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: GLUCOSYLCERAMIDE SYNTHASE REGULATES HEPATOCYTE REPAIR AFTER CONCANAVALIN A-INDUCED IMMUNE-MEDIATED LIVER INJURY
Review round: 1
Reviewer: 2

Output:
Basic reporting: The experimental study by Jian-Gan et al. has been designed to investigate the action of Glucosylceramide synthase (GSC), in the regulation of hepatocyte repair after concanavalin A-induced immune-mediated liver injury,  using 7-9-week-old pathogen-free male C57BL/6 mice. The authors performed an experimental design by GCS siRNA transfection before Con A administration. Authors report that administration of GSC in mice on Con A injection downregulated the hepatic expression of IL-4, IL-6, and IL-10.  Moreover, the authors also report that administration of GCS reduced ALT, AST levels. Authors conclude that GCS inhibition reduces Con A-induced immune-mediated liver injury in mice, which may be due to the involvement of GCS in the hepatocyte repair process after injury.  In addition, I consider that it should be improved the clarity and professional language of writing.

I suggested including “Con A” meaning in results from the abstract. Because of up to lines 98 and 283-284, it mentioned, “concanavalin A” and “Con A is a kind of plant lectin extracted from jack beans (Canavalia ensiformis)”.

The introduction provides a worthy, generalized background of the topic that gives the reader an appreciation of the importance of study the molecular mechanism of glucosylceramide synthase in glycosylation metabolism by regulating the hepatocyte repair process after injury. Although to make the introduction more considerable, the author may wish to provide percentages or data about immune-related chronic liver injuries, specifically autoimmune hepatitis prevalence or incidence to substantiate the claim made in the 60-65 lines. Besides updated references about this suggestion.

Articles proposal:
1.  Tunio NA, Mansoor E, Sheriff MZ, Cooper GS, Sclair SN, Cohen SM. Epidemiology of Autoimmune Hepatitis (AIH) in the United States Between 2014 and 2019: A Population-based National Study. J Clin Gastroenterol. 2020 Oct.
2. Sucher E, Sucher R, Gradistanac T, Brandacher G, Schneeberger S, Berg T. Autoimmune Hepatitis-Immunologically Triggered Liver Pathogenesis-Diagnostic and Therapeutic Strategies. J Immunol Res. 2019 Nov 25;2019:9437043.
3. Lv T, Li M, Zeng N, Zhang J, Li S, Chen S, Zhang C, Shan S, Duan W, Wang Q, Wu S, You H, Ou X, Ma H, Zhang D, Kong Y, Jia J. Systematic review and meta-analysis on the incidence and prevalence of autoimmune hepatitis in Asian, European, and American population. J Gastroenterol Hepatol. 2019 Oct;34(10):1676-1684.
Experimental design: The hypothesis is clearly defined in 78-80 lines. However, I suggested adding an aim “The purpose of this study was to…” in the introduction in line 60 line to provides more clarity and comprehension for the reader since the beginning of the article.

In Figure 2, the tissue picture of HE subsection B) is of poor quality. A better picture should be presented. Another suggestion is to include labels in the figure to improve clarity ( A-D represents the control group, Con A group, GCS siRNA+Con A group, and Genz + Con A group).

The experimental study by Jian-Gan et al. is novel and of potential interest in the field of immune hepatitis. Data and conclusions reported are convincing and I have just a minor request:

Authors should offer, morphological evidence that inflammatory infiltrate is increased in the Co A group compared to the Control group (immune-histochemistry for CD68, F4/80, for example).

Furthermore, after stating that the choice of flow cytometry technique for evaluating apoptosis rate is important, the author offers a clear explanation of why he chooses this method in the present work. I think the motivation for the present research would be stronger if the author could provide a more direct link between the importance of choosing an appropriate method and the results obtained.

In material and methods, I consider including the name of the image analysis program they used and the number of images captured in histological and immunofluorescent in line 146.
In line 150, there is an error of "electrophoresi" word, inside of electrophoresis.
I recommend improving the English language and redaction of 148-152 lines and 174-176 lines for more clarity and understanding from readers.
Validity of the findings: As suggested above, I think a more in-depth discussion of Figure 4 would be helpful. I consider this is an important result for this article, and therefore it merits more discussion.

No significant limitations are discussed. It may be worthwhile to mention the tradeoffs involved in choosing flow cytometry as opposed to some other method.
Additional comments: Did a pathologist confirm the histological analysis of the HE stain?
I recommend include arrows in figure 2 and figure 3, that represents explication in results.
More separate scale in figure 6B (example 0,1,2,3,4).  Homogenize graphics formats in "y axis".


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: GLUCOSYLCERAMIDE SYNTHASE REGULATES HEPATOCYTE REPAIR AFTER CONCANAVALIN A-INDUCED IMMUNE-MEDIATED LIVER INJURY
Review round: 2
Reviewer: 1

Output:
Basic reporting: The manuscript has been significantly improved.
Experimental design: The experimental design is correct.
Validity of the findings: Main findings have been effectively validated in the current revision version
Additional comments: The authors have responded to all reviewers´ comments. I think it is suitable for its publication in its current form


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: GLUCOSYLCERAMIDE SYNTHASE REGULATES HEPATOCYTE REPAIR AFTER CONCANAVALIN A-INDUCED IMMUNE-MEDIATED LIVER INJURY
Review round: 2
Reviewer: 2

Output:
Basic reporting: Authors have complied with my queries.
No comment.
Experimental design: Authors have complied with my queries.
No comment.
Validity of the findings: Authors have complied with my queries. No comments.
Additional comments: Authors have complied with my queries. No 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: ACTIVE BEHAVIOUR OF TERRESTRIAL CATERPILLARS ON THE WATER SURFACE
Review round: 1
Reviewer: 1

Output:
Basic reporting: I admit this has been a bit difficult ms to revise as there are overall some meaningful aspects brought to light. However, focusing on its logical structure and theoretical background, I have to highlight some formal and conceptual flaws that lead me to suggest for a full rejection because some decisive steps to streamline methodology with the premise and to bring interpretations in line with evolutionary thinking are lacking.

The ‘story’ starts from the empirical observation that if some fully terrestrial caterpillars happen to fall by chance into water, they are capable to “swim” on the surface, then to escape predation and safely reach the nearby vegetation.

The methodology then involves studies on such kind of locomotion by throwing into water caterpillars of several terrestrial species and recording those capable of “swimming”, either via undulatory or kick movements.

The analysis details aspects of such locomotion according to larval instars, body mass, shape etc.

The conclusion claims that such “swimming” attitude is a preadaptation which may explain colonization of aquatic environments by caterpillars not just of Noctuoidea but in general.

Granted that there are some known swimming caterpillars in Noctuoidea that regularly inhabit semi-aquatic or regularly flooded environments, above all those of the Neotropical genus Paracles (duly recorded in the ms), in this piece of research I see no relation between the main assertion in the premises, that of some typically terrestrial caterpillars can swim, and the methodology to proof such assertion. In fact, the methods just detail aspects of the locomotion giving for granted that this is a kind of swimming, but real swimming has yet to be demonstrated. Hence there is a logical leap in how reasoning develops.

I see an obvious difference between normal locomotory reactions shown by caterpillars placed under stressful circumstances and the proper concept of “swimming”. For instance, when they face a menace, many species show the so-called jerking behavior (see Stamp & Casey 1993. Caterpillars), which seems to exactly correspond to the undulatory movements seen in the videos accompanying the ms. The only difference would be that with normal jerking the larva holds onto the substrate with the false-legs, while in liquid this cannot happen and the undulations thus propagate towards the end of the body. Whilst the looping or semi-looping locomotion seems at the base of the kick movements seen in another video. I suspect then that there are much more parsimonious explanations accounting for such kinds of movements in the aquatic medium.
Experimental design: As just said, I see no links between the driving idea of the ms and methods. They are totally uncoupled.

I would like to see a methodology oriented towards probation of the initial really heavy assertion that such terrestrial species do swim, otherwise this would only remain postulated. I would like to see a kinematic comparison among exclusively terrestrial Noctuoidea caterpillars, supposedly/occasionally swimming ones and truly swimming ones, ideally also with anatomical analysis of their muscles.
Validity of the findings: Due to failure in probating the suggested evolutionary path, any assertion about preadaptations has to be challenged too. I think no preadaptation can be claimed to have been discovered unless it has been realized. Only when one adaptation has been achieved, some premise may be inferred to have represented a preadaptation.

However, let’s admit that the research unveiled a basic locomotory pattern which may explain swimming in other groups.

It becomes essential here to bring some phylogenetical thinking into the analysis, otherwise all variables and players would remain loose and ultimately unrelated. For sure there are no direct relationships between arctiine Paracles and the species of present trials, thus how can the latter reveal a preadaptation realized in the former? In theory it could be done, but the authors should be able to demonstrate that there is a common groundplan locomotory attitude shared by a monophyletic group of Noctuoidea which subsequently diversified into swimming by some lineages and jerking (or kicking) by others (or both).

In the ms instead, the conclusions are pushed even further, being extended not just to Noctuoidea, something that has to be demonstrated yet, but in general to aquatic caterpillars.

