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: SHIELDING EFFECTS OF MYELIN SHEATH ON AXOLEMMA DEPOLARIZATION UNDER TRANSVERSE ELECTRIC FIELD STIMULATION
Review round: 1
Reviewer: 1

Output:
Basic reporting: The paper was nicely written, but the mathematical equations were not well formatted and not consistent in font types:
Eq (1): Is there supposed to be an arrow (instead of a box) on top of the electric field symbol E?
Eq (3): Is there a partial derivative symbol missing?
Eq (8): ln, sin and cos, missing brackets, or non-italic.
Eq (10 & 13): cos should be round bracket
Eq(10): missing bracket
Eqs for term1,2,3,4: change of font size from main content, suggest move to appendix or footnote
Eqs for coefficients in appendix: unreadable, should be broken down at some point and organised neatly
Experimental design: The authors in this paper were trying to prove mathematically the capability of exciting neural axons with a transverse electric field, and to identify the influence of axonal geometric parameters on the excitation.
The paper in itself was nicely written and the deduction was good. The findings however were rather unsurprising, for the existence of the electric field, regardless of transverse or parallel, can result in local polarity change with the axon, which in turn causes voltage difference and may lead to substantial transmembrane voltage drop and result in depolarisation.
Due to the irregular trajectories of nerve fibres, it is rather difficult to pin-point whether the electric field is parallel or perpendicular to the fibres, or even at the particular segments; and chances are that both types should be present at the same time. Therefore, a more important/interesting question is, when a parallel electric field is present, is transverse electric field still important?
Validity of the findings: See comments to 2nd point.
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: SHIELDING EFFECTS OF MYELIN SHEATH ON AXOLEMMA DEPOLARIZATION UNDER TRANSVERSE ELECTRIC FIELD STIMULATION
Review round: 1
Reviewer: 2

Output:
Basic reporting: Minor issue: many of the equations presented are multi-term, complex mathematical expressions that appear to be parsed from a symbolic math toolbox - it would greatly improve the presentation if these were formatted in a professional math typesetter such as Latex or Word equation editor. This applies to lines 152-154, 162-164, 169-172 and 353-389.

Other typographical errors:
Line 99: in equation 1, the overscript for E seems incorrect - should it be an arrow to denote a vector?
Line 211: replace “did not significant affect” with “did not significantly affect”.
Line 220: replace “constructs of a lipid bilayer” with “is constructed of a lipid bilayer”.
Lines 420-421: Delete "[pii]" and format reference correctly (doi is on a separate line)
Lines 484-486: As above, delete "[pii]" and format reference correctly.
Line 496: Delete "Academic".
Lines 502-504: Delete "[pii]" and format reference correctly.
Lines 580-583: Delete "[pii]" and format reference correctly.
Experimental design: This paper presents theoretical derivations of transverse electrc field stimulation of myelinated axons, finding that the myelin sheath provides a significant resistance in this mode, effectively shielding the axon from activation. Although the mathematical derivations are interesting, the significance of this finding is hard to justify, particularly given that myelinated axons have closley-spaced unmyelinated nodes of Ranvier, and any applied transverse fields are likely to stimulus these first. Of more interest is the effect of transverse stimulation in this more realistic scenario, where the shielding effect of myelin is likely to be vastly diminished - is the stimulus threshold actually lower when compared to the unmyelinated axon? This would have been far more interesting and useful to explore theoretically.
Validity of the findings: Derivations are intersting - I have not seen these explicitly published before. However, significance of this work is not clear, as per my comments above.
Additional comments: See my comments 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: SHIELDING EFFECTS OF MYELIN SHEATH ON AXOLEMMA DEPOLARIZATION UNDER TRANSVERSE ELECTRIC FIELD STIMULATION
Review round: 2
Reviewer: 1

Output:
Basic reporting: 1. The equations between line 149 and 182 are confusing. Suggest a better formatting, such as everything belonging to the same equation stays to the right of the '=' sign, even after a line break.

2. Unable to find the paper Pourtaheri 2008. Citation information incomplete.
Experimental design: Despite the authors' best efforts, I still find they have not yet addressed to all the comments from the previous reviewers. For the foremost, the impact of the study.

