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

Input:
Title: IDENTIFICATION OF BIOMARKERS ASSOCIATED WITH CLINICAL SEVERITY OF CHRONIC OBSTRUCTIVE PULMONARY DISEASE
Review round: 3
Reviewer: 1

Output:
Basic reporting: Please find response in general comments
Experimental design: Please find response in general comments
Validity of the findings: Please find response in general comments
Additional comments: First of all I want to thank the authors for all the efforts they done to improve the manuscript.
Unfortunately, there are still several deficiencies in the logical structure, experimental setup and language expression. Moreover, the present manuscript revealed unpredicted errors in basic reporting, experimental design and validity of the findings.
The present manuscript is now reasonably comprehensible written; unfortunately there are still many general deficiencies in language expression.
For the first time, method descriptions like TLR-expression analysis are presented, but still information like the chosen housekeeping gene or the calibrator used to perform the delta/deltaCt method are missing. Moreover, in line 319 of the revised manuscript they describe that TLR2 expression seems to be cloth to the detection level (low Ct values and that this might led to experimental errors. There are still some uncertainties, the authors described that they isolated PBMC and analyze the mRNA expression of TLR-2 in the monocytes. However, PBMC consists of lymphocytes as well as monocytes, were the cell types separated?
The chosen method, analyzing TLR-2 expression on mRNA level, seems an inappropriate approach, comparing the expression of a surface receptor of the innate immunity. There could be wide differences between mRNA and protein expression. Here methods, like flow cytometry are recommended analyzing surface expression of the receptors. This analysis is mandatory, especially because experiments of others demonstrate no differences in these levels on blood monocytes of healthy, smokers and COPD patients [for example: Scheele, Respiratory Medicine, 2011].
The authors detected in the second revision that the showed wrong results for IL-10. IL-10 was not a hub gene in the PPI network. They excluded IL-10 from the introduction and the detailed description in the discussion. However, there are still parts in the discussion [line 297], describing IL-10 as a hub protein. Thank you for the disclosure of the wrong presented results. Unfortunately, this misanalysis in the central focus of the manuscript, the evaluation and detection of biomarkers in a preexisting dataset, gives a weak expression of the overall quality in the analysis process.
Moreover, the authors try to evaluate expression levels of CD79A. However, here no differences in their cohort were detectable.
Due to all this points revealed in the revision process and the fact that results and methods are still not clearly presented and improved upon three revision cycles I would recommend to reject 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: IDENTIFICATION OF BIOMARKERS ASSOCIATED WITH CLINICAL SEVERITY OF CHRONIC OBSTRUCTIVE PULMONARY DISEASE
Review round: 4
Reviewer: 1

Output:
Basic reporting: Please find response in general comments
Experimental design: Please find response in general comments
Validity of the findings: Please find response in general comments
Additional comments: As I mentioned in my last revision the present manuscript revealed several unpredicted errors in basic reporting, experimental design and validity of the findings during the revision process.
Some points were improved by the authors for example: Language Expression.
Still my concerns of the last revision are not completely eliminated and again new questions are raised.
a.	Why are only monocytes analyzed for TLR2 expression and not PBMC completely? TLR2 is also expressed on lymphocytes.
b.	The expression of this marker was only on mRNA level, and here expression was cloth to the detection level (low Ct values and that this might led to experimental errors (as mentioned by the authors themselves).
c.	The authors add information upon the housekeeping gene, but did not add any information upon the calibrator.
d.	The chosen primer seem to be specific for TLR2 transcript variant 2, why only analysis of this variant is chosen is not mentioned.


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: DEEP-SEA WATER DISPLACEMENT FROM A TURBIDITY CURRENT INDUCED BY THE SUPER TYPHOON HAGIBIS
Review round: 1
Reviewer: 1

Output:
Basic reporting: Writing is clear and professional throughout. Literature reference could be further extended (see review attached).
Experimental design: Method is rigorous with sufficient detail. Aims are clear.
Validity of the findings: Data seems sound. Conclusion well stated, but could be improved (see review attached).
Additional comments: see review attached


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

Input:
Title: DEEP-SEA WATER DISPLACEMENT FROM A TURBIDITY CURRENT INDUCED BY THE SUPER TYPHOON HAGIBIS
Review round: 1
Reviewer: 2

Output:
Basic reporting: The manuscript discusses direct observations of turbidity current events most likely induced by Super Typhoon Hagibis, in Suruga Bay, Japan.

The manuscript is well written in general and provides adequate literature references and context. I would recommend avoiding idioms such as for instance "wreaking havoc" in the abstract. I also recommend that the authors check the accepted date format in PeerJ. For instance, "14 October" should most likely be "October 14" instead.

Figures are clear and add value to the manuscript. A schematic of the different sensors mounted on Edokko Mark 1 could be added to figure 2, as it is not essential to the discussion in its present form. The caption for figure 6 should mention if this is for the AM3 or AM11 event.
Experimental design: Direct, in-situ measurements of turbidity current are always relevant and interesting, and the methods described in the paper are, to the best of my understanding, technically adequate. The turbidity current is observed in a single location, which makes it impossible to estimate its kinematic properties and to infer its propagation speed. This is obviously not something that can be addressed in the present manuscript, but I would be interested in the future to see the methods described here applied at different locations downslope from the possible release point of the turbidity event.
Validity of the findings: The paper clearly demonstrates the existence of the turbidity events, well supported by direct measurements. However, the authors additionally state that "The
temperature and salinity characteristics during these high turbidity events matched
independent measurements for shallow water layers in Suruga Bay at the same time,
strongly indicating that the turbidity current caused mixture of the bay’s water column". I find this statement confusing, as it suggests that the turbidity current generates vertical mixing throughout the water column, at the location where it is measured, which I do not believe to be the case. If my understanding is correct, the turbidity current transports warmer interstitial fluid from a shallow region of the Suruga Bay to the deeper region where Edokko Mark 1 is located, downslope of the canyon. This is made possible, as the authors point out, by the fact that the suspended particles are presented in high enough concentration for the mixture to have a higher density than the deep ambient fluid. Downslope transport of interstitial fluid by turbidity currents is completely expected and is, in my understanding of the term, significantly different from predominantly vertical mixing, which would refer to convection of warmer fluid directly above Edokko Mark 1 down to the seabed, which I do not believe is the case. I suggest that the authors revisit the statement quoted at the top of this paragraph and clarify what is meant by "vertical mixing".

Because of uncertainty with the salinity measurement during the turbidity event, it seems challenging for the authors to provide a more quantitative assessment of the density of the interstitial fluid, as well as overall mixture density, of the turbidity current. Because the time and location of release of the turbidity current is not known, it is also impossible to estimate the speed of the current front and thus to estimate its properties based on simple models.  Because this quantitative analysis cannot be performed, I strongly encourage the authors to improve their discussion of mixing and transport, as discussed above.
Additional comments: My understanding of the results presented is that the turbidity current does not generate a large scale vertical mixing of the water column in the Suruga Bay, but instead, as is expected of a turbidity current, transports interstitial fluid from the shallower release site to deeper parts of the bay, following the slope of the canyon topography. If so, the discussion should be modified accordingly. If there is indeed large scale mixing throughout the water column, the discussion should be improved to better demonstrate this statement, and show how the results prove this is the case.

Minor comment: The last paragraph of the discussion does not refer to the results presented in the results section, and reads more like an introduction. I recommend that the authors move this paragraph to the introduction, and develop the points identified above in my comments in the discussion instead.


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: DEEP-SEA WATER DISPLACEMENT FROM A TURBIDITY CURRENT INDUCED BY THE SUPER TYPHOON HAGIBIS
Review round: 1
Reviewer: 3

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: Kawagucci et al. present an interesting work studying a direct observation of the deep-sea turbidity current triggered by the Super Typhoon Hagibis (2019). The manuscript is thus worthy of such rapid publication but should be done after a few necessary revision works below:

Reads unclear whether the authors suggest canyon helps the turbidity mixing observed by, for example, "canyon flushing event." The authors should convey the mechanism on how Hagibis triggers and generates the turbidity current the authors observed. Several papers studying Japan and NZ below are helpful:
Kioka, A., Schwestermann, T., Moernaut, J. et al. Megathrust earthquake drives drastic organic carbon supply to the hadal trench. Sci Rep 9, 1553 (2019). https://doi.org/10.1038/s41598-019-38834-x
Mountjoy, J.J., Howarth, J.D., Orpin, A.R., et al. Earthquakes drive large-scale submarine canyon development and sediment supply to deep-ocean basins. Sci Adv 4, eaar3748 (2018). https://doi.org/10.1126/sciadv.aar3748

Specific comments

L42: "This" -> "A turbidity current"
L47: "matched" -> "agreed with"
L48: "strongly indicating" -> "indicating"
L52: "greatly contribute to" -> "contribute to"
L64-67: There is relevant literature studying direct observation of deep-sea turbidity current:
Paull, C.K., Talling, P.J., Maier, K.L., et al. Powerful turbidity currents driven by dense basal layers. Nat Commun 9, 4114 (2018). https://doi.org/10.1038/s41467-018-06254-6
L85: Yes I remember the cancellation was a great shock ...
L88: Should specify the time zone here. "18:00" should be "18:00 (UTC+9:00)" given the authors may use Japan standard time. Modify Figure 1 accordingly
L92-96: Long sentence
L119: Address the location more specifically (e.g., the mouth of Heda Canyon)
L123: I kannt find the Heda River in Figure 1. Could you please show it in Figure 1?
L164: "14 October" should be "16 October?"
L187-190: Hard to believe the temperature value is such high precise. Perhaps to better to round up at least to the second decimal place...
L262-265: This should be moved to the Discussion session because it should not be a conclusive message of the study.