Very weak are also claims that six of the seven "swimming" species occur in riparian habitats and make use of such preadaptation to rescue themselves, thus implicitly assuming that it is an adaptation proper. This is fully speculative and subjective, in the absence of a thorough analysis of the ecological distribution of such species, some of which (Spirama retorta, Hypopyra vespertilio, Dinumma deponens and Lymantria dispar) I can confirm are non-hygrophilous elements. Laelia coenosa, Naranga aenescens and Acosmetia biguttula are instead hygrophilous. So what is the relationships among them? How a same explanation can be extended to species with such different environmental requirements? Are the authors assuming that the "preadaptation" works already as an "adaptation" in some species? How all this stands together given their phylogenetic separation? Only explanation is that the attitude is homoplasous, but then it cannot be a synapomorphy, then there would be no shared preadaptation.
Additional comments: To sum up, I think the core part with the analysis of locomotion is meaningful but it does not really fit with the main scope of the research. At the present state of knowledge, the ‘pseudo-swimming’ shown by some terminal taxa seems more parsimoniously ascribable to known locomotory patterns expressed under unusually stressful circumstances. It should not be claimed to reveal a  preadaptation to ‘swimming’ in other far apart, not directly related terminal taxa.

I think also the authors may have really opened a window on some ancestral locomotory character and encourage them to carry on with their research born after a serendipitous observation, but everything should be demonstrated, while as of now, the work essentially suggests an analogy, which seems a too weak justification for deserving publication. Showing that such “analogy” is a homology would instead decidedly convert the work from exclusively speculative to a sound scientific one.

Speculation is welcome in the journal, but this should at least build on some achieved results, while at this state of the study I see admittedly none.


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

Input:
Title: ACTIVE BEHAVIOUR OF TERRESTRIAL CATERPILLARS ON THE WATER SURFACE
Review round: 1
Reviewer: 2

Output:
Basic reporting: This strikes me as a tight, careful study, and fully worthy of publication in PeerJ. The manuscript is concise, well edited, focused, well organized, and well referenced. The amount of information in the Table 1 is impressive. The finding that swimming ability increased with size (across instars) is novel and well documented. My comments are minor and mostly represent suggestions, rather requirements.
Experimental design: Very good. Experimental design, details of methods, statistical analysis, and figures appear to be strong.
Validity of the findings: See below. I have only minor quibbles.
Additional comments: The abstract should be recrafted to down-weight the mention of the evolutionary/comparative aspects of the study, which get little treatment, and are not (yet) carried out or discussed in a phylogenetically (rigorous) manner—see below. This sentence could be deleted or re-worded: “Because some aquatic caterpillar species belong to these moth families, we posit that the active swimming observed in these terrestrial caterpillars represents preadaptation to an aquatic environment.” (It’s fine to speculate about these matters in the Discussion. See also below.) To the Abstract they should make mention of some of their key experimental results, which are not yet mentioned, e.g., that the frequency of active swimming behaviors and speed of swimming increased across instars/body size, and perhaps other key findings.

Some mention of surface tension of water and its role in the observed floating and swimming is warranted, especially because there are caterpillars that swim below the surface of the water. This matter might be handled either in the Introduction or Methods. Somewhere they should state that they only dealt with taxa that swim at the surface of water (aided by water tension). (And while the physical properties of water tension are recognized as important, they are not treated in the study.) I wonder if the authors observed any species that broke through the water surface? What happened?

Table 3 and 4 appear unnecessary and that the essential data therein could simply be added to text.

The evolutionary arguments are a bit weak. While the authors claim that the observed swimming behaviors across four families represent a pre-adaptation, and they are probably correct, they did not carry out comparative tests, e.g., of whether related caterpillars of arid environments also had innate swimming behaviors. Or make comparisons between surface swimmers and fully aquatic taxa. No mention is even made of the locomotory movements of fully aquatic species. In a concluding sentence the authors state, “Active swimming will likely be found in other lepidopteran families (e.g., Crambidae) that include aquatic caterpillar species.” Which, to my mind, argues that swimming behaviors are selected for/under selection and thus seemingly (weakly) argues against swimming being a pre-adaptation. Regardless, I would tighten up the logic and language; perhaps simply suggest in the Discussion that the elongate bodies and undulating, locomotory actions of caterpillars on land appear to have pre-adapted Lepidoptera for active swimming behaviors. And these behaviors in turn, have pre-adapted some caterpillars to become fully aquatic.

Unless the arguments can be bolstered/expanded in the Discussion, I think the evolutionary claim needs to be downweighted in the Abstract. If it is retained in the Abstract the authors should give the basis for the conclusion, something akin to: The discovery of active swimming behaviors across four different families of Lepidoptera is suggestive that the elongate bodies and locomotory systems of moth larvae have pre-adapted the lineage for swimming.

I suggested edits and added these to a pdf version of the manuscript that I am returning.

In sum, I think this a strong effort that is novel, well documented, handsomely illustrated, and acceptable for publication with only minor revisions.

David Wagner

PS As a side note, I have observed woolly bear caterpillars (Arctiinae) swimming, and foraging underwater in the Neotropics—craziest thing I have ever seen! I think the caterpillars that I observed were members of the genus Paracles. I reared one in aquarium on submerged lettuce and lawn weeds! The fully fed larva came to the surface, formed a floating mat out of its own setae that it tore off its body with its mandibles, and then pupated on the floating raft of setae!


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: ACTIVE BEHAVIOUR OF TERRESTRIAL CATERPILLARS ON THE WATER SURFACE
Review round: 2
Reviewer: 1

Output:
Basic reporting: Having reviewed a previous version of this ms, I will be now very straightforward and state that I am very happy with the current draft. The findings are interesting, worth of being shared with the scientific community, all unsubstantiated speculations not even fitting with current knowledge on both ecology and systematics of the families involved have been removed, and from now on further research may be build upon the current findings.
Experimental design: All is clearly stated and replicable. I may just stress that is more usual to see the three dimensions of the plastic 'vessel' (possibly better 'container') in mm and not as mm3, as only after the multiplication is done the mm3 are got.
Validity of the findings: Ok.
Additional comments: I am recommending a truly minor revision to just adapt to the following suggestions:
1) per instar stage - looks redundant, as the stage is the larval one. only 'per instar' would be ok;
2) despite it can be used after the plural, behaviour is usually used as singular, especially in sentences like 'two types of behaviour', but also on other occasions. I guess it may be mostly employed at the singular throughout the ms;
3) last sentence of animal ethics - it should be areas;
4) ref Sugiura S, Takanashi etc. should be moved above Sugiura S, Yamazaki K.
5) see above for mm, mm3;
6) I would not say 'silk threads produced from spinnerets' as silk is produced by the relative glands whose secretion is emitted through the spinneret; better to use 'emitted', 'released' or some equivalent expression;
7) in using the silk threads for hanging down by the caterpillars, the important phenomenon of 'ballooning' to disperse at the larval stage has not been mentioned at all;
8) the ref 'van Griethuijsen LI, Trimmer BA. 2014' should better be placed as 'Griethuijsen LI, van, Trimmer BA. 2014' due to small 'v', also because that's the way after it is cited in the main text;
9) In the discussion, the sentence that 'Aquatic behaviours have been reported in some aquatic and semi-aquatic caterpillars' looks obviously circular. Furthermore, in the examples that follow it may look weird to many readers that Ostrinia penitalis is mentioned while the important aquatic group of other pyraloids such as Acentropinae/Nymphulinae (or Nymphulini) is omitted.


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: ACTIVE BEHAVIOUR OF TERRESTRIAL CATERPILLARS ON THE WATER SURFACE
Review round: 2
Reviewer: 2

Output:
Basic reporting: See below
Experimental design: See below
Validity of the findings: See below
Additional comments: This paper is original, includes novel observational from field and lab. It is focused, grounded, well-written. The figures and videos are great. Overall, I find the manuscript acceptable with minor revision.

There is substantial redundancy that can be eliminated—for example, I recall some six places where the authors mention that there are seven species that exhibit “forward movement on the water.” Geesh. The same ideas get repeated too many times. I recommend trying to shorten the Abstract and text by 10%.

The hypothesis in the introduction is forced, uninteresting, and unnecessary as written. Why does one need a hypothesis, “that some terrestrial caterpillars can exhibit forward movement on the water surface” when you already observed the behavior and filmed it? It’s just filler, if this is going to be the hypothesis. This is largely a novel observational study—it doesn’t demand a hypothesis.

I disagree with other reviewer that this is not swimming. According to Merriam Webster:
Definition of swim; intransitive verb”:
1a: to propel oneself in water by natural means (such as movements of the limbs, fins, or tail)
b: to play in the water (as at a beach or swimming pool)
2: to move with a motion like that of swimming : GLIDEa cloud swam slowly across the moon
3a: to float on a liquid : not sink

The text would read better if the action were called swimming. Other workers will NOT be able to find the work, if it is called something other than swimming. No one that is interested in swimming will be able to search and find “forward movement on the water surface.”

The larger matter is why they “swim” and I think this was the issue the other reviewer had with the first draft. Intent. We/you don’t know why they exhibit swimming behavior. The behavior could be multifunctional. Beyond helping to avoid predation, the behavior is necessary to reunite the caterpillar with its hostplant, it also prevents drowning, and may return the larva to substrate upon which it could/would settle until the time to feed again. If you place an erebid caterpillar on glass over white paper, many would move to a new site of safety where their coloration would more cryptic. Caterpillars see. They assess their light environments. In sum, I would call it swimming, but think you should backpedal on why they swim—that is unknown.