The transverse EF may be able to activate the cells, however, under what condition? The value of EF the authors used for their study is 20k-200k V/m, which they got from a study of electroporation, and thus achieved a depolarisation in a micron range. However, it's hard to imagine a neural stimulation in an ordinary situation using such a high stimulation intensity without damaging the nerve tissue. In terms of brain stimulation, the electro-shock therapy is the one using a relatively high stimulation, and the EF normally peaks at 200 V/m, resulting in significant side effects. Therefore, such an unrealistic value does not support the findings of this study. On the contrary, it suggested that only in very extreme and rare case, the transverse EF can cause depolarisation.

In addition, it is still debatable whether in the literature the authors referenced in this paper the transverse EF is the key player in exciting the nerves. For instance in cuff/cylindrical electrode, the excitation may be due to the parallel EF drop at the boundary of the electrode; and in the MS situation, it may be due to the uneven shape/trajectory of the nerves... none of these definitively supports that it is the transverse EF but not the parallel EF that plays a role in the excitation.

Indeed, 'with significant transverse stimuli, the cable model assumptions are known to be invalid', and I like the idea of modifying the cable eq to include transverse EF to make it more precise. However, the authors have failed to provide us with the necessity to do so.
Validity of the findings: Please see my comments above.
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: SHIELDING EFFECTS OF MYELIN SHEATH ON AXOLEMMA DEPOLARIZATION UNDER TRANSVERSE ELECTRIC FIELD STIMULATION
Review round: 2
Reviewer: 2

Output:
Basic reporting: This revised manuscript represents a substantial improvement from the previous submission. Issues I had raised earlier have largely been addressed in this revision, although the presentation of some of the more complex equations in the paper could still be improved, as per my suggestions below:
Line 156, Eq (10): Place large brackets around the final term [term1/(term2 + term3)].
Lines 159-161: This is a complex equation broken over several lines. It would improve readability by inserting an explicit multiplication operator x after the first term on line 159. Also line 161 appears to have an extraneous single quote, which could be misinterpreted as a differentiation operator.
Lines 162-162. As above, insert an explicit multiplication operator 'x' after the first term on line 162.
Line 170, Eq (11): Place large brackets around the term [term4/(term2 + term3)]
Line 176, Eq (12): Place large brackets around the term [(term5 + term6)/(term2 + term3)]
Lines 178-179. Insert an explicit multiplication operator 'x' after the first term on line 178.
Lines 180-181. Insert an explicit multiplication operator 'x' after the first term on line 180.

Other typographical errors are:
Line 15: Replace "an effect method" with "an effective method".
Line 19: Replace "electric filed" with "electric field".
Line 25: Replace "Polarization of both axons were" with "Polarization of both axons was".
Line 69: Replace "with is considered" with "which is considered".
Line 104: Replace "Inside the cylindrical coordinates" with "Using the cylindrical coordinates".
Experimental design: The authors have addressed my earlier comments on experimental design.
Validity of the findings: Derivations of transverse electric field contribution to axonal depolarization are novel and interesting. The authors have also addressed my initial concerns as to the significance of their findings.
Additional comments: As per above, my only recommendations are that the authors address improve presentation of their complex equations and correct some remaining typographical errors.


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: WHEN AND WHERE TO HATCH? RED-EYED TREEFROG EMBRYOS USE LIGHT CUES IN TWO CONTEXTS
Review round: 1
Reviewer: 1

Output:
Basic reporting: 'no comment'
Experimental design: Experiments description, in particular Hatching orientation section is a little confuse (lines 227-251) and could be improved for clarity. I suggest to divide this section (Hatching orientation) by following the two big questions tested i- To test whether environment provides cue for hatching timing and ii- to test for light-cued hatching orientation. I think authors should state at the beginning of the second question (ii-), as shortly and clearly as possible a description of Experiment 1 and Experiment 2. Perhaps, a figure showing the setup from dark and light source will help the reader to understand better the experimental design and interpret the results.
Validity of the findings: Regarding figures, I suggest to improve or show in a different manner results from Figure 3. It is a little confuse the inclusion of SE as shaded ribbons with different colors. And also will be fine to combine both, Fig 3 and Fig 4 in only one figure as is showed in Fig 5.

