on page 10, line 14 ... were observed ...
on page 10, line 18-22, you write ... both sludge and sediment elicited CAT... but on line 21it is unclear, no such relationship...
on page 12, line 3 ... soluble protein decreases during senescence... And how is it with effect of stress, e.g. caused by toxicity of compounds from sludge?
FIG 1.   ...... explain better, e.g. PCB added within the sediment .., or ...contained after treatment by...
FIG 2.  the capture might be more improved, what does it tell.


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

Input:
Title: THE RESPONSE OF CUCUMBER PLANTS (CUCUMIS SATIVUS L.) TO THE APPLICATION OF PCB-CONTAMINATED SEWAGE SLUDGE AND URBAN SEDIMENT
Review round: 2
Reviewer: 1

Output:
Basic reporting: The revised version of the Ms by Wyrwicka et al. was notably improved by properly addressing my remarks, especially those concerning the statistical data assessment. In accordance with the new approach in the statistical data elaboration, results presentation and discussion were satisfactorily modified making the Ms a sound work highlighting the multifaceted responses  of cucumber plants to the application of sewage sludge and urban sediments. Therefore, the Ms meets the requirement for publication.
Experimental design: The revised version of the Ms by Wyrwicka et al. was notably improved by properly addressing my remarks, especially those concerning the statistical data assessment. In accordance with the new approach in the statistical data elaboration, results presentation and discussion were satisfactorily modified making the Ms a sound work highlighting the multifaceted responses  of cucumber plants to the application of sewage sludge and urban sediments. Therefore, the Ms meets the requirement for publication.
Validity of the findings: The revised version of the Ms by Wyrwicka et al. was notably improved by properly addressing my remarks, especially those concerning the statistical data assessment. In accordance with the new approach in the statistical data elaboration, results presentation and discussion were satisfactorily modified making the Ms a sound work highlighting the multifaceted responses  of cucumber plants to the application of sewage sludge and urban sediments. Therefore, the Ms meets the requirement for publication.
Additional comments: The revised version of the Ms by Wyrwicka et al. was notably improved by properly addressing my remarks, especially those concerning the statistical data assessment. In accordance with the new approach in the statistical data elaboration, results presentation and discussion were satisfactorily modified making the Ms a sound work highlighting the multifaceted responses  of cucumber plants to the application of sewage sludge and urban sediments. Therefore, the Ms meets the requirement for publication.