Please discuss/mention the two families that did not exhibiting swimming behaviors—their failure to swim is important.

The larvae does not kick the anal prolegs to swim. Look at your videos. The caterpillars use the entire abdominal terminus, folding it under and then flicking it rearward. No less than segments A7-A10 are involved. This is flicking behavior, not kicking. This is more analogous to the snapping movements of fleas and Mexican jumping bean caterpillars than it is when kicking, i.e., where the appendages propel the individual. It is not definitely an error to state anywhere in the manuscript that these insects are kicking with their anal prolegs. Categorically false. Nor is the movement equivalent to the smooth upward and downward movements of cetacean swimming propelled by flukes. I am surprised I didn’t catch this the first time I read this paper—and apologize. This must be corrected before I would recommend the manuscript for publication.

I made a Word version from the pdf, and added many edits to text and figure legends. Many of the suggestions will help to make the text more concise and less redundant. Happy to help with a last read before resubmission or help in other ways.


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: ACTIVE BEHAVIOUR OF TERRESTRIAL CATERPILLARS ON THE WATER SURFACE
Review round: 3
Reviewer: 1

Output:
Basic reporting: Being already a third review round, please go directly to final commenting.
Experimental design: Ditto.
Validity of the findings: Ditto.
Additional comments: Unfortunately, the authors have taken the opportunity of a comment by the other reviewer that the locomotion observed may be termed “swimming”, and turned back to their original interpretation, which undoubtedly would make the title catchier. I am again here only to avoid that other scholars would make fun of the authors like it happened at a congress presentation that I attended many years ago. The presenter reported on the “swimming” behavior by terrestrial ground beetles (Carabidae) (note: terrestrial like the caterpillars of ours) that had been thrown in a pool to observe their “escape” strategy (note: chance events like those of ours in an overwhelmingly hygrophilous group). The audience burst into hearty laughter and sometimes, when participants to that congress meet, they still recall such episode with great fun despite over 30 years have elapsed. “Do you recall the silly talk of the swimming carabids?”

This work is perfectly twin to that research and I would not do a good job if I recommended to call the behavior as swimming. Authors: do you prefer a more fashionable title at the risk of damaging your reputation or would you just be satisfied with a nice article published in PeerJ avoiding any hindrance to your career? I would have no hesitation if I had to recommend a choice to my students.

Regarding terminology, it is a well-known fact that many terms in life sciences are borrowed from common speech, but they regularly have a more restricted meaning. In case of interest, there is only one word whose meaning is broader in biology (anthropology) than in common speech, that is “culture”. Accordingly, despite dictionary definitions, “swimming” in biology assumes a kind of locomotion made possible by a coordinated integration of muscles, nerves, proprioceptors and stimuli that could only have developed as an evolutionary response to some ecological needs (that’s why the original idea of “preadaptation” was unacceptable). These caterpillars are not aquatic, they fall in water by chance and at a ratio impossible to drive an evolutionary path, let alone the maintenance of such a complex anatomo-physio-ethological system as if real natatory behavior was involved.

If the authors want to pursue with their intent, I do not want to feel responsible for their reputational damage and am compelled to recommend again a full rejection.

Nothing in biology makes sense except in the light of evolution (Dobzhansky, 1973).


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: ACTIVE BEHAVIOUR OF TERRESTRIAL CATERPILLARS ON THE WATER SURFACE
Review round: 3
Reviewer: 2

Output:
Basic reporting: This is a clear, significantly shorter version, without much of the redundancy of the previous drafts. The authors have incorporated nearly all my suggestions from my two previous reviews, including my line edits to their text. So I have only a couple minor reservations.

The new paragraph on the evolution of the swimming in (terrestrial) caterpillars is speculative and based on extremely small sample sizes, but it is so short that I feel it is fine. But in three separate sentences, the authors make a sweeping statement that surely has exceptions. All three sentences require qualifying language--I have made some suggestions that the authors may find helpful. I have also offered suggestions to the Conclusion.
Experimental design: NA
Validity of the findings: The findings are clear, succinctly presented.  The videos provide a great rendering of the swimming behaviors
Additional comments: This is a clear, significantly shorter version, without much of the redundancy of the previous drafts. The authors have incorporated nearly all my suggestions from my two previous reviews, including my line edits to their text. So I have only a couple minor reservations.

The new paragraph on the evolution of the swimming in (terrestrial) caterpillars is speculative and based on extremely small sample sizes, but it is so short that I feel it is fine. But in three separate sentences, the authors make a sweeping statement that surely has exceptions. All three sentences require qualifying language--I have made some suggestions that the authors may find helpful. I have also offered suggestions to the Conclusion.


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

Input:
Title: GENOME-SCALE IDENTIFICATION OF PLANT DEFENSIN (PDF) FAMILY GENES AND MOLECULAR CHARACTERIZATION OF THEIR RESPONSES TO DIVERSE NUTRIENT STRESSES IN ALLOTETRAPLOID RAPESEED
Review round: 1
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: The manuscript entitled “Genome-scale identification of plant defensin (PDF) family genes and molecular characterization of their responses to diverse nutrient stresses in allotetraploid rapeseed” submitted by Liu et al shows great effort in elucidating plant defensin gene divergence in allotetraploid rapeseed and how these genes expression change in response to diverse nutrient stresses. This work is very important for the in-depth study of PDF function. However, several content organization need to be slightly rephrased to highlight importance of their work before it can be accepted for publication.

1.	Line 26: change into following should be clear. “Three distinct clades were identified in BnaPDfs phylogeny. Clade specific conserved motifs were identified within each clade respectively.” Please check your MEME results and denote those domains.

2.	Line 120-125: Please add more detail and explain how it work briefly.

3.	Line 142: format problem, please check.

4.	Line 217: “The shoots and roots were sampled separately and were…” What kinds of shoots and roots?? Please make it clear.

5.	Line 293-295, 299-300, 304-305, 315-317, 392-394, 498-400, 408-409: please move to introduction.

6.	Gene structure of your target gene exhibits great variety. Please check your Figure 4. The intron and exon length differs among your target genes.

7.	Line 459-467: Here is a repeat of your results, not discussion. Please rephrase.

8.	Line 510-511: This is rough. Please make your statement clear.

9.	The following articles about the function of defensins CAL2, AtPDF2.2 and AtPDF2.3 should be cited in the background.
(1)	Luo JS, Xiao Y, Yao JY, Wu ZM, Yang Y, Ismail AM and Zhang ZH. Overexpression of a Defensin-Like Gene CAL2 Enhances Cadmium Accumulation in Plants. Frontiers in Plant Science. 2020, 11:217.
(2)	Luo JS, Zhang ZH. Proteomic changes in the xylem sap of Brassica napus under cadmium stress and functional validation. BMC Plant Biology. 2019,19:280.


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

Input:
Title: GENOME-SCALE IDENTIFICATION OF PLANT DEFENSIN (PDF) FAMILY GENES AND MOLECULAR CHARACTERIZATION OF THEIR RESPONSES TO DIVERSE NUTRIENT STRESSES IN ALLOTETRAPLOID RAPESEED
Review round: 1
Reviewer: 2

Output:
Basic reporting: The manuscript submitted by Liu et al deal with the description of rather unknown family of proteins, defensins. This genome wide analysis is well performed, although I have several comments on the works.

The introduction have some spelling or English problems, as in line 50 and in line 72 (better to say Arg 38, is more scientific spelling). But my major problem is that the introduction needs more work about the role of defensins related to the experiment that are performed in the article. Authors perform experiments on nitrate, and other compounds that are not defined in the short introduction of this experiment.

Figures are extremely small, especially those that contain trees or domains (1, 2) and are almost impossible to follow.

The main fail of this paper is that many figures in the discussion present data that are impossible to see, like 11 A, B, D. 12 B, C (this even have a negativa caption in the axis). And there is statistic asterisk that doesn't make any sense. In every footnote, authors only claim that asterisk indicate a P<0.05 statistic difference but in the figures you include, indistinctly one, two or three stars, without indicate what that does mean!. Authors should improve the figures, like breaking the axes to star reviewing this data,
Experimental design: For the identification of brassica PDF I don't understand how the authors perform that. I don't get if the identification has been made only with the PDF domain or if the whole A.thaliana proteins have been used. Moreover, authors perform a whole identification of PDF in A. thaliana (even the chromosome identification), when this is not the scope of this work.

In the line 124 authors claim that if a gene is within a 100kb region are defined as tandem. This is a very large distance, and did not present any reference for that. Also, have you checked that this putative tandem genes are similar between them?
The reference used in line 228 about the AACt calculation is not accurate, as is not the one that explain that methods.

When authors performed phylogenetic tree, like in the figure 2B, they should include the bootstrap number moreover because they say they calculated It (line 133).

It is noteworthy that you haven't performed any curation on the sequences obtained. Other articles on genome-wide investigation have discovered genes that there is genes that may contain not complete domains or duplication that needed to be curated. Did you find some genes like that?
Validity of the findings: Between lines 245 and 250, author preform a identification of A.thaliana PDF, which is not the scope of this work.

In lines 261-263 authors claim that those genes are tandem, are those genes more similar between them than others located in other Chr?

When author perform MEME to identify domains in the sequence, have you checked if any of those domains are predicted in proteins databases as PDB?