Also, I have some minor commentaries about figures:
Figures 1 and 2 should be better resume in one.
I suggest to delete Figure 7. Results are well described and sustained in text and by statistics.
Additional comments: The manuscript is well presented and structured. Language is clear and unambiguous permitting the reader to fluently follow the text.
Beyond there is many information about how different cues shift hatching in amphibians, in this paper authors present novel data on how an amphibian species Agalychnis callidryas use external cues to inform when and where to hatch based on light cues.
Introduction and background resulted clear, and then situating the reader in perspective through relevant and pertinent literature about hatching timing as response of direct threats. Also signaling the scarce information available on the spontaneous hatching or absence of immediate threats. Again, the manuscript result very valuable at this point.
I consider that the structure of the manuscript conforms the standards required by PeerJ following the forms usual for these kind of discipline.
Data analysis is well explained and statistically sound and I thank authors for providing raw data; these supplemental files helped to check in more detail results presented and also better interpret the Figures.
The research questions are well defined followed by an appropriate experimental design to respond the questions.
Finally, this work has novel results relative to hatching responses to cues not yet explored in amphibians The results are very clear and discussion is well supported by literature. Conclusions are well presented, following the initial questions and focusing on the supporting results. Also, the investigation was performed following ethical standards and permits (experiments conducted under specific permits detailed in the manuscript).


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

Input:
Title: WHEN AND WHERE TO HATCH? RED-EYED TREEFROG EMBRYOS USE LIGHT CUES IN TWO CONTEXTS
Review round: 1
Reviewer: 2