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

Input:
Title: DEEP-SEA WATER DISPLACEMENT FROM A TURBIDITY CURRENT INDUCED BY THE SUPER TYPHOON HAGIBIS
Review round: 2
Reviewer: 1

Output:
Basic reporting: Clear and unambiguous, although I have added some minor textual suggestions in the pdf attached.
Experimental design: original primary research within aims and scope of the journal.
Validity of the findings: Conclusions are well stated, linked to the original research question and limited to supporting results.
Additional comments: I really enjoyed reading the manuscript again. I have provided some small suggestions in the sticky notes on the attached pdf, but I do not see any issue that would prevent me to recommend publication of the manuscript. Looking forward to see more results coming from this new mooring!

All the best,
Matthieu


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: DEEP-SEA WATER DISPLACEMENT FROM A TURBIDITY CURRENT INDUCED BY THE SUPER TYPHOON HAGIBIS
Review round: 2
Reviewer: 2

Output:
Basic reporting: My comments have been addressed, nothing to add.
Experimental design: My comments have been addressed, nothing to add.
Validity of the findings: My comments have been addressed, nothing to add.
Additional comments: The authors have addressed my comments and concerns  throughout the manuscript, which has been greatly improved.


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

Input:
Title: EXPERIMENTAL LUNG INJURY INDUCES CEREBRAL CYTOKINE MRNA PRODUCTION IN PIGS
Review round: 1
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: Kamuf et. al. investigate cerebral cytokine production in an experimental lung injury model induced by oleic acid injection in pigs. They found an increase in hippocampal IL-6 mRNA in the OAI group compared to control group without histological evidence of neuronal damage.

- The study is well written with recent literature cited in the text.
- A detailed and critical disscussion is made and limitations of the study are included.


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: EXPERIMENTAL LUNG INJURY INDUCES CEREBRAL CYTOKINE MRNA PRODUCTION IN PIGS
Review round: 1
Reviewer: 2

Output:
Basic reporting: Paper is generally clear throughout.

Please proof read the paper as some minor english corrections need to be made. e.g. Line 102 "were" should be "was"
Line 216: "only a trend towards higher levels of TNFalpha" add "was observed".
Please subscript all of the "2s" in FiO2 throughout the manuscript.
Experimental design: The statistics are appropriate only for the inflammation measurements. Physiological measurements should be examined using a 2-way Repeated Measures ANOVA and a post-hoc test will determine time points that are significant.
Do you mean 10% formaldehyde which is the normal fixation strength (i.e. make 4% paraformaldehyde into 10% formaldehyde solution)?
Validity of the findings: The assessment of microglia conducted was rather simplistic as you simply showed that the number of microglia was not different, which isn't overly surprising, however the state of the microglia, whether it is active or not, is important. If done correctly the histology of the microglia will tell you whether they are ameboid or ramified and that will tell you whether an inflammatory response is taking place within the regions. Further, microglia in inflammatory states often form aggregations and a thorough examination would include the total area of aggregations within the region of interest, % coverage of aggregations, etc. Simply counting will miss these inflammatory signals.
Lines 223-227 - IBA1 stains microglia, whether they are active or not. Did you try to distinguish between them?
Additional comments: Line 261-62: "Even though the pulmonary damage score was significantly
 higher in the lung injury group, this indicates an improvement of the lung injury" - this is worded poorly. How can increased injury indicate an improvement of lung injury?
Did you measure any anti-inflammatory cytokines which may explain the reduction in cytokines or injury discussed?


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: EXPERIMENTAL LUNG INJURY INDUCES CEREBRAL CYTOKINE MRNA PRODUCTION IN PIGS
Review round: 1
Reviewer: 3

Output:
Basic reporting: No comment
Experimental design: No comment
Validity of the findings: No comment
Additional comments: Kamuf et al., investigated the mechanisms by which acute lung injury induces cognitive impairment in a pig model of Acute respiratory distress syndrome (ARDS). The authors suggest that acute lung injury induces peripheral inflammation (increasing plasma IL-6 and TNFa) which would mediate an increase of IL-6 and TNFa mRNA synthesis in hippocampus.
The topic of research is interesting and well introduced, the manuscript is clearly written and the used methods are appropriated. However, in my opinion some aspects need to be deepened and better discussed to improve even more the quality of research. These are addressed section by section, below:

UNDER METHODS:
-	How long were the brains  fixed in 4% formaldehyde solution?
-	How were the HE and IBA1 immunohistochemistry quantified? Please, briefly explain it in methods.
-	From which body’s part were taken the blood samples?

UNDER RESULTS:
-	To study the effects of acute lung injury on neuroinflammation the authors quantified the number of microglial cells in CA2 region of hippocampus but no differences between groups were found. Activation of microglial cells is not necessarily accompanied by proliferation (as the authors also discussed). Thus, in my opinion, to increase even more the quality of research, the authors should quantify eventual morphological changes in microglial cells (perimeter of microglia or their cell body size). Furthermore, considering the small size of CA2 hippocampus region, it would be interesting and more appropriate analysing the number and morphological changes in the whole hippocampus.
-	The authors should also perform the morphological/proliferative analysis of microglia in cortex, as they did the content of mRNA cytokines.
-	The authors also state: “there were no differences in the number of neurons”. These results are not so unexpected considering the experimental design (animal were sacrificed 18 hours after inducing lung injury). The authors should analyse other parameters to evaluate the neuronal damage such as nuclear pyknosis and eosinophilic degeneration of the cytoplasm as in in Heuer et al., (cited by the authors). The authors should also analyse other hippocampal regions (CA1, CA3, and DG).
-	The authors only analysed the content of mRNA cytokines in hippocampus and cortex but not their protein. An increase of mRNA is not necessarily accompanied by an increase of the protein. To increase the quality of their research and better support their conclusions, the authors should also analyse the content of the protein. At least, a note of caution should be added discussing their results.

MINOR CONCERNS
-	In my opinion, the main title “Experimental lung injury induces cerebral cytokine production in pigs” is misleading. In fact, the authors only have analysed the content of mRNA but not the protein of cytokines. Please change or support it experimentally by analysing the content of cytokines besides the mRNA.
-	Most of graphs are lacking of units of measurement. Please add them where lacking.
-	Under figure 1, the authors should also include one representative photo per group of HE stain and IBA1 immunohistochemistry.


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: EXPERIMENTAL LUNG INJURY INDUCES CEREBRAL CYTOKINE MRNA PRODUCTION IN PIGS
Review round: 2
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: The author has adequate addressed my concerns.


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

Input:
Title: EXPERIMENTAL LUNG INJURY INDUCES CEREBRAL CYTOKINE MRNA PRODUCTION IN PIGS
Review round: 2
Reviewer: 2

Output:
Basic reporting: No comment
Experimental design: No comment
Validity of the findings: No comment
Additional comments: The authors have satisfactorily addressed most of the comments but there are still a few aspects of manuscript that in my opinion could be improved to increase even more the quality and the fluidity of the manuscript:

UNDER METHODS:
-	The authors should add in the text a sentence about the time of fixation of the brains in 4% formaldehyde solution
-	The authors should add a sentence in the text about from which body’s part were taken the blood samples

UNDER RESULTS:
-	Why did the authors delete the IBA1 graph from the figure 1? Please, restore it including the new data obtained from the quantification of CA1, CA3, CA4 and DG regions. Please, also add a graph with the quantitative data from the morphological analysis and cite them in the text.

UNDER FIGURES:
-	Under Suppl. Figure 1, the authors should also include one representative photo per each analysed group and region (CA1,CA2, CA3, CA4, DG) of HE stain and point some example of pyknotic and eosinophilic degeneration on it.
-	Under Suppl. Figure 2, please point some example of activated and resting microglia.


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: ASSESSMENT OF THE EFFECTS OF ISCHAEMIA/ HYPOXIA ON ANGIOGENESIS IN RAT MYOFASCIAL TRIGGER POINTS USING COLOUR DOPPLER FLOW IMAGING
Review round: 1
Reviewer: 1

Output:
Basic reporting: The paper is written in a clear English, even if some corrections are needed.
Literatrure references are provided to illustrate the background, even if the authors fail to adeguately quote them in the text.
The structure of the article is well designed except the organization/numbering of figures which does not correspond to the text.
In detail:
1)	In the keywords please correct: Ischemia – Hypoxia.
2)	Figures: there is no correspondence between the numbering in the text (Figures 1-6) and the single figures (numbered 1-16).  Please provide numbering and lettering of panels and appropriate titles and legends of the 6 figures.
3)	References at lines 296-297 have no correspondence in the reference list. Please check names/surnames for accuracy.
Experimental design: The paper by Jiang and co-workers reports that trauma and overuse induced ischemia-hypoxia conditions are involved in the formation of myofascial trigger points (MTrPs), then responsable of muscular pain. Color doppler flow imaging (CDFI) is associated with neovascularization markers as HIF-1a and VEGF. The paper demonstrates that CDFI by assessing the blood flow signal through a not bloody external procedure can detect the features of angiogenesis in or surrounding MTrPs.
The paper is interesting and the results reported sound enough.
Major comments:
1)	Could the authors give a rationale for the time used in the model? “All rats in the model group were treated in this way for 8 weeks with a subsequent 4 weeks of rest, while the rats in the control group did not undergo any intervention in this period.” Did the author performed a time course study to evaluate neovascular responses following injury? The time for angiogenesis occurrence is usually shorter (few days-1 week) and probably the authors uasing shorter times can reach more striking results respect to the ones reported here (i.e. figure 6 and table 2).
2) Soon after the muscle trauma (1200 g stick freely dropped from a height of 20 cm!) it is supposed to have tissue damage due to compression, a strong inflammatory reaction and platelet activation. The authors should consider the role played by angiogenic factors released by inflamamtory cells and platelets, again at short times after the injury.
3)	HIF-1a and VEGF measurements by ELISA: please check since in the MM section it is reported that they are done on tissue homogenates, while in results it is reported serum levels.
Validity of the findings: The findings have enough impact and novelty.
All the data reported statistically sound.
Conclusions are well stated on the base of the reported data. Authors try to speculate and give possible explanations about the less robust data.
Additional comments: Please provide rationale and explanation to the above requests and improve the editing/organization of the mansucript.