In the results section, when describing the qPCR data, you perform an introduction, like in lines 377 to 383 that are more a discussion rather than results. You should move these description to the discussion.

In line 455, how can be the extracellular matrix the site of PDF biosynthesis?

Lines 463 and 465 why are this lines here? What do you want to say with that? It the same with the lines 468 to 471, you need to discuss more this section

In lines 505 to 509 you made a nice study of PDF related to potassium channels. Have you find any of this sequences in the brassica PDF sequences related with potassium?

As stated in the basic reporting part, authors should improve the qPCR figures to continue the revision.
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: GENOME-SCALE IDENTIFICATION OF PLANT DEFENSIN (PDF) FAMILY GENES AND MOLECULAR CHARACTERIZATION OF THEIR RESPONSES TO DIVERSE NUTRIENT STRESSES IN ALLOTETRAPLOID RAPESEED
Review round: 2
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: Though, the authors have made a great effort in improving the manuscript. Still, I am not satisfied with the revised version. There is a scope for another round of revision. Please see the attached file without track changes for line numbering. Some of the comments are as follows:
	Line 27-28, please mention the common and scientific name together on the first appearance in the abstract and the main text, i.e., rapeseed (Brassica napus L.).
	Line 34, enriched elements. Please add enriched cis-elements, and the word “cis” should be in italic throughout the text.
	Line 37, please specify the tissues and specific genes.
	Please carefully check the entire text and make sure the genes name should be in italic, but not the protein.
	Line 97, Blackshaw et al., 2011 can be replaced with a recent review https://doi.org/10.1007/s00344-020-10231-z.
	Line 119-120, please mention the threshold e-value used to identify the AtPDFs using the Hidden Markov Model (HMM V 3.0) profiles.
	The legend is missing in Fig 1 (A and B).
	In table 1 and 2, please do not merge the Arabidopsis and rapeseed genes. Arrange them in order, firstly Bna followed by AtPDFs.
	In the methodology sections, there is no information about the statistical analysis used in Fig 7-12. Please add a separate section of “statistical data analysis”.
	Further, please rearrange the graphs (Fig 7-12). Remove the green and orange legend for each graph and keep it at the end near to the last graph, no need to repeat it for each graph. Further, there are so many extra spaces between the graphs. In one line, 3-4 graphs can easily be adjusted. So, please check and revise all graph figures.
	Did the authors try to predict the putative miRNA targeting BnPDF genes and GO annotation analysis? If no, I suggest adding it to the current version to add additional insights into the current work. This work will not take much time based on my own experience but greatly improved the work strength. For miRNA prediction, the authors can try with psRNATarget database http://plantgrn.noble.org/psRNATarget/. If you can identify any miRNA targeting any of your genes, you can add the results accordingly. If there are no miRNAs, please omit them. However, you can try GO annotation results.
	Line 460, 476, this paragraph is just the repetition of the results part. Please try to compare your findings with the previous ones and support your results.
	Line 591-595, make a separate conclusion section and update the conclusion with future directions.
	The English language needs improvement.


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

Input:
Title: GENOME-SCALE IDENTIFICATION OF PLANT DEFENSIN (PDF) FAMILY GENES AND MOLECULAR CHARACTERIZATION OF THEIR RESPONSES TO DIVERSE NUTRIENT STRESSES IN ALLOTETRAPLOID RAPESEED
Review round: 3
Reviewer: 1

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


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

Input:
Title: GENOME-SCALE IDENTIFICATION OF PLANT DEFENSIN (PDF) FAMILY GENES AND MOLECULAR CHARACTERIZATION OF THEIR RESPONSES TO DIVERSE NUTRIENT STRESSES IN ALLOTETRAPLOID RAPESEED
Review round: 3
Reviewer: 2

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: Dear Authors,
Thank you for revising the MS according to the proposed comments and suggestions. Though the authors have addressed most of the comments. Still, I feel there is a scope for further improvement. I believe these minor corrections will help in improving the overall work presentation.

	In the abstract, add a brief line describing the miRNA targeting PDF genes.
	Add “miRNA” in the keywords.
	Supplementary Fig 7 (GO results) and Table S1 (miRNA) are not cited in the text.
	In supplementary Table S1, the vital information is missing. Please add the following information in different columns 1) miRNA name 2) Target gene ID 3) Gene name 4) Expectation	5)	miRNA_start 6) miRNA_end	7) Target start 8) Target end 9) miRNA_aligned_fragment 10)	alignment 11) Target_aligned_fragment 12) Inhibition. The miRNA result table should comprise of these 12 columns. From the given table, readers cannot identify this vital information of miRNAs.
	Please add a subheading for GO and miRNA results, “GO annotation and miRNA prediction”. Also, add the GO terms ID in the text.
	In the discussion, please support the finding of miRNA targeting PDF genes. Is there any previous report of PDF genes in any organisms targeted by different miRNA families?
	Please give a subheading to the last paragraph. “conclusion”.


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

Input:
Title: SUBSTRATE TYPE AND PALAEODEPTH DO NOT AFFECT THE MIDDLE JURASSIC TAXONOMIC DIVERSITY OF CRINOIDS
Review round: 1
Reviewer: 1

Output:
Basic reporting: The hypotheses presented are sound, however, there is no research question/problem that is stated in the introduction to indicate the need to test these hypotheses. It is not until the discussion section that the problem is mentioned (i.e. the hypotheses posited by Hans Hess). The introduction can be greatly strengthened by explicitly stating the problem that the hypotheses would address. Additionally, lines 70–72 suggest that there might be distinct crinoid provinces in addition to those observed in ammonites. However, there is no mention of possible crinoid diversity differences between provinces in the results or discussion. Perhaps adding if/how crinoid diversity, paleodepth, and substrate occurrence differ between the two provinces would strengthen the overall effectiveness of the paper.

The use of “reservoir” in the context of this study is problematic. Reservoirs in a geologic context refer to accumulations of fluids (such as hydrocarbons and groundwater) within rocks. I believe “reservoir“ is used here to refer to depositional system (i.e. carbonate vs. siliciclastic dominated). I suggest avoiding the use of “reservoir” in this context.

The English language could be improved throughout the manuscript so that readers can clearly understand the information conveyed. I have annotated the manuscript in areas where there are grammatical mistakes with suggestions to improve for clarity.
Experimental design: Looking over the supplemental material, I see that comprehensive lithological data are missing. I don’t question the interpretation of water depth the authors have made as their results are consistent with other studies, but are there any other lithological data to strengthen the interpretations?
Validity of the findings: Conclusion section can be strengthened by linking results back to the research problem. The results of study suggest that some crinoids can be used to infer water depth while others can not. Additionally, there is no explicit summary for how water depth and substrate affect crinoid diversity as stated in the title. Furthermore, the discussion section could be strengthened by linking results back to the research problem more directly as well. Perhaps a final paragraph that summarizes whether crinoids can indeed be used to infer water depth and how water depth and substrate affect crinoid diversity.
Additional comments: Overall, this study is an intriguing test of whether crinoids can be reliably used to infer substrate type and water depth using the fossil occurrences from the Middle Jurassic of Poland. The authors are to be commended for their rigorous taxonomic approach as well as diligently presenting background information, methodology, and discussion to support their conclusions. Additionally, as far as I can tell, their taxonomic descriptions are sound. With that being said, this manuscript suffers from a few structural and grammatical issues that weaken the presentation of the study.

An option to consider to strengthen the paper further (but not required) would be to use quantitative methods to test whether various crinoids indeed have a habitat preference (i.e. water depth, substrate). If there are comprehensive lithologic data, they can be combined with ordination techniques to graphically demonstrate the association of crinoids and water depth and substrate. Ordination techniques such as non-metric multidimensional scaling or detrended correspondence analysis could be used, which are relatively easy to use. This approach could also be used to test whether there is evidence for crinoid provinciality within the Central European Basin. In case the authors are not familiar with these techniques, they find more information on ordination techniques, particularly, non-metric multidimensional scaling here: https://www.palass.org/publications/newsletter/palaeomath-101/palaeomath-part-13-mds-and-ordination and here: https://strata.uga.edu/software/pdf/mdsTutorial.pdf


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: SUBSTRATE TYPE AND PALAEODEPTH DO NOT AFFECT THE MIDDLE JURASSIC TAXONOMIC DIVERSITY OF CRINOIDS
Review round: 1
Reviewer: 2

Output:
Basic reporting: This MS is clearly written with good English. It appears rushed in places. Some English is informal. For example, they site Hunt & Underwood (2009) when it is Hunter & Underwood (2009). Hunt is distinct from Hunter as a family name. Balanocrinus subtrees does not exist maybe its Balanocrinus subteres? In addition, lots of key references are missing.

The MS is well cited throughout. The title suggested a broad study, However there is a bias towards the Polish literature and to the Middle Jurassic.

The structure of the confusing in its current state, the title and abstract suggest a study with a wide general significance. However, the introduction and data analysis is very specific and the discussion reads more like a literature review rather than focusing on the very useful data-set.

The data from the cores is certainly interesting, but the MS appears to be a mixture of limited new data from cores and a literature

It is not clear how the new data is relevant to the extensive discussions of previous research on Middle Jurassic. The figures and tables are limited for example Figure 2 “Model showing distribution of selected Callovian crinoids.” Why selected? Does this mean data has been omitted?
Experimental design: Part of this MS is “Original primary research within Aims and Scope of the journal” however, as parts of the data is limited is relies heavily on a literature review of previous research.