Output:
Basic reporting: The language is ok but the manuscript suffers from a lack of clarity at several places (detailed in the “experimental design section”).
I would recommend the authors to present more deeply the findings coming from the same lab on hatching behaviour of red-eyed treefrogs. Particularly, I would recommend giving a more exhaustive review of environmental factors influencing hatching time and synchronicity in the introduction section (e.g. for example, the reference Salica et al., 2017 is missing). The background is very general and important informations on the biological model are missing.
I would recommend the authors to re-organise the introduction section in order to improve fluidity and the articulation between hypotheses. To that end, I would recommend removing the subheading “study organism and hypothesis” and, information given lines 97-113 should be placed after lines 72-73.
Lines 111-113: the hypotheses regarding hatching time and synchronicity should be clarified. Why the authors expected earlier hatching in the context of continuous dark? Does it means that the lack of light cues may accelerate embryonic development or engender hatching at an earlier stage of development? In the same direction, why did the authors expect an influence on the embryonic development of embryos in the context of continuous light? If an endogenous rhythm is expected by the authors (line 110), why did they not expect similar patterns of hatching between the contexts? This is confusing.
Lines 115-116: the authors should state more clearly that they investigated the influence of light in the specific context of environmental-induced (hypoxia) hatching and not just “when exiting the clutch”.  The hypotheses tested should be clarified. The authors state line 92; “we examine hatching orientation and test the role of light as a possible orientation cue” and line 194: “To test if the natural clutch environment provides a cue….”. I did not get the relevance of using both embryos in clutches and in individual tubes. In my opinion there are too many uncontrolled environmental factors between the two conditions to make reliable comparisons. What was the hypothesis tested? Did the authors expect a higher success in whole clutches than in single embryos and why? If the authors just wanted to test the effect of light, then the focus should not be between “treatments” but within each treatment.
The images should be revised and their number reduced. I would recommend using a boxplot representation and not curves for the data presented Fig 3. The photographs presented in Figures 1 and 2 are nice but not informative enough. Schematic representations or pictures of the whole dispositive (and not just embryos) should be presented. As it is, it’s very difficult to understand how the embryos were maintained, particularly for the part related to hatching orientation. Fig 7 should be revised. The data should be presented as boxplots and the division of data in two sets (experiments 1 and 2) is confusing.
Raw data are supplied except for whole-clutch experiments.
Experimental design: The research is original and within the scope of the journal.
As explained above, the research questions are not well defined.
The methods are partly questionable and the paper generally suffers from a lack of clarity.
Possible methodological flaws need to be addressed.
Line 162: how were the embryos manipulated?
Lines 170-171: what is the link between the present study and the study mentioned (Warkentin et al., 2017)? Were the embryos exposed to other manipulations than those presented in the present paper?
Lines 173-174: was this protocol previously used?
Lines 176-190: the method is unclear. Was each treatment attributed to a specific room? Were the 12 clutches per treatment prepared and observed simultaneously? If different trials were conducted, this has to be mentioned and included in the analysis.
There is no indication of the temperature and the humidity within each room. This information is crucial since the results could be explained by a simple room-effect. As temperature and humidity can affect hatching time, how were these parameters controlled? As it is, it is not possible to disentangle room effects from treatment effects.
In several oviparous species, like fish, the spectral composition of light has an influence on embryonic development. The spectral composition and intensity of the light has to be mentioned. Was this type of light used in previous experiments and was it controlled for effects on embryonic development?  What was the distance between the light and the embryos? Was it a source of extra heat which could have an influence on humidity? Was this distance equivalent between light and photoperiod treatments.
Lines 183-190: this part is unclear. For the photoperiod treatment, were the bins wrapped and unwrapped daily? In that case, did this group received extra manipulations compared to the others? The authors mentioned line 101 that undisturbed eggs hatched after 6 or 7 days. Did the present embryos hatched earlier than expected? In Salica et al., 2017, it is stated that a stress like dehydration engenders earlier and more synchronous hatching. Could the authors rule out the hypothesis that differences in vibrations and/or humidity, temperature and manipulations may have affect the results?
Lines 192-251: This part needs to be revised. The relevance of observing whole-clutch or individual-egg flooding needs to be scientifically explained with clear hypotheses.
I did not get why the authors gave details regarding embryos tested in 2014. If I understood well, these embryos are not included in the present paper. In addition, the method is not exactly the same than for 2015. This is very confusing and should be removed.
Lines 215-220: very few details are given regarding flooding of whole clutches. As it is, it is not possible to replicate and to understand what was done. How were light-cue orientation tested on clutches?
Lines 222-251: A schematic representation should be given to understand the protocol. If I understand well, the protocol was changed during the experiment (wrapping with black tape (line 232) and change in the light distance (line 245)). Why? Was the exposure to light not controlled enough during the first part? The authors have to test more properly the potential effects of these factors before conducting statistics on pooled data. The authors present two experiments related to these changes (e.g. fig 7) but I would bot called this “experiment” since nothing is tested.
As a control for side bias (or laterality), the authors used 20 additional embryos which were not exposed to light.  In my opinion, this is not a good control because of environmental differences. All tested embryos were exposed to light and they may have used environmental cues present in the environment to orient their behaviour. The authors have to mention if the sides “light” or “dark” were counterbalanced between the right and the left between trials. And, the occurrence of “hatching to the right” or “hatching to the left” has to tested against chance level.
The intensity of the light should be mentioned. Was it comparable to what is found in natural conditions?
Validity of the findings: Statistical analysis: lines 255-277: there is a mix between parametric and non-parametric statistics. The authors should clearly state if the requirements to use parametric statistics were met.
Lines 259: the sentence is not clear. Does it mean proportions? Does per “clutch” refers to the split-clutch (7 or 10 eggs)? I would recommend the authors to present data as box plots with the different time-points or specific points like the time points presented Fig 3 (4.5, 5, 5.5, 6; 6.5 and 7 days of age). Hatching success should also be mentioned. The authors focused on specific time points around 5.75, 5.67, 5.83 days but it does not provide a more general view of the process (onset of hatching, 50% hatching, hatching completion). Hatching synchrony must be defined more properly. How was the criteria “maximum proportion” defined? Why a specific 2h window (line 268-269) and not 1 or 3 hour-window? What is commonly observed in undisturbed embryos under natural conditions?
Line 271: what is meant by “compared the distribution of these modal hatching times across treatment”. This is unclear.
I would recommend the authors to provide first, a descriptive view of the course of hatching in each treatment (e.g. time of onset, 50%...). And secondly, to provide comparisons between groups.
Lines 280-281: The authors mentioned: “ based on the null hypothesis that the probability of hatching into open water by chance was equal to the natural proportion of exposed surface area of embryos”.  I did not get the meaning. The same for the hypothesis of “the proportion of hatching into the glass in individual trials was equal to the observed average incidence of embryos hatching into the jelly in whole clutch..”
I did not get these hypotheses. If the authors expected embryos to hatched towards the light, then they should just focus on the comparisons of the proportion of embryos hatched towards the light compared to chance level 50%. Laterality bias should be tested properly.
Why the authors expected an effect of the side of insertion in the tube? Does it influence the orientation of the head of the embryo at the start of the experiment?
Lines 334-340: the authors mentioned a high proportion of hatching “failure” in individual embryos. Were the tubes used to maintain embryos previously used in the lab? How is this explained?
The discussion section needs to be revised according to the points raised. In addition the authors provide very general information which are not based on their data.
The discussion is speculative and not supported by data. For example, there is only once sentence lines “449-450” related to the results of the experiment on hatching direction. The authors state that the embryos use light cues but this is not demonstrated. The authors should discuss the parameters observed.  They should also discuss the limitation of their study. Validity and accuracy of the data should be discussed. The hypotheses mentioned in the introduction section should be discussed in light of the results. The present data should also be compared to data obtained previously in the lab.
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: WHEN AND WHERE TO HATCH? RED-EYED TREEFROG EMBRYOS USE LIGHT CUES IN TWO CONTEXTS
Review round: 2
Reviewer: 1