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: ASSESSMENT OF THE EFFECTS OF ISCHAEMIA/ HYPOXIA ON ANGIOGENESIS IN RAT MYOFASCIAL TRIGGER POINTS USING COLOUR DOPPLER FLOW IMAGING
Review round: 1
Reviewer: 2

Output:
Basic reporting: I think the manuscript might meet the standard after major revision.
Experimental design: Methods: Microvessel density- it is written ‘MVD of the gastrocnemius muscles of rats from the two groups was detected by hematoxylin150 eosin (HE) staining’. MDV was analyzed by CD31 immunostaining. Such mistakes are not good for paper. What is the hotspot method, and how it was used in a tissue section to determine MVD?
Validity of the findings: No comment
Additional comments: Title: The effect of ischemia – hypoxia in rats on angiogenesis in myofascial trigger points assessed by color Doppler flow imaging
In this study, Jiang et al. investigated about the myofascial pain syndrome, which is characterized by the presence of myofascial trigger points (MTrPs). Specifically, their aim was to check the hypothesis proposed by Simons regarding the development of MTrPs in MPS. In the hypothesis proposed by Simons, local hypoxia plays an important role in the development of MTrP. In this paper, the authors showed increased angiogenesis at the areas of MTrP. Since increased angiogenesis suggests better tissue perfusion and MTrPs are developed due to hypoperfusion, I think the findings shown in the paper contradict Simons hypothesis. In the areas of MTrPs, vessels show some changes. The authors might investigate such vascular changes and correlate with angiogenesis event to explain the development of MTrPs or should fully explain what’s mechanism is involved there compared with the previous knowledge.
There are also some serious flaws in the paper:
1.	HIF-1α ELISA: in the result section, the authors stated that ELISA was done with serum. But in the Methods section, they stated that muscle tissue was used for ELISA. Such discrepancy is not good for a paper. Also, the authors need to mention the tissue lysis method, because that might affect the quantity of target protein.
2.	Description of results: Description of results is very incomplete. It is very difficult to correlate what is written in the result and the figures.
3.	Figures: Figures are usually arranged in a paper according to the story. One figure tells 1 section of the story. In a figure, several sections are arranged as (A), (B) or (C) etc. Here 17 figures are shown in a random way, which is very difficult to follow. Some figures do not corroborate with the results. For example, in the results of HIF-1α ELISA, figure 6 is referred. But figure 6 is ‘the screenshot of the sonoelastography modes’.
4.	Figure legends need to be amended. First, arrange the figures more carefully according to the story, and then write the figure legends. Figure legends should explain how we should understand the figures.
5.	Discussion should be re-written completely.


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: ASSESSMENT OF THE EFFECTS OF ISCHAEMIA/ HYPOXIA ON ANGIOGENESIS IN RAT MYOFASCIAL TRIGGER POINTS USING COLOUR DOPPLER FLOW IMAGING
Review round: 2
Reviewer: 1

Output:
Basic reporting: The manuscript is cleary written and hypothesis and experimental data reported and quoted according to conventional scientific procedures.
Experimental design: The experimental desing is clearly reported according to ethycal and technical standads. Methods are described in a sufficient manner.
Validity of the findings: All the data are robust, controlled and statistically sound. Conclusions are well stated
Additional comments: All the referees' requests and comments have been satisfied and the mansucript corrected for English style and grammar.
The revised version is thus improved.


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

Input:
Title: ASSESSMENT OF THE EFFECTS OF ISCHAEMIA/ HYPOXIA ON ANGIOGENESIS IN RAT MYOFASCIAL TRIGGER POINTS USING COLOUR DOPPLER FLOW IMAGING
Review round: 2
Reviewer: 2

Output:
Basic reporting: There are sometimes careless mistakes.
Experimental design: Time-course analysis should be necessary.
Validity of the findings: It is difficult to evaluate whether the results are reasonable.
Additional comments: Title: The effect of ischemia – hypoxia in rats on angiogenesis in myofascial trigger points assessed by color Doppler flow imaging
In this paper, the authors showed increased angiogenesis in the areas of MTrP. However, they speculated that the initiation of the MTrP is hypoperfusion, which was not verified in the present study. Since time-course analysis was not scheduled, it is very difficult whether their insisted theory is reasonable or not. They can investigate every parameter, especially blood flow with doppler flow imaging, from before and after making the disease model.
The relationship between blood flow signals and other parameters should be shown in the scatter graph because the correlation might be very weak.
It is unbelievable to see the title name is wrongly written.


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

Input:
Title: ASSESSMENT OF THE EFFECTS OF ISCHAEMIA/ HYPOXIA ON ANGIOGENESIS IN RAT MYOFASCIAL TRIGGER POINTS USING COLOUR DOPPLER FLOW IMAGING
Review round: 3
Reviewer: 1

Output:
Basic reporting: Grammar and word mistakes are all improved.
Experimental design: The authors should include about time-course change in this model and the necessity of it will be discussed in Discussion. It may be added to the limitation.
Validity of the findings: Further sound investigation strengthens this study's evidence.
Additional comments: No more comments.


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

Input:
Title: ONCOLYTIC EFFECTS OF THE RECOMBINANT NEWCASTLE DISEASE VIRUS, RAF-IL12, AGAINST COLON CANCER CELLS IN VITRO AND IN TUMOR-CHALLENGED NCR-FOXN1NU NUDE MICE
Review round: 1
Reviewer: 1

Output:
Basic reporting: The manuscript entitled “Oncolytic Effects of the Recombinant Newcastle Disease Virus Strain AF2240-i Expressing Interleukin-12, rAF-IL12, against HT29 Colon Cancer Cells In Vitro and in Tumor-challenged NCr-Foxn1nu Nude Mice (#47395)” by Najmuddin et al, describes the effect of recombinant NDV that expresses IL-12 on the colon cancer cell line as well as tumor-challenged nude mice. The recombinant virus is replicating well in the cancerous cells in a stable manner. The recombinant virus induces apoptosis in vitro and also reduces tumor growth in vivo without any deleterious effect on normal cells. The authors also described the safety assessment of the recombinant virus for various organs including lung, liver, spleen and kidney. The effect on the cytokine’s modulation along with the increase in the expression of apoptosis-related genes has been observed. Overall this is an interesting paper. The experiments are carefully executed and well described. However, the manuscript needs some improvements.

1.	The title seems too descriptive. It can be improved by removing the names of virus strain, cell line.
2.	The abstract needs to be modified for better organization of sentences. For example, in line 41, it is difficult to differentiate for the readers whether it is about previous background study or the authors started describing current one.
3.	The discussion section seems a bit unorganized too. At some places it looks like a repetition of the results (for example line 416-456), while at others, a lot of literature has been described without connecting the literature to the current study. For instance, line 477: the paragraph starts with “It has been postulated that there are two major modes of oncolysis taken by NDV; one is by selective infection and killing tumor cells, while the other is by indirectly eliciting/invoking specific host immune responses against the tumor tissue”. However, no further connection of these two mechanisms has been discussed later. It should be discussed which possible mode of oncolysis authors find in the current study.
4.   The authors described the literature in introduction in details. However, the recombinant NDV studies are not mentioned that used various cytokines expressing virus.
Experimental design: 1.	The exact same recombinant virus has already been shown as an inhibitor of breast cancer. This is another study of different type of cancer. Is it possible that this recombinant virus has the capability to reduce a number of different kind of tumors? Also, the research has been cited only for the construction of the recombinant virus. More details should also be included in abstract, introduction and discussion.

2.	Several studies are available showing the tumor inhibitory effect of recombinant NDV expressing different cytokines. Was there any specific reason for the use of IL-12 among other cytokines? Is IL-12 better for the tumor inhibition as compared to other cytokines?

3.	An important study of the survival rate of cancerous mice in untreated and treated groups is missing here.

4.	The study shows the inhibition of tumor growth in mouse when treated with recombinant NDV (Figure 6). Does this or any other study have evidence of the reduction in tumor growth after treating mice when the tumor is fully grown. To make it a strategy for the treatment of cancers, the studies should be there which proves the treatment after tumor development as in some cases it is hard to identify individuals before disease progression. For example, what happens if the mice are treated after 14, 21 or 28 days after tumor cells injection. Will they survive?

5.	Figure 1: The authors showed the viral replication using qPCR for the quantitation of viral RNA. The growth kinetics should have been performed for the calculation of infectious virus particles.
Validity of the findings: 1. Lines 295 and 301: Figures 3 and 4 are showing different results for the similar effect on apoptotic cell population in control, virus treated and recombinant virus-IL-12 treated cells. These both are exact same experiment; the only difference is the use of two different techniques. A result is confirmed only when same results are obtained by using different methods. I do not see any reason for different results here when only the methodology is different. 20 % cells are viable according to figure 3 while 80% cells are viable with different method used in figure 4. This does not look like because of the variability within the experiment as the error bars are too small. How does authors explain this?