The core premise of the MS is to cast doubt that crinoids are, with a few exceptions, benthonic organisms are generally considered as good indicators for determining environmental conditions. This is an important question in the study of crinoids. This is a useful case study, but I am not sure the quality and preservation of the data is enough to fulfil the two very bold questions.  Do Callovian crinoids occur equally and frequently in both deep and shallow water environmental settings (palaeo-depth dependency)? and (b) Do Bathonian and Callovian crinoids occur equally and frequently in both carbonate and siliciclastic facies (substrate dependency)? It is not clear from the discussion if these questions are answered. The results of the paper get lost in a long discussion of other studies. The conclusions are very general for example “Almost all Bathonian and Callovian crinoids recorded from Poland dominate in relatively shallow environments”. This observation is certainly not new. “The only exception are cyrtocrinids, that are equally common in both shallow and deeper settings.” Again, this is well known. “Isocrinids, most commonly recorded, occur frequently in both Carbonate and siliciclastic rocks” Again very general statement and not a new idea. The Research question needs to be better defined there is no discussion on where previous studies have failed to answer these questions. In contrast the methods on core analysis are useful can could be expanded.
Validity of the findings: All underlying data has been been provided but it is not robust in that the facies are generalised. The discussion reads too much like a literature review rather than a detailed discussion of the data provided in the tables. As a result, the conclusions need to be revised.

I am a bit confused as the MS confidently, says that “isolated remains of isocrinids, consisting of columnals, pluricolumnals, cirrals, cup plates, and brachials, are classified at the specific level.” Yet authors then discuss the  Problems with isolated isocrinid classification Most of this section does not take into consideration recent literature on Pentacrinites

I have big problems with the “Distribution of crinoids within particular facies. Does it really work?” In that it compares species from very different environments are paleobiogeographic provinces and in some ways is oversimplified and is over focused on the Middle Jurassic. For example, Balanocrinus subteres might be a cosmopolitan crinoid in Poland. But this might not be the case in the UK or North America.
Additional comments: In Conclusion this is an interesting study, that questions if crinoids as possible indicators of Paleo Depth. As the authors point out there is ample research that demonstrates a link between robust proxies for inferring changes in salinity and sedimentation rate and for inferring substrate type. I am supportive of this MS and I believe it should be published in some form after a major revision. However in its current state I fail to see how the current MS challenges this observation with limited new data and a discussion of previous published data.

1. The title is a bit misleading, it suggested a global study, when in-fact it’s a local cases study from (a) archival Bathonian-Callovian (Middle Jurassic) crinoid occurrences from Poland and (b) newer finds from five borehole data from eastern Poland.

2. It is from a limited timescale Bathonian-Callovian. Despite this it makes a bold conclusion that use of crinoids as reliable proxies for inferring reservoir depth (paleodepth) and substrate type, should be considered with caution. Yet make these conclusions there needs to be discussion of crinoids outside the Jurassic. Such as the extensive literature on the Cretaceous and Palaeogene.

3. The sedimentology is over-generalised into “near shore close to beach” or shore face carbonate ramp” or Offshore/open shelf. This is does not take into consideration local variations within these defined facies. Sadly this is one of the  issues in using borehole data.

4. The title uses the word reservoir, which should only be used in its correct context within Petroleum geology. I think indicators of palaeodepth are more appropriate

5. One of the achievements of studies such as Hunter and Underwood (2009) was that it looked a diverse set of Formations grouping these into lithofacies and in Hunter and Underwood (2009) tapho-facies. These facies covered a limited time interval of the Bathonian. However this study lumps data from the whole Middle-Late Jurassic.


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: SUBSTRATE TYPE AND PALAEODEPTH DO NOT AFFECT THE MIDDLE JURASSIC TAXONOMIC DIVERSITY OF CRINOIDS
Review round: 1
Reviewer: 3

Output:
Basic reporting: The manuscript is exhaustively referenced, with many recent, high-quality publications cited.

The regional geologic background is provided and explained clearly, as is the basis for identification of crinoid elements.  The taxonomic status of the major crinoid groups encountered is discussed, with classification issues addressed.

Data are provided, both in the form of novel data collected from analysis of new borehole sampling and a literature review.

There are a number of minor grammatical and formatting errors throughout the manuscript.  It would be best to go through the manuscript thoroughly after making any substantive revisions to "clean up" any wording issues.  Some examples are provided below:

1. Line 19: the first sentence needs to be re-worded (perhaps "...benthonic organisms THAT are generally..."

2. Line 34: Balanocrinus subteres is mis-spelled ("subtrees").

3. Line 52: an extra parenthesis is present.

4. Lines 73-76: this paragraph needs to be divided into several sentences or have some modified punctuation in order to be grammatically correct.

5. Line 261: "high dynamic" should probably be "highly dynamic".

6. Line 293: "alsoi.e."

7. Lines 404, 406, 412: The Hunter and Underwood article is listed in the references as 2009, not 2010.
Experimental design: This contribution represents original primary research.  It also serves as a novel compilation and synthesis of previously described crinoid occurrences.

The techniques used to collect new data are described in sufficient detail for replication.

The criteria used to identify and classify recovered crinoid remains are outlined, consistently applied, and appear systematically sound.

The knowledge gap filled by this research and its broader significance is outlined, although the scope of this study is somewhat under-stated.  For example, the discussion of taxonomic standardization and the systematic issues relevant to some Jurassic crinoids--which comprise a large portion of the manuscript--are not mentioned in the beginning of the paper.
Validity of the findings: In general, conclusions are supported by all available data.  Interpretations are directly linked to data.

There are only a few substantive issues that I wanted to raise:

1. On lines 240-241, it is stated that all paleo-depth interpretations should be taken as accurate, as all crinoid remains are autochthonous.  However, on lines 255-257, it is stated that the high diversity of asteroid ossicles in the Lukow glacial drift reflects transportation of elements into deeper water.  This makes it seem as though at least some of the crinoid elements may have been allochthonous as well.

2. It is not clear why the description and discussion of the Lukow glacial drift is included on lines 251-272.  What does this add to the manuscript?

3. At the end of the abstract (lines 37-38), it is stated that the use of crinoid remains for paleoenvironmental interpretation should be considered with caution.  However, the results of the study demonstrate a number of consistencies and corroborate a number of previously suggested paleoenvironmental preferences.  Moreover, the need for caution is not really brought up significantly in the discussion section and is not mentioned in the conclusions section.  The fact that this statement comprises the end of the abstract makes it seem as though the need for caution is the most important take-home message of the paper, but this does not appear to be the case.

4. This is less significant, but (1) I don't like the use of the word "reservoir" in the title and throughout the manuscript, as it is not clear how this word is being used; and (2) the word "Polish" or "Poland" should probably occur within the title, as this is not a global survey of Middle Jurassic crinoids.
Additional comments: This is a highly ambitious contribution, and I commend the authors on their work.  In particular, I appreciate the detailed research on primarily disarticulated specimens and isolated ossicles, which are generally under-studied.


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: SUBSTRATE TYPE AND PALAEODEPTH DO NOT AFFECT THE MIDDLE JURASSIC TAXONOMIC DIVERSITY OF CRINOIDS
Review round: 2
Reviewer: 1

Output:
Basic reporting: With the change of title and the introduction this MS is much improved. This is a very useful study and should be published. It means the apparent bias towards the Polish literature fits well with the introduction to the MS. The revisions mean that the hypothesis is now clear with the twin objectives clearly spelt out.
Experimental design: I have no further issues with the experimental design as limitations of using data from boreholes his clear.
Validity of the findings: I commend the authors for a very good revision that re-frames the MS in the Polish context but now makes the MS significant case study that can be built on using other examples of crinoids from the Middle Jurassic and as the authors have said, this can now be applied to other parts of the stratigraphy. Before publication I would like to see a short discussion on the influence of time averaging, abrasion and transportation on the samples. They also point out that Hunter and Underwood used quantitative analysis for inferring crinoid distribution patterns. But actually, the samples were exhaustively sampled so that ALL the ossicles from the beds sampled and therefore taking into account the bias from preservation and time averaging. I look forward to seeing this published!
Additional comments: They have included Salamon, M.A., Gorzelak, P., Zatoń, M., 2010. Comment on “Palaeoenvironmental control on distribution of crinoids in the Bathonian (Middle Jurassic) of England and France” by Aaron W. Hunter and Charlie J. Underwood. Acta Palaeontologica Polonica, 55, 172–173.....Can they also include my reply as its relevant to the preservation of the crinoids?


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: CATOSTYLUS TAGI (CLASS: SCYPHOZOA, ORDER: DISCOMEDUSAE, SUBORDER: RHIZOSTOMIDA, FAMILY: CATOSTYLIDAE) LIFE CYCLE AND FIRST INSIGHT INTO ITS ECOLOGY
Review round: 1
Reviewer: 1

Output:
Basic reporting: As a not native English speaking person I cannot appreciate true value of the language, but my opinion is the English can be improved.
The rest is Ok, except the figures, some of which need editting in lebels.
Experimental design: No comment - everithing is Ok
Validity of the findings: Everything is Ok, but discussion should be improved.
Additional comments: ABSTRACT
The last paragraph of the abstract describes the methodology and results in short, excessively short. It must present more results in more details.