Output:
Basic reporting: After reading carefully the suggestions proposed by Reviewer 2 and myself I found that authors were very open to the revision and I celebrate that they responded with extreme detail to all the questions and suggestions expressed by the reviewers.

However, in my opinion, with the intention to reinforce and explain in detail why and when some of the methods were employed Discussion resulted too long. Particularly, those sections that identify caveats to the major findings and interpretations of the results (Discussion, “data interpretation and limitations of experiment” and “Hatching orientation – data interpretation, evidence, and limitations”), then it could be abbreviated and or moved to Materials and Methods.
Experimental design: Experimental design was improved, by including photographs and figures as was suggested. Also, I suggest to move information that was included in Discussion to improve the experimental methodology
Validity of the findings: This manuscript provide novel data about the rol of light shifting hatching timing and orientation in an amphibian on undisturbed conditions. Methods resulted adequate to the proposed questions; statistics is correct and in this new version authors worked hard to explain experimental design in detail. I consider that Discussion needs some changes. Conclusions are appropriate and conneted to the obseved results.
Additional comments: I think authors did the best to improve the manuscript, I appreciate that; however, in my opinion, some of the information included in the new version of the manuscript needs to be worked.

I would recommend to the authors:

Section: Diel pattern of hatching – data interpretation and limitations of experiment
1)	To move this entire paragraph to M&M:
“A limitation of our experiment, imposed by field-season logistics, is that we only had one light-controlled compartment per treatment; thus, any other environmental variation between compartments was confounded with light treatment. In general, variation in temperature and humidity can influence the hatching timing of amphibians via effects on development rate (Duellman & Trueb 1986; Seymour & Bradford 1995). In A. callidryas these variables may also directly affect embryos’ hatching decisions; both drying and heating can induce hatching (Salica et al. 2017). For instance, during the warm El Niño conditions in 2015, embryos in our laboratory developed faster and hatched earlier than in other years (see Warkentin et al. 2017). All treatments were run simultaneously, on split-clutches, and eggs in different compartments were separated by only a short distance and thin plastic sheet. Thus they were subjected concurrently to any overall temperature fluctuations in the room. We used LED lights to minimize heat from the bulbs and manual misting to maintain humidity. In our few spot checks, all compartments were within a range of 0.6°C, but we do not have continuous temperature data through the experiment.”

2)	To delete the following paragraph, this information does not contribute with testable or comparable data in the discussion.
“Furthermore, these experimental results are consistent with unquantified prior observations of A. callidryas in many different contexts; continuous light generally appears to delay hatching and hatching often peaks shortly after natural dusk or experimental lights-out (Warkentin, personal observations”.

Section: Hatching orientation – data interpretation, evidence, and limitations
I suggest to move to Materials and Methods the following paragraphs:
1)	“Their poorer orientation in glass cups suggests that any potential memory of their prior oxygen gradient is insufficient as a cue, and that visual cues may be relevant. Our phototaxis experiment was designed to test for a contribution of light or visual cues “.

2)	“The way that we constructed the cups precluded an equivalent “both sides light” control to test for side-bias due to aspects of the visual environment. Thus, our experiment does not address whether embryos might tend to orient hatching toward or away from any particular visual stimulus, simply whether they orient toward light and visual cues rather than their absence. We did not measure light intensity in this experiment, nor do we have field measurements of underwater light intensity in A. callidryas ponds”.