2.  Line 283: The authors has described the difference between the EC50 values of two viruses as significant. Though these values look pretty similar and it can be due to the variability between two different experiments. Also, in Fig 2, error bars are missing so it is hard to interpret the variations in the cell viability for three independent experiments.

3. Line 327: The authors mentioned that the virus is disseminated in vital organs. Lung and spleen show a significant amount of viral copy number though. Is it possible for the authors to check the infectious virus particles for these organs as well?
Additional comments: 1.    Line 372: Both viruses show different effects on the cytokine levels. Is it possible that they follow two different mechanisms/pathways to induce apoptosis?
2.     Line 459: “detected in kidney and lung”. Authors did not detect viral RNA in kidney and liver.
3.	Figure 3: How many cells were calculated in total?
4.	Line 470: “However, the enzyme levels were restored back almost to their normal level in the rAF-IL12-treated group, rendering the intra-tumoral injection of rAF-IL12 to have very minimal/limited toxicity towards liver and kidney function.” It has to be rephrased as the enzyme levels were not lowered from a higher one in the same group of mice. It was an independent experiment where authors found lower enzyme levels.


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: ONCOLYTIC EFFECTS OF THE RECOMBINANT NEWCASTLE DISEASE VIRUS, RAF-IL12, AGAINST COLON CANCER CELLS IN VITRO AND IN TUMOR-CHALLENGED NCR-FOXN1NU NUDE MICE
Review round: 1
Reviewer: 2

Output:
Basic reporting: Najmuddin and co-workers studied the oncolytic effects of the recombinant NDV Strain AF2240-i expressing IL-12 (rAF-IL12) against HT29 colon cancer cells in vitro and in tumor-challenged NCr-Foxn1nu Nude mice. Their study proves that the recombinant NDV had better cytotoxicity effects than wild-type and could potentially be ideal for treatment of colon cancer in near future. The study comprises of an extensive set of experiments providing conclusive results. The literature and background content required to understand the scope of the study and its importance is clearly mentioned. They have also included evidences for choosing IL-12 as an immune-enhancing cytokine. In Line 106, the T cell differentiation information is not required as far as the study in concerned. It may require more experimental evidence to support that in the study and thus can be removed from the Introduction. The Materials and Methods section is very lucid and is understandable. The assays are clearly mentioned. The results and discussion have good coherence. The authors can try to shorten the discussion section a little bit.
Experimental design: Najmuddin and co-workers strategically designed the experiments which falls within the scope of the journal. The authors have pointed out the significance of generating the recombinant strain NDV as well as choosing IL-12 to be included in the construct. The authors can show the difference between the two constructs – wild-type and recombinant. It is missing in the introduction section. All assays performed are 72 hours post-infection and the authors did not mention any literature reference to back up. For Figure 1, the authors claim that viral copy number in HT29 is significantly higher in rAF-IL12 infected cells compared to wild-type AF2240-i. But their graph does not reflect any significant change. For Figure2, the graph does not show any significance value associated with it.  For Figure 3, 4, and 5, the results section mentions Control while the figures talk about Negative Control. The labeling of samples used should be consistent throughout the article. For Figure 4, the quadrants are not explained well. They do not indicate what each quadrant is for. For Figure 7, the authors talk about the capability of rAF-IL12 to replicate inside tumor and organ tissues. The authors do not mention the reason behind the decision of choosing lung, spleen, kidney, and liver. Other vital immune organs can also have a strong influence which has not been reported in the study. Lines 327-332 also does not clearly mention what does dissemination show although their discussion section talks about it. Its better to be consistent with the flow of the results and observations. For Figure 8-11, the authors must re-write the result section. They need to clarify the outcomes of the observation. Currently, the result section just has the observation and no explanation on the findings. For Figure 12, the authors should explain their decision behind choosing the specific cytokines. Overall, all Figures in the images have faintly visible and missing error bars (Figure 7) and do not have the visually convincing error bars. The authors should put the images in high-resolution with visible error bars wherever probable.
Validity of the findings: The research findings are very convincing and promising. Authors have delved into each aspect of how a oncolytic viral particle can be tested. It will be great if the authors can explain why the recombinant variant has better viral copy number or has better response than the wild-type variant. It will be great if the authors can also perform a study showing the other possible variants of rAF-1L12 as IL12 plays a significant role in production of IL12 cytokine stimulating the immune response towards cancer.
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: ONCOLYTIC EFFECTS OF THE RECOMBINANT NEWCASTLE DISEASE VIRUS, RAF-IL12, AGAINST COLON CANCER CELLS IN VITRO AND IN TUMOR-CHALLENGED NCR-FOXN1NU NUDE MICE
Review round: 2
Reviewer: 1

Output:
Basic reporting: This is a revised submission of the manuscript entitled “Oncolytic Effects of the Recombinant Newcastle Disease Virus, rAF-IL12, against Colon Cancer Cells In Vitro and in Tumor-challenged NCr-Foxn1nu Nude Mice”, concerning the anti-tumor effect of the recombinant newcastle disease virus, rAF-IL12. Authors have answered all the comments/concerns pointwise and improved the manuscript accordingly. The abstract, introduction and discussion sections are improved very well.

In the revised version of the manuscript, authors have added the use of recombinant NDV-IL12 strain for the oncolytic activity in previous studies. Same construct of the virus and IL-12 has been used for the inhibition of breast cancer. Another recombinant NDV strains have been published for colon cancer. The current study confirms these findings and is a good advancement from previous studies. However, few questions still remain unanswered.
Experimental design: Figure 5: I do understand author’s decision about performing the tumor treatment experiment at later time points in near future. However, is it possible for authors to check re-growth of the tumor after 28 days if the recombinant virus is not injected? Also, have authors tried any another route of injection if it makes any difference in anti-tumor activity?
Validity of the findings: Figure 1: This change in chart type does not answer the original question. On the basis of presentation, the previous graph was also fine. However, the question was regarding the calculation of infectious virus particles. Since qPCR gives signal with both infectious as well as defective virus particles, it can be only a supportive experiment, not a full proof. Authors should have the samples saved I assume. It should not take much time and effort to perform a plaque assay or TCID50 calculation to measure infectious virus particles.

Figure 12: Have authors also tested the gene expression levels of Fas, caspase-8, BID, BAX, and SMAD3 in non-cancerous cell lines, or normal mice? That will further prove authors finding that the recombinant virus does not cause any apoptosis in non-cancerous cell lines.
Additional comments: Line 505-509: It is not important to discuss the differences in different methods in discussion. These lines can be deleted.

I understand that in Conclusion section authors have mentioned all the experiments they wish to perform in near future. However, it does not seem appropriate to mention a list of experiments to be performed later. Specially, for very simple experiments such as plaque assays. It would be better if authors remove these future experiments from conclusion and mention either in results or discussion wherever the related experiment is discussed.


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: ONCOLYTIC EFFECTS OF THE RECOMBINANT NEWCASTLE DISEASE VIRUS, RAF-IL12, AGAINST COLON CANCER CELLS IN VITRO AND IN TUMOR-CHALLENGED NCR-FOXN1NU NUDE MICE
Review round: 3
Reviewer: 1

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


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

Input:
Title: INSIGHTS INTO DOG OWNER PERSPECTIVES ON RISKS, BENEFITS, AND NUTRITIONAL VALUE OF RAW DIETS COMPARED TO COMMERCIAL COOKED DIETS
Review round: 1
Reviewer: 1

Output:
Basic reporting: Language is excellent.
Some remarkable research papers in the field were not cited and discussed.
The article is well structured; however, the discussion section does not follow the results section, and this creates come confusion. Some figures are redundant. No copy of the survey was provided, thus making it difficult to fully understand results source.
The submission is self-contained.
Experimental design: Further details could be provided according to CHERRIES guidelines for e-surveys.
Validity of the findings: This study is interesting, provides new data in a little explored field, innovative use of word clouds for displaying data.
The presentation of data sometimes is too shallow, and some precise numbers are missing.
Conclusions are ok.
Additional comments: TITLE
I think “responsible raw?” is misleading and should be removed from the title. Responsibility could not be deducted from this survey.

INTRODUCTION
L57 and throughout: Seen the recent history of pet food, “traditional” is a term that should be avoided when speaking of canine nutrition.

M&M
Further details about the survey creation may be reported following the CHERRIES guidelines on e-surveys.

RESULTS
-	L93-100: Please give precise numbers about the owners demographics
-	Was the signalment of owners’ dogs asked?
-	Even when “most” and “majority” terms are used, please report the precise n/% in brackets
-	Please consider writing all results using both numbers and percentages (n/N, n%)
-	please be consistent using one decimal after comma
-	please verify that percentages sum is 100%; if needed, report the % of “other” option whenever available to reach 100%

DISCUSSION
-	Please respect the same topics order in the results section
-	L202-206: balancing canine diets is not “difficult”, it should be emphasized that this should be done by board-certified veterinary nutritionists whose consultation should always be recommended; usually you do not write the name of universities when citing papers in the main text
-	L226-247: it may be pointed out that interviewed owners were mainly concerned about Salmonella (probably because it is the most talked-about bacterium in general?), even if many studies showed that RMBDs are contaminated by many pathogens
-	There are two recent studies that deserve to be added and widely discussed: Raw meat-based diets for dogs: survey of owners’ motivations, attitudes and practices (Morelli et al., 2019); Owners’ perception of acquiring infections through raw pet food: a comprehensive internet-based survey (Anturaniemi et al., 2019)
-	Commercial RMBDs were shown to be even more contaminated that human grade meat and byproducts (Zoonotic bacteria and parasites found in raw meat-based diets for cats
and dogs; Van Bree et al. 2018; Evaluation of microbial contamination and effects of storage in raw meat‐based dog foods purchased online, Tuberculosis due to Mycobacterium bovis in pet cats associated with feeding a commercial raw food diet, O’Halloran et al. 2019; Morelli et al. 2020); I would add some discussion about safety perception in home-prepared RMBDs vs commercial RMBDs

TABLES
Such tables and values are not effective in displaying Likert-type scores. Please choose a more appropriate style or graph

FIGURES
Figures 5 and 6 seem redundant to me as they show the same results of figures 1 and 2. I would suggest removing them

OTHER
Please provide a copy of the survey to help me understand the source of the results.