In the Results it is a long and detailed description of the ephyra development (growth) stages, missing the temporal parameters. And no discussion of these data in Discussion. The authors need either discuss (compare) their data with the similar descriptions of other species, or remove such detailed descriptions.

The final part - the spatial and seasonal distribution of the species (in the Results and Discussion) - in its present description sound poorly connected with the rest of the manuscript. One possibility - is to connect the revealed ecological preferences of the species with its records in the region. At the moment it sounds vice-versa.

lines 1, 38, 42, 348, Table 4 - Rhizostomida -you should clearly define the taxonomic classification used in the study. According to WoRMS taxon names are different. Moreover sometimes the spelling is different in different parts of the manuscript: Rhizostomida or Rhizostomidae...

Other comments you will find in 'comments' in the file.


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: CATOSTYLUS TAGI (CLASS: SCYPHOZOA, ORDER: DISCOMEDUSAE, SUBORDER: RHIZOSTOMIDA, FAMILY: CATOSTYLIDAE) LIFE CYCLE AND FIRST INSIGHT INTO ITS ECOLOGY
Review round: 1
Reviewer: 2

Output:
Basic reporting: This research, even if it contains original data of undoubted interest, has some criticalities.
English would need to be refined.
References ok.
Article structure should be improved; the morphological / morphometric description is sufficient, but the section concerning the experiments is a bit confusing and unclear.
Fig. 7 is unclear and confusing
There are also some doubts about the experimental design system, see next point.
Experimental design: The authors tested the effect of temperature and salinity on planula development, and this is OK, but why didn't they perform the same type of experiments on the asexual reproductive rate of the polyps?
The authors limited themselves to analyzing the effect of temperature and feeding on asexual reproduction, but this section is somewhat confusing and unclear.
As for the statistical analysis, some doubts fall in the methodological approach adopted. Following the authors’ rationale, the nature of repated measurements (line 194) have not been considered in the models. GLMM or LMM would be appropriate in such type of data. Furthermore, in relation to the performed models, I think that, once a Poisson GLM model was overdispersed, the Authors have to opt for a quasipoisson distribution of error (just changing the overdispersion parameters) since prediceted values (integers) are more realistic than those predicted by a negative binomial distribution.
In general, the whole section describing statistical methods can be improved and Authors choices better justified.
Validity of the findings: I do not doubt that the observations made by the authors in the final discussion are correct, but the problems indicated above make them poorly supported.
Additional comments: I would suggest to refine the research as indicated above, perhaps adding an experimental part on the asexual reproductive rate as a function of the main environmental variables, temperature and salinity, and not only as a function of nutrition. Also review the statistical analysis section, as indicated above.


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

Input:
Title: CATOSTYLUS TAGI (CLASS: SCYPHOZOA, ORDER: DISCOMEDUSAE, SUBORDER: RHIZOSTOMIDA, FAMILY: CATOSTYLIDAE) LIFE CYCLE AND FIRST INSIGHT INTO ITS ECOLOGY
Review round: 1
Reviewer: 3

Output:
Basic reporting: 1) I would strongly recommend this article to be revied by a native speaker. English, which is used here, is ambiguous. Abstract is especially hard to understand due to the fact that sentences are not connected with each other. Moreover, there are multiple typos and some mistakes in terminology.
Problems with language, that I can define:
Lines 1.38.42. “Rhizostomida”, the preferred name of this suborder is Dactyliophorae. Line 1. Also, it is strange to name class and suborder (Scyphozoa, Rhizostomida) without naming the order (Scyphozoa, Rhizostomeae, Dactyliophorae). Maybe naming only class and order will be more appropriate (Scyphozoa, Rhizostomeae).
Lines 32-33 are unreadable. What do you mean by “jellyfish outbreaks are … natural events”? What do you mean by “environmental… activities”? How the first leads to the second?
Line 34. “different jellyfish life stage” – it should be: “different jellyfish life stages”
Line 34.385. “its” – “it’s”
Line 35. “have its full life cycle understood” – I’m not sure the word “understood” is a good fit for the scientific article.
Line 38. “scyphistoma presents 18 tentacles” – The word “presents” doesn’t look like a good choice.
Line 39. “Only podocyst production was observed.” – This sentence should be expanded. In the current state it conflicts with the next sentence, where you talk about strobilation, that you also observed.
Line 41. “ephyrae… at liberation” – The word “detachment” or “release” is usually used.
Lines 52-53. “Tagus estuary” is used twice.
Lines 56-57. What do you mean by “The outbreaks often have severe… ecosystems”? Please rephrase.
Lines 57-60. The sentence is too complicated and thus very hard to understand.
Lines 63.115.157. I would recommend to use the word “attached” instead of “fixed”.
Lines 64-65.224-225.326-327.332-333. “(e.g. budding, swimming buds, podocysts, fission, strobilation) (Arai, 1997)”; “lateral budding by stolon, lateral scyphistoma budding, or pedalocysts”; “various budding modes (e.g. lateral budding, budding from stolon, motile bud-like tissue particles, etc.) and podocysts (Arai, 1997)”; “lateral budding, lateral budding through stolons, reproduction from parts of stolons/stalks, motile bud-like tissue particles, podocysts) (Schiariti et al., 2014)” – The unification of the names of modes of asexual reproduction is required, especially while you citate the same article on this lines.
Line 68. “both phases integral to the study” – I’m not sure the word “integral” fits here.
Lines 112.119.148.149.150.154.271.273.277.280.282.350.366.377.Figure 5.Gigure 6. Please use the sign ‰ , when you specify the salinity.
Line 115. “given to planula choice to” – please rephrase.
Lines 140.230.237-238.249.320.Table 1.Figure 1.Figure 2. “ephyra lappet elongated”; “Ephyrae typically had eight marginal arms with a pair of antler palm-like rhopalial lappets and a single rhopalium per lappet” – unification of the terms is needed. Which part of ephyra do you call “marginal arm”, “marginal lobe”, “lappet”, “marginal lappet”, “rhopalial lappet”, “rhopalia lappet”?
Line 142. “MDD/CL 9 100” – I believe it was supposed to be “MDD/CL x 100”.
Lines 143.144. “CDD/TBD 9 100”, “RLL/TMLL 9 100” – the same.
Line 148. “three temperature” – “three temperatures”.
Line 161. “The effect of temperature and feeding on asexual reproduction” – “The effect of temperature and feeding regime on asexual reproduction”.
Line 162. “two temperature levels” – “two temperatures”.
Line 163. “three feed regimes” – “three feeding regimes”.
Lines 163-164. “Eighteen polyps were tested in each of the six combinations.” – This sentence is ambiguous and thus should be rephrased.
Lines 164-165. The sentence is very hard to understand. Please rephrase.
Line 215-216. 218. “total body length (TBD)” – I believe it was supposed to be “total body length (TBL)”.
Line 218-219. “was distinctly longer than wide” – seems to be a poor choice of words, specifics are required.
Line 227. “strobiles” – “strobilae” is preferable.
Lines 229-230. “calyx upper part” – How do you define “up” and “down” in strobila? It would be correct to use terms “oral”-“aboral”.
Lines 245-248. “The first oral tentacles develop on the distal ends of the manubrium. … The first oral tentacles develop around the manubrium opening” – information seems to be doubled.
Line 258.Table 2. “two other canals develop horizontally” – the word “horizontally” doesn’t describe the position of the structure in the jellyfish body. Please rephrase.
Line 260. The word “canals” is missing.
Line 359. “up to” is used twice.
Table 1. “ep:ephyrae” – “ep:ephyra”.
“ephyrae:500μm” - “ephyra:500μm”
“ephyrae” - “ephyra”
“•18 filiform tentacles •Long, club-shape and flexible” – I believe it was supposed to be “•18 filiform tentacles; long, club-shape and flexible”
“•MDD ≈ 44 % of the TBL” – Is everything correct here? Diameter in relation to length?
Table 2. “Development stages” - “Developmental stages”.
“• Rhopalia canals are sli forked, rounded points • • rhopalial canals slightly forked with rounded points” – information is doubled.
Figure 3. “Enlargement of the mouth development of Catostylus tagi.” – “Enlargement of the mouth of Catostylus tagi.”
“at the lips mouth in (stage 1)” – the content is not clear.
Figure 5. “Stage 1: 1-3 tentacles” – but on the lines 157-158 “Stage 1: 1-4 tentacles”.
Figure 7. “C. tagi asexual reproduction under temperature and diet regimes.” – the word “different” seems to be missing.
“ephyra. strobilation-1” - “ephyrae.strobilation-1”.