In general I am pleased with the changes made on this new version of the Manuscript, but Discussion needs to be shorten and I suggest that some of the paragraphs could be moved to Materials and methods.


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: CORAL DISEASE PREVALENCE ESTIMATION AND SAMPLING DESIGN
Review round: 1
Reviewer: 1

Output:
Basic reporting: The study ‘Coral disease prevalence estimation and sampling design’ simulates belt-transect surveys of varying sizes on a simulated coral reef to determine how well current survey methodologies are able to accurately estimate disease prevalence. Their results indicate that it is difficult to accurately estimate disease prevalence with high confidence even when surveying large reef areas using the belt-transect method, which is currently the most commonly used method in field studies for studying coral disease. This study highlights an important issue in coral disease field studies and suggests that a probabilistic approach would allow for more accurate estimates of population level disease estimates. The authors provide sufficient background and context, reference the literature appropriately, and provide results that are relevant to their hypotheses. Overall, I recommend moving some figures and tables that are currently in the main text to supplemental information, and potentially adding a new figure if possible (see comments below). The writing includes several spelling mistakes and some grammatical errors, several of which I indicate in the comments below. I recommend considerable editing for clarity in the introduction, methods, and results sections, which would greatly improve readability of the manuscript and better highlight the value of this study. I recommend the authors re-submit this article with revisions according to the comments below.
Experimental design: The experimental design of this study is appropriate and the authors provide code and data to reproduce their results. The authors should reference supplementary information in the main text so readers know where to find associated code and results for each section of the study. The one issue I think the authors should consider in terms of their experimental design is that they created several scenarios (simulated coral reef landscapes) with specified coral densities, disease prevalence levels, and disease colony aggregations, yet selected a single scenario for the rest of this study based on the scenario that mostly closely estimated coral densities and disease prevalence in their field belt-transect surveys, yet their results suggest belt-transect surveys are unable to accurately estimate these measurements (see comment on lines 111-122 for further details). I do not think the study needs to be re-designed, but the motivation for selecting this experimental design should be more carefully considered and explained.
Validity of the findings: This is a novel study that highlights an important issue that is often ignored in coral disease field studies and provides well supported and validated results. This study was performed using simulated data and statistical analyses, which are made available in the supplementary information. The conclusions are well stated and link to the original research question.
Additional comments: Introduction
Line 37: Emergent diseases on coral reefs indicates that diseases only started arising since the 1970s on coral reefs and coral associated organisms. This sentence should be restructured to clarify that observations have only been reported since the 1970s although diseases were likely always part of the system.

Lines 37-62: This first paragraph can be written more concisely, there is not much information that is critical to the paper.

Line 53: the evidence of an association between coral disease and ocean acidification is spurious, consider removing.

Line 67: “logistic constrains” should be “logistical constraints”.
General: Species names should be italicized

Methods
Lines 82-89: Please edit this paragraph for clarity so that it is completely clear that the authors conducted 24 belt-transect surveys (6 surveys per sampling station, 4 sampling stations) and that abundance and prevalence estimates from those surveys were used to parameterize simulations.

Lines 82-83: change “parametrize” to “parameterize”, “estimates” to “to estimate”, and “reef build coral” to “reef-building coral”.

Line 84: Include the months when the surveys were conducted in 2006.

Line 87: Be consistent with writing the area of belt-transects: “25 by 2 m” versus “25x2 m”.

Lines 99-100: While Jolles 2002 argues that randomly aggregated diseased colonies could be associated with water-borne disease transmission, water-borne disease transmission is also hypothesized to be highly directional due to currents, tides, stream flow etc. I would recommend including additional conditions which would result in a random aggregation of diseased colonies, such as transmission by a vector that has a home range equal to or larger than the survey scale.
Lines 103-106: Does this approach mean that each diseased colony in the scenarios with contagious disease transmission was assigned a different probability of having a diseased nearest neighbor, or that there were multiple simulation scenarios and within each scenario diseased colonies had a single probability of having a diseased nearest neighbor? This information needs to be more clearly explained here. In addition, if the former approach was used (each colony was randomly assigned a different probability) I’m not sure this makes sense biologically because it would indicate that a single disease has five different transmission rates and that those differences in transmissibility are random across colonies. While it is great that the authors are considering that coral disease transmissibly given close distances among colonies is unknown within a range of possibilities, assuming that transmissibility varies by colony for a single disease is likely to produce less realistic scenarios rather than more realistic scenarios. Thus, if this approach was used I would suggest the authors re-run the simulations selecting a single nearest neighbor distance percentile or for each nearest neighbor distance percentile individually.