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: INSIGHTS INTO DOG OWNER PERSPECTIVES ON RISKS, BENEFITS, AND NUTRITIONAL VALUE OF RAW DIETS COMPARED TO COMMERCIAL COOKED DIETS
Review round: 1
Reviewer: 2

Output:
Basic reporting: This article is well written, clear, concise, and easy to follow. References to contemporary researched background information are appropriate.

Within the results, please include a table of respondent demographics. As the information is currently presented, the reader is unable to determine what other options were available to the respondent other than female, omnivorous, and not working as part of the animal industry. Additionally, which animal industry is being referred to? Please provide more information here. Please also include the number of participants from each country.

While reporting the proportion of respondents ranking 4 to 5 out of 5 in the different categories does convey appropriate information in the results, please include in the tables the prevalence of each rank, not just 4 to 5 out of 5.

The use of the word clouds is a relatively novel way to report concepts identified by the respondents. However, there are some improvements required for these to be more precise. For example, in Figure one, there are multiple concepts that have been reported separately, such as "poop", "poops", "stools" and "allergy", "alergens". Please review the data and combine these similar words into single words so that their impact can be more accurately evaluated. In Figure two, there appear to be concepts that have been separated and are not being depicted clearly. For example, "getting" is unlikely to be a risk that was reported, and this data should be reviewed and the world cloud presented in a manner in which the entire concept is displayed. These recommendations extend to all the word cloud figures.

In figure 6 there is a mistake in the title and description as these do not match. The title is the most commonly cited RISKS but in the description it includes the prompt BENEFITS. Figure 7 has a similar mistake. Figure 8 the prompt does not make sense: "if you feed your dog a raw diet, why not" - likely it was intended to read "if you DO NOT feed your dog a raw diet, why not".
Experimental design: The article presents original primary research with a well defined research question. The background clearly demonstrates the relevance and meaningfulness of the study.

The methodology is vague and does not describe the study with adequate detail to replicate. It is unclear how data regarding dog diet was collected as described in the results section. Within the section "Establishment of Diet", the 'main diet' of the dogs represented in the study is described, but there is no description of how this information was obtained. Please include a more detailed description of how this information was obtained in the methods section and clearly indicate what "predominantly" and "main diet" refer to within the context of the dog diets.
Validity of the findings: The conclusions reported by the authors are consistent with the results. The findings presented within this article will be of practical value to veterinary practitioners. Some novel inferences were made and identify areas for further research.

Most of the data is reported as frequency and proportion, for which no statistical tests are required. However, when comparisons between RMBD and CCD feeders were made, statistical differences were determined using chi-squared testing. This test may not be the most appropriate for ranked outcomes (Tables 1 - 3) and a different statistical test should be considered.

In the discussion it is mentioned that raw feeders do not make a distinction between nutritional quality of homemade and commercial RMBD (Lines 210-211). Please include a brief comment regarding the nutritional quality of commercial RMBD, as there have been studies indicating that commercial RMBD may fare no better than homemade RMBD in terms of nutritional content and balance. This may vary geographically as regulations controlling pet food manufacture and sales practices differ.
Additional comments: This is an interesting article that presents some new insights and reinforces previous findings regarding the behaviours surrounding feeding RMBD to dogs. In general the article is well written, there are just some minor revisions required particularly regarding the methods and results.


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: INSIGHTS INTO DOG OWNER PERSPECTIVES ON RISKS, BENEFITS, AND NUTRITIONAL VALUE OF RAW DIETS COMPARED TO COMMERCIAL COOKED DIETS
Review round: 1
Reviewer: 3

Output:
Basic reporting: The figure labels don’t appear to be listed.
Within lines 33 and 156, one of the most repeated words for risks has been reported as “bacteria(1)”. Relevance of the number 1 here is unclear and needs to be clarified.
Experimental design: Methods are clearly described, using sufficient information to enable reproduction by another investigator, however it would have been valuable for a copy of the questionnaire to have been supplied by the authors. It would also be useful to know how many Facebook pages/groups the questionnaire was posted to initially.
Validity of the findings: Impact of the study has been assessed, with findings clearly reported and predicted to be of interest and value to a wide variety of stakeholders. Further detail regarding the number of survey participants hailing from the USA and UK versus the other countries would have been interesting to include.
Additional comments: This is an insightful and informative article that demonstrates originality, helps to fill the acknowledged knowledge gap in this field and is a pleasure to read.


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: INSIGHTS INTO DOG OWNER PERSPECTIVES ON RISKS, BENEFITS, AND NUTRITIONAL VALUE OF RAW DIETS COMPARED TO COMMERCIAL COOKED DIETS
Review round: 2
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: Dear authors, it was a pleasure for me to revise your manuscript, which has greatly improved after the revisions. I think your work deserves to be published in PeerJ, I just leave a few final comments. Nice job, best regards!

THROUGHOUT THE TEXT
Please be consistent using one decimal after comma: Sorry if this was not clear (I try my best but I am not native speaker), I meant that it would be appropriate to 1) write just one decimal number (e.g. 93.57% becomes 93.6%), as this is commonly enough for surveys and you do not need more overly precise results 2) report the same quantity of numbers of decimals (e.g. 20% becomes 20.0%)

INTRODUCTION
L44: Google with capital letter

M&M
L80: “section 11 consisted of a thank you”, is it too informal? Sorry again, I am not native speaker, it just looks not very elegant to me. Please consider rephrasing if deemed appropriate

RESULTS
L108-109: “One entry consisted only of the consent and no other data so it was not included in the analysis of results.” This sentence serves no purpose and can be removed

DISCUSSION
L189-190: “for reasons that remain unclear” I do not think this is a “scientific mystery” (probably women take more care of the family meals, pets included) and I would avoid this phrase, but feel free to disagree with my opinion

FIGURES
Figures 1: For each parameter, the upper line is the judgement of cooked feeders and the lower line is that of raw feeders? Because it is difficult to understand from the box on the right (whose colors are just black and grey which I cannot find in the figure). Please put a clearer graph legend about owners or just specify this in the figure title/description


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: INSIGHTS INTO DOG OWNER PERSPECTIVES ON RISKS, BENEFITS, AND NUTRITIONAL VALUE OF RAW DIETS COMPARED TO COMMERCIAL COOKED DIETS
Review round: 2
Reviewer: 2

Output:
Basic reporting: No comment
Experimental design: Description of the experimental design has been greatly improved from the first submission.
Validity of the findings: No comment
Additional comments: My comments were addressed appropriately.


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: INSIGHTS INTO DOG OWNER PERSPECTIVES ON RISKS, BENEFITS, AND NUTRITIONAL VALUE OF RAW DIETS COMPARED TO COMMERCIAL COOKED DIETS
Review round: 2
Reviewer: 3

Output:
Basic reporting: The article is well written with clear and unambiguous use of English language throughout. Text is technically correct and the article conforms to professional standards of courtesy and expression.

Sufficient introduction and background provided, demonstrating how the work fits into the broader field of knowledge. Additional current and relevant literature has been incorporated and cited correctly, providing sufficient context.

The revised figures and tables are relevant to the content of the article and are of sufficient resolution. The addition of a table outlining details of respondent demographics is helpful. The use of a diverging stacked bar chart in figure 1 is an acceptable method for presenting rating scale data, however the layout could be improved. The percentage figures are hard to read and identifying which results relate to cooked versus raw feeders could be made more explicit.
Experimental design: The study is within the aims and scope of Peer J and the research objectives are well defined, relevant and meaningful.

Methods are clearly described. Additional information regarding the experimental design and survey details has been supplied, together with a copy of the survey, enabling reproduction by another investigator.
Validity of the findings: Impact of the study has been assessed and additional details have now been added, providing a more comprehensive overview and clearer reporting of the findings.

In general, results have been reported using either (n=X, X%) or X% (n=X). Please adopt this same format throughout the manuscript and ensure consistency; the results reported in tracked lines 327 and 328 (untracked lines 268 and 269) differ. Please also provide both numbers and percentages. The percentage is provided but the corresponding number is missing from tracked lines 210 and 283, however this detail has been included in the untracked version. There is an n missing from tracked line 113 (untracked line 111) - currently 306, 73.21% instead of n=306, 73.21%.
Additional comments: Thank you for re-submitting your manuscript. The revisions have enhanced your article which is predicted to be of great interest and value to readers of PeerJ, however comments have been provided for your consideration in relation to figure 1 and the overall reporting of results.

Your survey has generated a substantial amount of data, some of which has not been reported or fully explored in this manuscript and will hopefully result in the production of further publications in the future.