2) The background is present, but it lacks the known information on ephyra development. This work is not the first to show the development of gastric system and margin of the Scyphozoan young medusa. For example:
•	Holst, S. (2012). Morphology and development of benthic and pelagic life stages of North Sea jellyfish (Scyphozoa, Cnidaria) with special emphasis on the identification of ephyra stages. Marine Biology, 159(12), 2707–2722. doi:10.1007/s00227-012-2028-0
•	Holst, S., Sötje, I., Tiemann, H., & Jarms, G. (2007). Life cycle of the rhizostome jellyfish Rhizostoma octopus (L.) (Scyphozoa, Rhizostomeae), with studies on cnidocysts and statoliths. Marine Biology, 151(5), 1695–1710. doi:10.1007/s00227-006-0594-8 (Which is by the way cited in the text).
Lines 61-62. “With some exceptions (e.g. Pelagia noctiluca), Scyphozoan species are meroplanktonic with a bipartite life cycle.” - There is a lack of references.
Lines 322-325. “This anatomic characteristic of C. tagi distinguishes the species from the other two Rhizostomatidae, Rhizostoma luteum (Kienberger et al., 2018) and Rhizostoma octopus (Holst et al., 2007), occurring in the same geographical area.” – Please specify, what is known about velar canals of Rhizostoma luteum and Rhizostoma octopus, how exactly are they differ.
Line 363. “(Gueroun, unpublished data)” – the date is also required.
Lines 363-368. It would be great if you’ll describe the dynamics of salinity and temperature changes during the year in Tagus estuary.

3) Structure of the article is clear. There are just few notes:
Abstract lacks the conclusion.
Between the lines 244 and 245 there is a lack of introduction. Stages of what are you going to describe?
Lines 308-310. “Our observations of adult gonads from individuals collected in the Tagus estuary (Portugal) showed the absence of planulae” – This information wasn’t mentioned in “results”.
Lines 359-360. “C. tagi scyphistomae were repeatedly strobilated twice a month (≥18°C), producing up to up to 15 ephyrae (6.8 ± 3.6)” – The same.

Table 1 and Table 2 would be better in a form of complex Figures with letters on each photo. Thus you will be able to make more concrete references in the text.
Also margins of the photos in Tables 1 and 2 should be corrected: less black space is needed and the color of the background must be well matched (in the current state the slight difference between colors is seen).
Table 4 is very informative, it is a pity that the information in this table isn’t discussed in the text.
The necessity of Figure 2 is questionable.
The color coding on Figure 7 is ambiguous.
Experimental design: Some notes on Materials and methods:
Line 112. “containers of ultraviolet treated artificial seawater (salinity 35) at 18°C” – What artificial seawater was used in experiments? What brand or the exact composition if it is self-made?
It is unclear, how much food was used per feeding.
What was the approximate number of planulae, used in experiment? – it should be mentioned in the text.
Lines 172-173. “Specimens in the unfed treatment never received food.” – Was the protocol of water exchange the same for this group?
Validity of the findings: How many fully developed polyps had 18 tentacles? It doesn’t seem plausible that this number was constant, because according to the literature (for example, articles that you cite in Table 4) Scyphozoa polyps usually have number of tentacles, which is multiple of four. Some individuals have extra tentacles or lack few tentacles but most have 16 or 32 tentacles. Please check your data.
Lines 245-267 and Table 2. Please add the timing of the stages. Most likely it isn’t the exact timing, but rather time ranges, nevertheless they should be given.
Additional comments: The research is important, well established and informative (Only the number of tentacles is questionable and the timing of the stages is missing). But unfortunately, the text of the article is poorly written and definitely should be revised.


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: SEX- AND STAGE-DEPENDENT EXPRESSION PATTERNS OF ODORANT-BINDING AND CHEMOSENSORY PROTEIN GENES IN SPODOPTERA EXEMPTA
Review round: 1
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: In the MS, authors identified odorant-binding proteins (OBPs) and chemosensory proteins (CSPs) in and established the expression patterns in Spodoptera exempta based on transcriptome analysis. It seems that the analysis is ok, but there are several major problems which need to be solved carefully. If not, the MS cannot be considered for the next step of the submission. As follows:
1.	In the work, authors identified 35 OBPs and 29 CSPs which contain intact open reading frames based on transcriptome analysis. I wonder that did the authors clone the 35 OBPs and 29 CSPs and sequence them by Sanger sequencing? I did not find the information of the primers, and I am not sure the authors conducted above things. From the perspective of myself, the clone and Sanger sequencing are essential for the identification. Please make it clear for me.
2.	About the expression analysis of the OBP and CSP genes, in the MS, authors worked on the OBP and CSP genes of S. exempta, but, in the line 131 to 133, why “the expression of the identified OBPs and CSPs genes was calculated based on the TPM values from the transcriptome data of S. exempta, S. frugiperda, and S. littoralis”?
3.	About the Sex- and stage-dependent expression patterns of OBP and CSP genes in the MS, the authors identified the differentially expressed genes (DEGs) by the TPM values, but after that, no further work of validation was performed. In my opinion, real-time quantitative PCR needs to be conducted and then, the DEGs can be validated. Why the authors neglected the validation?


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: SEX- AND STAGE-DEPENDENT EXPRESSION PATTERNS OF ODORANT-BINDING AND CHEMOSENSORY PROTEIN GENES IN SPODOPTERA EXEMPTA
Review round: 1
Reviewer: 2

Output:
Basic reporting: Dong et al. investigated the OBPs and CSPs in a major global crop pest (Spodoptera exempta), sequence alignment indicated that 30 OBPs and 23 CSPs completely contained the conserved cysteines. Their data with whole bodies indicated that all highly expressed OBPs and CSPs in adults were male biased or were not diﬀerent between the sexes, suggesting diverse functions of OBPs and CSPs in adult insects. However, there are several issues that must be addressed before it can be accepted for publication.

1. L32, L43, some contents are repetitive and need to be modified.
2. L75-77, the expression is not clear, please add relevant references to support this conclusion.
3. L82, add the two references: Zhang et al., 2013, plos one, 10.1371/journal.pone.0069715b; Zhang et al., 2017, peerj, 10.7717/peerj.3157.
4. L87, add the two references: Zhang et al., 2017, FIP, 10.3389/fphys.2018.00432, Li et al., 2021, BER, 10.1017/S0007485321000109.
5. Figure 1, Lack of signal peptide sequences. I suggest labeling OBP with complete ORF and different types of OBPs: classic, minus-c, plus-c…
6. Figure 2, I suggest labeling CSP with complete ORF.
7. Figure 3 and 4, the non full-length ORF genes should be deleted.
8. I suggest that the authors randomly select some genes for quantitative PCR to verify the data in Figure 8 and 9.
Experimental design: Dong et al. investigated the OBPs and CSPs in a major global crop pest (Spodoptera exempta), sequence alignment indicated that 30 OBPs and 23 CSPs completely contained the conserved cysteines.
Validity of the findings: Their data with whole bodies indicated that all highly expressed OBPs and CSPs in adults were male biased or were not diﬀerent between the sexes, suggesting diverse functions of OBPs and CSPs in adult insects.
Additional comments: Dong et al. investigated the OBPs and CSPs in a major global crop pest (Spodoptera exempta), sequence alignment indicated that 30 OBPs and 23 CSPs completely contained the conserved cysteines. Their data with whole bodies indicated that all highly expressed OBPs and CSPs in adults were male biased or were not diﬀerent between the sexes, suggesting diverse functions of OBPs and CSPs in adult insects. However, there are several issues that must be addressed before it can be accepted for publication.

1. L32, L43, some contents are repetitive and need to be modified.
2. L75-77, the expression is not clear, please add relevant references to support this conclusion.
3. L82, add the two references: Zhang et al., 2013, plos one, 10.1371/journal.pone.0069715b; Zhang et al., 2017, peerj, 10.7717/peerj.3157.
4. L87, add the two references: Zhang et al., 2017, FIP, 10.3389/fphys.2018.00432, Li et al., 2021, BER, 10.1017/S0007485321000109.
5. Figure 1, Lack of signal peptide sequences. I suggest labeling OBP with complete ORF and different types of OBPs: classic, minus-c, plus-c…
6. Figure 2, I suggest labeling CSP with complete ORF.
7. Figure 3 and 4, the non full-length ORF genes should be deleted.
8. I suggest that the authors randomly select some genes for quantitative PCR to verify the data in Figure 8 and 9.


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: SEX- AND STAGE-DEPENDENT EXPRESSION PATTERNS OF ODORANT-BINDING AND CHEMOSENSORY PROTEIN GENES IN SPODOPTERA EXEMPTA
Review round: 1
Reviewer: 3

Output:
Basic reporting: The English language need to be improved by the native speakers or professional science editing service.
Experimental design: This is a gene sequence analysis and expression pattern comparison manuscript based on the previous transcriptome dataset without any new experiments conducted in this study. The analysis methods are suitable and well-rounded.
Validity of the findings: This study did the sequences analysis of S. exempta OBPs and CSPs based on previous transcriptome dataset and compare the expression patterns of them in different development stages. The draft may can provide the sequence info of SexeOPB/CSPs for further functional characterization of these proteins.
Additional comments: In this study, Dong et al., investigated the S. exempta OBP/CSPs sequence features and expression patterns in different development stages. 40 OBPs and 33 CSPs  were identified based on the previous transcriptome dataset. The result provide a molecular basis for further functional characterization of these genes and also pest management control strategies. Please find the comments below which may help improve the draft.
Line 61-64: is this true? In my understanding, the OBPs function remain unclear. There are several models of the roles of OBPs in olfactory system. I would not like to say how OBP bind to odor molecules and help olfactory perception in sensillum lymph.
Line 91-92: change "fifth instar larvae on the first day of this stage larvae" to " the larvae on the first day of the fifth instar stage".
Line 127-128: change the sentence "...samples of S. exempta from single pairs, including the larvae on the first day of the fifth instar stage, pupae, and adult males and females,..." to "...samples of S. exempta from single gender mixed pairs, including the larvae on the first day of the fifth instar stage, pupae, and adult,..."
Line 135: it should be "one-way ANOVA" and at 0.05 significant level?
Line 164: change "thirty" to "30".
Line 169: change "twenty-three" to" 23".
Line 181-182: could you please add the principal component analysis method in the method section? and explain how you conduct the PCA.
Line 214: change "fourteen" to "14".