Lines 106-108: 1) Add 1-2 sentences to explain how Riply’s K estimates clustering and how the Bezag’s (Besag’s?) transformation of Ripley’s K accounts for greater variability associated with larger areas. 2) “Ripleys’ K” should be “Ripley’s K”. 3) Were Bezag’s transformation of Riply’s K plots created for all six scenarios? Based on this sentence it seems like they were, yet Figure 1 only shows results for one scenario (random clustering of colonies and random distribution of diseased colonies). Figure 1 should include these plots for all six scenarios. Figure 1 should also be moved to supplemental information as the information is important to ensure the simulated scenarios were created correctly, but do not provide any important information for the central question in the study.

Figure 1: The figure 1 title and caption need more information so that readers who are not familiar with Ripley’s K can easily and quickly interpret the results. Information should be added to briefly indicate the definitions of R, r, and L, what a departure of the black line from the shading would represent (clustering versus inhibition). In addition, I believe the y-axis represents L(r)-r rather than L(r) which cannot be discriminated with the current y-axis label, please add this information to the y-axis label.

Lines 111-122: This paragraph should be edited to make it clear that the goal of this comparison was to identify which colony distribution and disease transmission scenario most closely represented the coral reef where in situ estimates of colony density and disease prevalence were observed in order to identify one scenario to perform multiple simulated surveys for comparison in the rest of the study. I think this approach raises an important point for consideration. The authors have made several scenarios which were created based on specific criteria for colony density and diseased colony presence/aggregation (i.e., simulated coral reefs with known colony densities and disease prevalence), yet then selected the simulation that most closely resembles the coral reef density and disease prevalence estimated from in situ surveys, which they are arguing such methodologies may not be able to adequately capture estimates of coral density and disease prevalence. It seems like a very circular argument to me and could make the readers question the value of the study. Careful thought in how to present this approach could considerably improve the study motivation, approach, and value of results.

Lines 114-116: What does the scenario with fewer trials mean? How many trials? The first sentence seems to indicate that in each scenario 30 simulated surveys were conducted.

Lines 116-122: Explain model formulation more clearly here. Were the covariates simply the abundance or prevalence values observed in the belt transect surveys? Were the random effects field site or simulation scenario? It is not entirely clear here how this data was analyzed.

Table 1: This table should be moved to supplementary information. While it provides the motivation for the use of simulating surveys using the belt-transect method and what area should be covered in those simulated surveys, the information is not pertinent to the major results of this study.

Lines 138-139: Is there a reason for selecting two of every 100 surveys to assess power or was this number chosen arbitrarily?

Lines 145-147: Permit information could be probability be moved to “additional information and declarations”.

Results
Line 164: Change “100x100m” to “100 m x 100 m” or “100 m2”.

Figure 3: change “dat points” to “data points”.

Lines 185-186: I think this finding (“..for a given effect size, the power of the test increased from smallest to the largest sample unit as sample size increased”) is important and could be valuable for readers. Authors should consider adding a figure of these results to the main text for a few select effect sizes (maybe a low and high effect size, or low, medium, and high effect sizes).

Lines 187-188: Please indicate the number or percent of tests that had power values above 0.5 and below 0.4.

Discussion
Overall, the discussion section is very nice.

Line 245: change “costume” to “custom”.


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: CORAL DISEASE PREVALENCE ESTIMATION AND SAMPLING DESIGN
Review round: 1
Reviewer: 2

Output:
Basic reporting: Overall Comments: The authors nicely and succinctly establish both the importance of coral disease and the methods used to study coral disease. Establishing the time of the first reports of coral disease emphasizes the idea that coral diseases at the levels observed is a more recent development, especially when coupled with the rise of other diseases. The establishing of the methods is also critical for the understanding the question posed by the paper. The figures and tables used to support the findings provide a nice visual to ensure that the methods used are both appropriate for the questions asked (figure 1) and provide a representation of the power that ecological methods can have for disease studies.

Use of English: Throughout the provided code and data, there are multiple instances where a mix of English and Spanish are used to annotate code. This should be standardized to English as per PeerJ’s standards and converting all language to English will also better allow readers to understand and interpret the methods that the authors used.