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: DENSITY-DEPENDENT CONDITION OF JUVENILE PENAEID SHRIMPS IN SEAGRASS-DOMINATED AQUATIC VEGETATION BEDS LOCATED AT DIFFERENT DISTANCE FROM A TIDAL INLET
Review round: 1
Reviewer: 1

Output:
Basic reporting: No comment
Experimental design: No comment
Validity of the findings: No comment
Additional comments: This is a well-written study in which the authors report density-dependent effects in two species of penaeid shrimp inhabiting seagrass beds located at different distances from a tidal inlet in Laguna Madre, Mexico. This was a field study, shrimp samples were taken in triplicate through three tows in each seagrass bed. Salinity, temperature and dissolved oxygen were recorded in each seagrass bed. The sampling method seems correct, as well as analytical and statistical methods.Their results show a negative relationship between body condition and density, with differences related to the proximity of the tidal inlet.
The authors discussed that the proximity of the tidal inlet is a crucial factor in shrimp density, in which the nearby seagrass bed showed higher density and poorer body condition than the distant seagrass bed.
While the proximity of the tidal inlet could indeed be a factor, the authors also report significant differences in salinity and macroalgal biomass between the two locations, but no analysis was performed (or at least it is not shown) to see if any of these factors also play a significant role in density and condition. I would suggest performing this analysis with the data presented in supplementary files 1, 3, and 5 just to know if other factors (besides proximity to the tidal inlet) participate in the density-dependent effect observed in this study.


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

Input:
Title: DENSITY-DEPENDENT CONDITION OF JUVENILE PENAEID SHRIMPS IN SEAGRASS-DOMINATED AQUATIC VEGETATION BEDS LOCATED AT DIFFERENT DISTANCE FROM A TIDAL INLET
Review round: 1
Reviewer: 2

Output:
Basic reporting: The Introduction, Methods and Results are well presented.  The grammar is clear are well structured.  Some sentences would be improved with a minor restructure if the grammar.  I have annotated a few examples in the PDF of the paper.

The grammar in the Discussion is less well structured.  Portions of the discussion drift and seem to suffer from a lack of clear points to reference.  The authors could define two to three critical themes and construct the discussion around them.

The first paragraph (lines 220-224) discusses shrimp condition, then paragraph 5 (lines 254-270) discusses shrimp condition again, and relate the current study to past studies.  These two paragraphs could be consecutive. The reasons for different shrimp density (differing postlarval advection and settlement) and the intraspecific and interspecific competition (food scarcity) can then be discussed.  But see comments on settlement, recruitment and shrimp size class elsewhere in this review.

Paragraph 4 (lines 245-253) is unconvincing.

The Conclusions are generally good, however, the statement that proximity to the inlet positively influenced the abundance of shrimp is not able to be supported by these data.  Proximity is related to shrimp abundance only.

The References comply with Peer J format.
Experimental design: The experimental design is clear.

A criticism of the design is that the data series was conducted for one year only; two or three years would have been more appropriate.
The most robust data gathered by this work are the extensive length/weight data sets for each species caught in each seagrass bed; the ‘distant’ and ‘nearby’ seagrass beds.  These data, together with the density data for each seagrass bed, are enough to support the investigation and conclusions of this paper.  These data should be the bedrock of the paper.

The month-to-month, size step-up analyses undertaken are the least robust data presented.  These data would benefit from a longer sampling period.  Two years of data would provide 23 data points; three years would provide 35 data points.  Each case would provide a more robust correlation series than the 11 data points currently analysed.

A description of the tide range and the likely tidal currents in Laguna Madre is needed (or data of available).  This would assist understanding the capacity for within-lagoon mobility by postlarvae using tidal currents.  For example, if tide range was small and if tidal exchange and current strength was low, then the case put by the authors would have more support.

As well, a description of the extent and possibility of microhabitats of the seagrass community in Laguna Madre would benefit the paper.  Has habitat mapping in general and the seagrass extent within Laguna Madre been undertaken?  Do seagrasses occupy different depth strata?  Have previous studies demonstrated that the current seagrass habitats sampled are the key settlement habitats for shrimp postlarvae?

Sampling Gear
The study techniques (net gear) do not capture the postlarval life-history stage of the shrimp as they would escape the 13 mm mesh.  Loneragan et al. (1994) and Vance et al. (1990) sampled postlatval and juvenile shrimp in seagrass and mangrove habitats and used a 2 mm mesh net (1 mm mesh cod end) in which prawns from 1 mm CL to about 12 mm CL were caught efficiently.  Hence, if the authors were attempting to sample ‘recruitment’ at each of the ‘distant’ and ‘nearby’ seagrass habitats they don’t achieve their aim.  Currently their net configuration allows the 1 to ~5 mm CL animals to escape the net through the mesh.   The unsuitability for the 13 mm mesh nets to catch recently settled postlarvae and very small juvenile shrimp is clear.  In escapement of small shrimp from the nets has been documented by Pérez-Castañeda and Defeo (2005) who state that <30% of shrimp < 8 mm CL are retained by this type of net.

Note that the size structure of shrimp populations in shallow coastal seagrass communities is highly skewed to the smallest size classes.  In Australia, coastal populations of a seagrass-dwelling shrimp (P. semisulcatus) demonstrate that 60-70% of the population is in the 1-3 mm CL range (80-90% is in the 1-6 mm CL range) (Vance et al. 1996, Figure 7; Loneragan et al. 1994, Figure 6), yet this size range is not efficiently sampled with the gear used during the current study.  These 1-2 mm CL shrimp are the critical first recruits to inshore seagrass habitats.

Clearly, shrimp in the 1-6 mm CL size range could have settled in high abundances in each of these seagrass habitats and post-settlement processes (predation, competition, migration, mortality) could have reduced numbers differentially so that by approximately 6-8 weeks after settlement whereby the shrimp reached 7-10 mm CL, their abundance had nothing to do with recruitment, but other processes.  Perhaps there is strong and equivalent recruitment at each seagrass location, yet a collapse of the 2-5 mm CL size range of shrimp at one site and not the other.  The 13 mm mesh nets would not detect these possible aspects of a skewed population structure.
In addition, penaeid shrimp postlarvae can use microhabitats that are closeby to habitats used by larger size classes, but they do not inhabit the exact same spatial distribution, and it may take years to identify the non-overlap (Vance et al. 1990).  Within the same seagrass bed, the shallowest portions of the habitat (0.5 m deep at low tide) can be the critical settlement habitat of postlarvae where they are exclusively abundant.  While medium-sized juveniles are found in both the shallower and deeper seagrass extents (Loneragan et al. 1994).  It is wise to sample a range of depth strata in seagrass habitats to ensure all shrimp size ranges are sampled and critical habitats that are limited in extent and scope are identified and sampled.  Apart from seagrass species and aboveground abundance, only depth of the two seagrass habitats in a range of 1.0 to 1.5 m is mentioned as to the fine scale description of the seagrass habitat.  It is possible that, not only were postlarvae escaping the nets, the critical depth strata of postlarval settlement and recruitment may not have been sampled.

As a consequence, this paper does not clearly show that distance from the mouth of this lagoon is critical to recruitment difference between the two seagrass sites.  Distance is related to the difference, but not necessarily determinant.  Nets set in the water column to catch pelagic larvae entering the lagoon and moving ‘up-lagoon’ in the vicinity of each site (1 km and 25km) would elucidate quanta of potential recruitment at each site.  The sampling of 1-2 mm CL recent recruits also would provide critical information.

This facet highlights a point made clearly in this review.  The length /bodyweight weight relationship is the key dataset that should underpin this paper.  The authors have 1000’s of measured and weighed shrimp; samples from each habitat and a difference in condition between the two habitats separated by 25 km.  Robust determinations and conclusions can be made from these data, and should be.  Relating month-to-month size categories to growth and cohort progression at different shrimp densities from these data is the least conclusive aspect of this study.

Raw data
The most robust datasets described in this paper are the ~2000 and ~ 1000 length/weight data for the two species of penaeid shrimp.  There are 685 individual P. aztecus from the distant seagrass bed and 1426 individual P. duorarum from the nearby seagrass bed.  Similarly, there were 258 individual P. aztecus from the distant seagrass bed and 686 individual P. duorarum from the nearby seagrass bed.  Yet these data are not emphasized in the results or the discussion.  The stated aim of this paper could be achieved using these data alone.  Other analyses are subsequent to the establishment of a difference in shrimp condition.

However, this analysis is mentioned at line 155 of the document.  The exploration of mean abundance of ‘small’ and ‘large’ shrimp by month, and their supposed contribution of the abundance of ‘small’ shrimp to the abundance of ‘large’ shrimp the next month is mentioned in line 135 of the text.

The order of mention of these analyses should be reversed.  The paper depends of a difference in length/weight relationships within the ‘nearby’ and ‘distant’ seagrass beds as a precursor to subsequent analyses.

Several analyses are described in lines 162 to 173; improved delineation and explanation of the logic behind these (at least) three analyses is required.

Conduct cohort analyses such as O’Brien (1994) in each of the ‘distant’ and ‘nearby’ seagrass populations to investigate the growth rates in each habitat type.
Validity of the findings: Similar to the perspective from the methods, the bedrock dataset for this paper is the length/weight data form the two species of shrimp.  Previously, length weight relationships have been used to asses prawn ‘condition’ seasonally and in different habitats (Pérez-Castaňeda, R. and Defeo, O, 2002, Ochwada-Doyle et al. 2011).  The presentation of data by Ochwada-Doyle clearly demonstrates the primacy of the length/weight data to establish the relationships that underpin assessment of condition in distinct habitats.  Their Figure 1 clearly shows the information content of the data to demonstrate   Ochwada-Doyle et al. (2011) used the accumulation of each individual shrimp’s length and weight to describe and compare relationships for shrimp condition between habitats.  That is, linear correlations were determined for shrimp caught in each habitat type and hundreds of animals were included in each correlation.  The large number of data points provided a robust relationship between length and weight and the slopes of the linear relationship could be compared (R > 0.9 in all cases).