The transcriptome dataset access number should be provided somewhere in method section.

In the discussion section, could you add more deeper discussion of the function difference of OBP/CSPs among different species mentioned in the text? such as recent OBPs CRISPR KO studies. Can you make a table/figure like SI table3, with the info such as how many OBP/CSPs in different species mention in discussion and which group they belonged to？

The figure legends must be enriched. 1. For example, in figure 3/4 legend, please add what the x-axis showed? and how you get the Seqlogo (which software?) 2. in figure 5/6, what is other species in the tree? how many OBP genes in each species? It will be better provide more info in figure legend, not only in SI 1. 3. the insect name can be abbreviated in figure legends.


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: SEX- AND STAGE-DEPENDENT EXPRESSION PATTERNS OF ODORANT-BINDING AND CHEMOSENSORY PROTEIN GENES IN SPODOPTERA EXEMPTA
Review round: 2
Reviewer: 1

Output:
Basic reporting: good
Experimental design: good
Validity of the findings: insufficient
Additional comments: In the revised MS, more results have been added and some of my problem have been solved. However, I still have some problems which confused me a lot.
In line 205-206, "For the OBPs,11 (SexeOBP9, 13, 15–16, 18, 26, 29–31, 35, and 37) of 40 were relatively highly expressed", and in the figure S2, the authors only showed nine of the OBPs, and why did the authors do that? Also, in line 225-226, "For the CSPs, 16 (SexeCSP2, 4–5, 8–10, 12–13, 17, 19, 23, 25–26, 28, 30, and 33) of 33 were relatively highly expressed",  and in the figure S3, the authors only showed fifteen of the CSPs, and why?
Most importantly, what I mentioned in the last general comments letter about the validation is not just validating highly expressed OBPs and CSPs. What the authors need to do is to validate the results of DEGs which means that the authors should conduct qRT-PCR work on all the OBPs and CSPs from DEGs. Besides, in this work, as many published papers in this journal or others, the results of qRT-PCR validation is really important and reliable, in my opinion, the authors cannot simplify it.
One more question about the material of qRT-PCR samples, the authors did not describe how they collect the samples. Based on the descriptions in the MS, I am not sure that the authors used the samples from the original ones for the DGEs? Or the authors collected new ones for the qRT-PCR work. Please explain it clearly.


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: SEX- AND STAGE-DEPENDENT EXPRESSION PATTERNS OF ODORANT-BINDING AND CHEMOSENSORY PROTEIN GENES IN SPODOPTERA EXEMPTA
Review round: 2
Reviewer: 2

Output:
Basic reporting: NA
Experimental design: I suggest that the author add details of insect rearing and tissue collection in the Materials & Methods section.
Validity of the findings: NA
Additional comments: I suggest that the author add details of insect rearing and tissue collection in the Materials & Methods section.


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: SEX- AND STAGE-DEPENDENT EXPRESSION PATTERNS OF ODORANT-BINDING AND CHEMOSENSORY PROTEIN GENES IN SPODOPTERA EXEMPTA
Review round: 2
Reviewer: 3

Output:
Basic reporting: The authors have revised the manuscript according reviewers' comments. The revised version is clear and in good shape.
Experimental design: no comment
Validity of the findings: no comment
Additional comments: The authors have revised the manuscript according reviewers' comments. The revised version is clear and in good shape.


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: SEX- AND STAGE-DEPENDENT EXPRESSION PATTERNS OF ODORANT-BINDING AND CHEMOSENSORY PROTEIN GENES IN SPODOPTERA EXEMPTA
Review round: 3
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: no further 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: BIOGEOGRAPHY RATHER THAN SUBSTRATE TYPE DETERMINES BACTERIAL COLONIZATION DYNAMICS OF MARINE PLASTICS
Review round: 1
Reviewer: 1

Output:
Basic reporting: The paper by Coons and colleagues describes a very interesting dataset obtained from experiments conducted with the same setup and analysis protocol in different oceanic regions and continents, in the framework of the GAME project. All experiments were meant to understand whether microbial community composition is driven by different plastic substrates and/or different geographical regions.
I have no comments on the abstract and introduction. The references are very good and sufficient background is provided. Figures are informative and well represented. The paper and the results well address the hypotheses but I wish it could go a little bit further into the discussion.
Since I can only choose between minor and major revision, I check the box of major revision. However, I wish to clarify to the authors and to the Editor that I suggest a moderate revision of the discussion section and that the manuscript in general reads very well.
Experimental design: I found the paper interesting and novel, advancing our current knowledge of the plastisphere community. The text is well written and the methods section is comprehensive, with a detailed and sound statistical approach. The experiments, analysis and statistical tests as well as results are fully described. The experimental design is robust and I think the comparison of different regions with the same setup is of particular interest.
Validity of the findings: The findings are novel and worth of being disseminated within the scientific community as these new evidences advance our knowledge of the topic and may help future research to focus on specific hypotheses on the bacterial community on plastics.
Conclusions are well stated, however I think the discussion could be expanded to include a broader perspective. I think the authors should dare to provide some new ideas arising from their findings. I would not consider that speculation but rather an advancement of the significance of their findings for processes related to plastics' fate in the marine environment, as well as to marine biogeochemistry.
Additional comments: I have some general comments/questions that I think should be considered in the discussion before publication, to expand the findings and reason some possible phenomena. These comments should not be considered as criticism, but rather that I found the paper very interesting and it has raised many questions and curiosity that I hope the authors can address. Please see below:

-In general, what is the motivation for the choice of plastic types? For example, polystyrene is very abundant and frequently found, but not included. Was there a technical motivation? As of this, the reason for choosing both HDPE/LDPE is the abundancy of these two types of plastic? Do you think that the bacterial community (even if the same) on HDPE/LDPE may affect these materials’ buoyancy differently?

-On buoyancy: clearly the materials you used have different properties. Even if they were all incubated at 1 m depth, I think that some discussion on how the microbial community may affect buoyancy and vertical as well as horizontal distribution of these plastic types, when found as particles, should be added. This would give some insights into the possible reasons behind the biogeographical differences. Maybe plastic properties play a role. The ones that are more widely dispersible can reach different marine compartments/depths than other types of materials. This does not change the results of your experiments, which are very precise, but I wonder whether we would find the four plastic types used, in surface waters, and for five weeks to the same extent to be able to conclude that plastic type does not influence much bacterial community composition. Just some thoughts about it.

-Plastic surface properties may play a major role in the attraction of microbes and on the later possibility of being further broke down in smaller debris. Is it possible that the melting of the plastic polymers onto the glass slides changed somehow hydrophobicity or other properties of the plastics, changing their “reactivity” to marine bacteria or their attractiveness to microbes? It is also possible that the treatment of the plastic coating modified the chances of biofilm-adhesion, thereby affecting embrittlement and microbial degradation of the polymers? How would you compare these surfaces to virgin materials or aged debris that can be both found in the marine environment? Moreover, do you think that melting of polymers may have affected the possible leaching of carbon from plastics thereby influencing microbial adhesion and/or composition? Just some ideas to expand comparison possibilities.

-Why did you choose to deploy the plastic coated slides and control slides at one meter depth? How does this influence the bacterial community?

-Lines 328-329: why? Could you give some examples of natural substrates? However, in lines 350-351 there is a somehow contrasting statement: if in a 5 weeks old biofilm you did not notice significant differences between polymer types and between polymers and control slides, the I guess that on natural substrates it may actually happen the same, that the succession of communities in the 5-weeks biofilm won’t be much diverse, that is, the “substrate core” won’t probably make much difference afterwards. Could you expand this?

-What was the reason for 5 weeks incubation? Comparison with other studies, timing of biofilm formation…just curious.

-Line 387: you discuss about pathogens. Although vibrio in your study was not classified as human pathogen, I was thinking that maybe you could spend a few words on how distinct bacterial communities across biogeographic regions may spread to other environments, possibly carrying along pathogens or threats to environments where they were not present before. This may go together with the horizontal dispersal of plastics. It is true that biogeography played a role in bacterial community composition to answer your research question, but since plastic is floating, it would be a nice addition to discuss on potential “cross-contamination” of the plastisphere community. Do you think there are some species that you would certainly not find in some of your locations of study? Again, just some thoughts on expanding the discussion section.

-Line 401: you discuss about a potential functional core microbiome. What kind of microbiome would you expect, and how could this core microbiome, or certain species that you observed, influence the fate of plastics, like their buoyancy and distribution in the water column? For example, are there certain bacterial species associated to specific organic compounds found on plastic particles that may modify their buoyancy? Or certain species that may be more active in degrading the polymers? I found this paper interesting
(Urbanek et al., 2018 https://doi.org/10.1007/s00253-018-9195-y) and maybe you could add some hypotheses on how distinct biogeography may affect the bacterial communities’ capability of not just adhere on plastic, but to actually modify it?