Currently, the authors of this paper (Blanco-Martínez et al.) focus attention on their month to month density estimates; presuming a cohort of ‘large’ shrimp represents a cohort of ‘small’ individuals that have grown in size from the previous month.  The annual sampling provides 11 data points.  The analyses rely on the 11 data-points from which to determine a relationship.  For both the ‘distant’ and ‘nearby’ habitats, the scatter plots of the data show a cluster of points in the lower left of the plot, and a scatter of points over the rest of the plot.  These data represent a single year’s sampling.  The analyses determine significant relationships among the data, however, the R2 for each correlation is not strong, both <0.5.  The analyses would benefit from another year‘s data  (or two years (~30 data points)) to put rigour into the relationship.

The focus of the Results should be the ~2000 and ~ 1000 length/weight data for the two species of penaeid shrimp.  There are 685 individual P. aztecus from the distant seagrass bed and 1426 individual P. duorarum from the nearby seagrass bed.  Similarly, there were 258 individual P. aztecus from the distant seagrass bed and 686 individual P. duorarum from the nearby seagrass bed.  The stated aim of this paper could be achieved using these data alone.  Other analyses are subsequent to the establishment of a difference in shrimp condition.

The research reported in the paper fails to measure true recruitment to the ‘distant’ and ‘nearby’ seagrass habitats in Laguna Madre.  There is an extensive literature on settlement and recruitment; and the exact definition of each process remains discussed.  In part, it depends on the taxa being described.  Generally for penaeid shrimp, settlement refers to the morph from a pelagic lifeform to a benthic lifeform; while recruitment can refer to the ‘success’ of recently-settled individuals surviving an initial time-period as benthic individuals.  Either way, in the case of penaeid shrimp postlarvae/early juveniles, recruitment involves animals in the 1 to 3 mm CL size range.  These are true recruits (even 1-2 mm CL individuals) that are new residents in a seagrass habitat and soon afterwards will be subject to predation, competition, migration etc. within the seagrass.

Juvenile P. aztecus and P. duorarum will be subject to ~10-20% mortality wk-1 (Minello et al. 1989, O’Brien 1994, Wang and Haywood 1999) in seagrass habitats prior to their reaching the 7-15 mm CL size range that is the focus of this paper.  They recruit at 1-2 mm CL, survive predation and competition for > 4-6 weeks and may move microhabitat before they are sampled as part of this study.

The authors need to rewrite the Discussion acknowledging these issues.

Figures

Figure 5 is nonsensical.  It plots predicted mean weight against total abundance for the ‘distant’ and ‘nearby’ seagrass habitats grouped, and suggests a relationship is evident.  The basic premise of the study is that there is no weight/abundance relationship in the ‘distant’ seagrass bed, yet there is a relationship in the ‘nearby’ seagrass bed; it is nonsensical to use these data in the same plot and explore a relationship.
Additional comments: This study provides robust data to assess the condition of two species of shrimp in two seagrass habitats in a relatively narrow, elongated lagoon with a single narrow opening to the ocean.  Condition is defined as the relationship between shrimp length (mm CL) and body weight, where a heavier shrimp for a given length is in better ‘condition’.  One seagrass bed is closeby to the inlet, one is 25 km distant; they are similar in the vegetated found at each site, the authors identify distance from oceanic source of shrimp postlarvae as the key factor determining different characteristics of the shrimp population between the two sites.

Lines 82-91 of the introduction make distance from the ocean inlet, the source of pelagic postlarvae, as a key determinant of ‘recruitment’ and hence shrimp abundance.  Higher or lower abundance then is postulated as the driver of shrimp condition.

However, the study does not sample shrimp recruitment.  The key size classes of postlarval recruits (1-2 mm CL) escape from the relatively large meshed nets (13 mm) used during the study.  Dall et al. (1990) include P. aztecus and P. duorarum among their Type 2 or 3 penaeid crustaceans with an offshore spawning, pelagic larval and postlarval phase that advect inshore to settle to a benthic existence in critical shallow coastal nursery habitats, and subsequent ontogenetic emigration back offshore.  The current study highlights 7 to 10 mm CL and 12-15 mm CL shrimp as the focus size ranges analysed.  Shrimp less than about 7 mm CL are poorly represented among the size classes of the catches.  Hence the critical ‘settlers and recruits’ are missed by the gear used to sample the shrimp.

Shrimp in the 7-15 mm CL size range are residents of seagrass habitats, not recruits.  15 mm CL animals are upcoming candidates for ontogenetic emigration to adult habitats.  These are large juveniles that have been resident on the seagrasses for at least 6-8 weeks, and subject to predation, migration, mortality etc.  Zimmerman and Minello (1984) and Minello et al. (1989) studied the population dynamics of juvenile P. aztecus in the Gulf of Mexico in the 1980s and showed that shrimp from 10-30 MM total length (TL) (~1 to 4 mm CL) form the bulk of the population on a seagrass bed.  The basic biology of these species demonstrates that these sizes would be present and the critical phase for recruitment in Laguna Madre.  For a similar seagrass-dependent penaeid shrimp on the other side of the world, O’Brien (1994) provides a detailed breakdown of the size range of juvenile P. esculentus on a seagrass bed (Figure 6), including modal growth analysis of a series of cohorts during the recruitment season of the species.  The dominance of the smallest size classes of shrimp is clear (1-4 mm CL) and the quick demise of each cohort as the individuals grow and reach the size classes that are the focus of this paper (8-15 mm CL) is repeated for subsequent cohorts through the spring/summer seasons.

Despite these criticisms, the current study provides valid data on prawn condition in two seagrass habitats.  It indicates that distance from the oceanic source of the earliest phase of their life history is related to condition and abundance (density) of shrimp within each habitat.  The current study cannot attribute causality to this relationship and does not measure differential recruitment between the seagrass habitats.  The authors need to reconsider the themes underpinning their paper and be clear about what their data demonstrate.

There is a history of juvenile shrimp population studies in coastal lagoon habitats of the Gulf of Mexico that use nets with a relatively large mesh size to sample juvenile shrimp (Pérez-Castañeda and Defeo 2002, 2005).  This gear may be appropriate for the aims of many studies, but for the current study that concludes that recruitment is the key driver of density and hence shrimp condition, the gear is not appropriate as it does not sample the recruits.  It samples mid-aged resident juveniles, the population of which is the result of many post-settlement processes.  The authors need to make this point clear, then continue with their description of condition in two seagrass habitats and then it is valid to hypothesize that distance from the source inlet may contribute to variation in density and hence condition.

Rather than emphasizing the supply of postlarvae alone as the source of variation in abundance and condition between the distant and nearby habitats, the shrimp size classes represented here could couched as representative of the interrelation of growth, mortality and migration and this ‘successful recruitment’ to these habitats of individuals who have survived and likely will contribute to the offshore adult population.  The second main analysis employed in this study attempts to validate this type of theme.  It relates subsequent monthly cohorts of two shrimp size classes as a statistically valid progression.  That is, shrimp in the 7-10 mm CL size class were supposed to grow to form the 12-15 mm CL size class in the subsequent month.  The monthly data plotted to represent this relationship are correlated.  While there is logic to this relationship, only 11 data points are analysed (1 year’s data) and the correlation R2 is < 0.50.  This analysis would have benefitted from a two or three year data series to provide a more robust dataset to correlate.  These analyses are secondary to the length/weight relationships.

The discussion of the paper requires at least two distinct themes to build a narrative around.  The authors need to justify why they do not sample the ‘true-recruits’ within these habitats.  Reference to the early studies of Minello and Zimmerman (see Minello et al. 1989, Figure 1) and their colleagues provide explicit detail on the structure and dynamics of one of the species studied here, including quantification of the full range of size classes found in inshore habitats (see Figure 1), yet the mention of this historical cache of literature is scant.  Studies by Haywood and Staples (1993), O’Brien (1994), Haywood et al. (1998) and Wang and Haywood (1999), provide descriptions of robust analyses of growth and mortality of seagrass-dependent penaeid species (and others), yet only one paper is mentioned briefly.  Best to address the issue of what is recruitment and build an argument from that.  Ochwada-Doyle et al. (2011) provide a clear example of the use of condition assessment in determining the ‘health’ of prawns that are released as part of artificial stock enhancement.  Yet this is a theme that is not explored.  These animals are medium-sized juveniles as well, similar to the size classes investigated in this paper.

This paper has merit; yet the authors need to build a basis for their concept of recruitment, back-up their focus on the size-class-limited medium-size juvenile shrimp and crystalise an argument around these themes and the usefulness of the outcome of their study.

Literature referred to:
Dall, W., Hill, B. J. Rothlisberg, P.C. and Staples, D. J. (1990). The biology of the Penaeidae. Advances in Marine Biology 27.

Haywood, M. D .E., and Staples, D. J. (1993). Field estimates of growth and mortality of juvenile banana prawns (Penaeus merguiensis). Marine Biology 116, 407.16.

Haywood, M. D .E., Heales, D. S., Kenyon, R. A., Loneragan, N. R. and Vance, D. J. (1998). Predation of juvenile tiger prawns in a tropical Australian Estuary. Marine Ecology Progress Series 162, 201-214.

Loneragan, N. R., Kenyon, R. A., Haywood, M. D .E. and Staples, D. J. (1994). Population dynamics of juvenile tiger prawns (Penaeus esculentus and P. semisulcatus) in seagrass habitats of the western Gulf of Carpentaria. Marine Biology 119, 133-143.

Minello, T. J., Zimmerman, D. J. and Martinez, E.X. (1989). Mortality of young Brown Shrimp (Penaeus aztecus) in estuaries nurseries. Transactions of the American Fisheries Society 118, 693-708.

O’Brien, C. J. (1994). Population dynamics of juvenile tiger prawns Penaeus esculentus in south Queensland, Australia. Marine Ecology Progress Series 104, 247.56.

Ochwada-Doyle, F., Gray, C.A., Loneragan, N.R., Taylor, M. D. and Suthers, I.M. (2011) Spatial and temporal variability in the condition of postlarval and juvenile Penaeus plebejus sampled from a population subject to pilot releases.  Aquaculture Environment Interactions 2, 15-25.

Pérez-Castañeda, R. and Defeo, O., 2002. Morphometric relationships of penaeid shrimpin a coastal lagoon: spatio-temporal variability and management implications. Estuaries, 25(2), 282–287.

Pérez-Castañeda, R. and Defeo, O., 2005. Growth and mortality of transient shrimp populations (Farfantepenaeus spp.) in a coastal lagoon of Mexico: role of the environment and density-dependence. ICES Journal of Marine Science, 62(1), 14–24.

Vance, D.J. Haywood, M. D .E. and Staples, D. J. (1990). Use of a mangrove estuary as a nursery area by postlaval and juvenile banana prawns (Penaeus merguiensis) de Man, in northern Australia. Estuarine, Coastal and Shelf Science 31, 689-701.

Vance, D.J., Haywood, M. D .E., Heales, D. S. and Staples, D. J. (1998). Seasonal and annual variation in abundance of postlaval and juvenile tiger prawns Penaeus semisulcatus and environmental variation in the Embley River, Australian: a six year study. Marine Ecology Progress Series 135, 43-55.

Wang, Y.-G., and Haywood, M. D. E. 1999. Size-dependent natural mortality of juvenile banana prawns Penaeus merguiensis in the Gulf of Carpentaria, Australia. Marine and Freshwater Research, 50: 313-317.

Zimmerman, D. J. and Minello, T. J (1984). Densities of Penaeus aztecus), , and other natant macrofauna in a Texas salt Marsh. Estuaries 7, 421-433.


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: DENSITY-DEPENDENT CONDITION OF JUVENILE PENAEID SHRIMPS IN SEAGRASS-DOMINATED AQUATIC VEGETATION BEDS LOCATED AT DIFFERENT DISTANCE FROM A TIDAL INLET
Review round: 2
Reviewer: 1

Output:
Basic reporting: In this revised version of the manuscript the authors made major modifications and it improved substantially. The English language is correct and professional, and references are appropriate and sufficient for the contents of the study.
Experimental design: The experimental design is well explained, with sufficient detail, as well as methods and statistical analysis.
Validity of the findings: Findings are supported by the data, the discussion is well presented and adequate according to the findings, it is not speculative, conclusions are well stated.
Additional comments: This revised version has improved compared to the previous one, the authors have answered my questions and I am satisfied with the answers. The manuscript is well written although I spotted two minor mistakes (line 335, “Shrimp abundance peaked at the beginning…” (instead of beginnin), and line 540, “However, it would be necessary…” (instead of It). Other than that I believe the article is ready for publication.


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

Input:
Title: DENSITY-DEPENDENT CONDITION OF JUVENILE PENAEID SHRIMPS IN SEAGRASS-DOMINATED AQUATIC VEGETATION BEDS LOCATED AT DIFFERENT DISTANCE FROM A TIDAL INLET
Review round: 2
Reviewer: 2

Output:
Basic reporting: The Introduction, Methods and Results are well presented.  The grammar is clear are well structured.  A few suggestions have been made as tracked changes in a Word version where previous tracked changes have been accepted.

The grammar in the Discussion is much improved and mostly is well structured.

The Discussion should be edited to improve its grammatical structure and composition (minor issues).  A few suggestions have been made as tracked changes in a Word version where previous tracked changes have been accepted.

The References comply with PeerJ format.
Experimental design: The experimental design is clear.
A criticism of the design is that the data series was conducted for one year only; two or three years would have been more appropriate.

The most robust data gathered by this work are the extensive length/weight data sets for each species caught in each seagrass bed; the ‘distant’ and ‘nearby’ seagrass beds.  These data, together with the density data for each seagrass bed, are enough to support the investigation and conclusions of this paper.  These data have been analysed and discussed as the bedrock of the paper.

A description of the tide range and the likely tidal currents in Laguna Madre has been provided.  This information assists to understand the capacity for within-lagoon mobility by postlarvae using tidal currents.

At the project conception phase, a strategy that successfully sampled recruitment of pelagic postlarvae to their benthic stage and their short-term success colonising each of the closeby and distant seagrass habitats would have benefitted the paper.
Validity of the findings: The focus of this paper’s Results has been shifted to be the ~2000 and ~ 1000 length/weight data for the two species of penaeid shrimp.  There are 685 individual P. aztecus from the distant seagrass bed and 1426 individual P. duorarum from the nearby seagrass bed.  Similarly, there were 258 individual P. aztecus from the distant seagrass bed and 686 individual P. duorarum from the nearby seagrass bed.  The stated aim of this paper has been achieved using these data alone.

The critical data for this paper are the length/weight data form the two species of shrimp.  Previously, length weight relationships have been used to asses prawn ‘condition’ seasonally and in different habitats (Pérez-Castaňeda, R. and Defeo, O, 2002, Ochwada-Doyle et al. 2011).  The presentation of data by Ochwada-Doyle clearly demonstrates the primacy of the length/weight data to establish the relationships that underpin assessment of condition in distinct habitats.

The Discussion is much improved and reads well.  It provides a good examination of the results of the current study relative to past work.  It also suggests that the outcomes of this research assist an understanding of the contribution of different spatial locations of ‘occupied juvenile shrimp habitat’ within Laguna Madre to the oceanic populations of P. duorarum and P. aztecus.  This relationship allows further studies to be conceptualized.

The Discussions also highlights the gap-in-knowledge about actual postlarval settlement and recruitment within Laguna Madre and how both tidal circulation/movement and seagrass habitat structural complexity may influence the resultant population of juvenile shrimp in these lagoon seagrass habitats, and hence their condition, survival and emigration offshore.  This knowledge gap allows further studies to be conceptualized.
Additional comments: The authors have responded to review by addressing the issues raised.  They have removed the contention that ‘recruitment’ was measured in each seagrass habitat separated by distance from the ocean inlet and its presumed source of pelagic postlarvae.  In addition, they have removed the month-to-month cohort progression and the statistical justification of that size-progression link.

In its current form, this paper has merit and meets a standard of scientific rigour for the experiment undertaken.  The authors have focused on the size-class-limited medium-size juvenile shrimp and presented an argument for the usefulness of the outcome of the assessment of shrimp condition from their study.  The body of work described in the paper would have benefitted from additional sampling of smaller stages in the life history of penaeid shrimp to conclude a more comprehensive and cutting-edge study.  At the conceptual stage and determining the hypotheses to be tested, the limitation of field gear that efficiently caught shrimp > ~ 7 mm CL should have become evident.  The limitation being that first-recruits of penaeid shrimp (~2-3 mm CL) would not be retained in the net by 13 mm mesh gear.  The experiments described in the paper would have benefitted from a sampling regime that did sample recruitment at each seagrass habitat, as well as the condition of shrimp encountered.

The authors could have undertaken pelagic plankton tows in the vicinity of each of the seagrass habitats in part to test their hypothesis that the potential for ‘recruitment’ was significantly greater closeby to the ocean inlet.  They could have used a small mesh net (~2mm mesh) in conjunction with the 13 mm mesh net that they used to sample the postlarvae and smallest juvenile phases of the shrimp on the seagrasses.  Field data that estimated actual recruitment of 2-3 mm CL shrimp or potential recruitment (1-2 mm CL Postlarvae) would have complemented the data presented in the paper and ensured much more interest in the author’s body of work from shrimp biologists worldwide.


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: DEVELOPING A MACHINE LEARNING MODEL TO IDENTIFY PROTEIN–PROTEIN INTERACTION HOTSPOTS TO FACILITATE DRUG DISCOVERY
Review round: 1
Reviewer: 1

Output:
Basic reporting: It is fine. No comment.
Experimental design: It is fine. No comment.
Validity of the findings: There are many many algorithms and machine learning approaches to predict protein-protein interaction, hot spot, drug binding affinity, etc. Most of these methods differ slightly in various metrics. The new features added into this study ( features related to amino acid composition, dipeptide composition, etc., to the already pre-existing data) should be listed in a Table. Also in the final results as in Figure 1, are the new features in the top list, and how the adding of new features change the overall distributions of feature importance.

The second question is the identification of the potential medications listed in the paper. Figures 5-8 should be combined into a larger figure with four panels. More description about the identification and selection should be provided. Details for top screened drugs is better in main text, with additional entries to prove that these drugs are outstanding from virtual screen. What are rankings of these drugs among hits. Otherwise, it is dubious that these drugs could be hand picked.
Additional comments: Overall, it is important to improve accuracy of machine learning in predicting various protein-protein interaction. This paper represent a rigorous approach to add more relevant features to screen hot spot related factors.