Use of references: The use of references throughout the paper is well done in most cases, except in lines 65-68. Here, the authors explain how coral disease prevalence is obtained using modified ecological techniques without referencing papers that exhibit this method. This can be simply solved by referencing Table. The references should also be checked for completeness, as the reference for Lamb et al 2018 is incomplete and missing 5 authors (Lines 363-364).
Experimental design: Overall comments: The authors employ a simulation strategy that, given the available information, best represents a ‘realistic’ scenario. The use of simulating different distribution patterns and comparing simulated samples to the parameters used to create the simulation allows following results to be credible based on the given parameters. The following tests for accuracy are valid given the types of data simulated and the checks for the appropriateness of the models used is statistically sound. This paper also falls nicely in line with the aim and scope of PeerJ as a biologically motivated paper.

Use of a single coral density and disease prevalence: The authors used a single estimate of coral abundance and disease prevalence from which they based their simulations on. For this, they chose a large, reef-building coral with low abundance. This creates a naturally low sample size, and would thus result in the presented results (Button et al. 2013). The use of this then could be considered cherry picking, as there are other areas of the world such as the Coral Triangle that have a much greater abundance (using cover as a proxy) and diversity of corals (Knowlton & Jackson 2008) and thus would have a greater sample size for a given transect length. This also includes the idea that not all studies survey for a single coral species, and surveying all corals on a smaller transect could lead to larger sample sizes than only surveying a single species on a longer transect. Given the data presented in the paper about Orbicella and Agaricia, the 25x2m transect had on average of 277 corals for just those two species compared to 34 Orbicella. Assuming this average, an area of approximately 400 meters squared (a 200x2 meter transect) would need to be surveyed in one transect to reach the same sample size as studying two species of corals. increasing to a complete survey of all corals present would thus increase the sample size even more. Thus, the authors need to account for variations in abundance to accurately investigate the use of ecological methods to estimate the prevalence of coral disease as in cases of highly abundant corals, smaller transects may be sufficient. (More on this is presented in section 3, validity of the findings).

Pooling of transects: The authors report that a ‘common practice’ of is to pool the counts of diseased and non-diseased corals for the five belt-transects, which leaves each site as a data point. While this practice is employed (eg. Miller 2016), another practice for the analysis of coral disease data from belt-transects is to treat each transect as individual data points (eg. Pollock et al. 2014, Lamb et al. 2018). Here, prevalence is calculated at the transect level and thus provides more data points per site/station with fewer corals per data point. I would suggest that the authors test this alternate method of analysis to better contextualize their study to the literature. Pooling of the transects into a single data point for each 'site' also changes the interpretation of the results as then the unit of study is an area of the reef using transects as a convenient method. With this, 5 10x2 meter transects is equivalent to 5 20x1 meter transects in the amount of area surveyed and thus the results should be similar.

Completeness of methods: In reporting the methods used to estimate disease prevalence, the authors pool the transects in a single station as is reported as ‘best practice’. The reporting of this methodology occurs in the results section (Lines 169-170) and would be better reported in the methods section in the appropriate areas. Another area that should be better represented in the code is the use of the generalized linear models to compare the simulated abundances and prevalence to the original parameters.

Button, K. S., Ioannidis, J. P., Mokrysz, C., Nosek, B. A., Flint, J., Robinson, E. S., & Munafò, M. R. (2013). Power failure: why small sample size undermines the reliability of neuroscience. Nature Reviews Neuroscience, 14(5), 365.

Knowlton, N., & Jackson, J. B. (2008). Shifting baselines, local impacts, and global change on coral reefs. PLoS biology, 6(2), e54.

Lamb, J. B., Willis, B. L., Fiorenza, E. A., Couch, C. S., Howard, R., Rader, D. N., ... & Harvell, C. D. (2018). Plastic waste associated with disease on coral reefs. Science, 359(6374), 460-462.

Miller, M. W., Karazsia, J., Groves, C. E., Griffin, S., Moore, T., Wilber, P., & Gregg, K. (2016). Detecting sedimentation impacts to coral reefs resulting from dredging the Port of Miami, Florida USA. PeerJ, 4, e2711.

Pollock, F. J., Lamb, J. B., Field, S. N., Heron, S. F., Schaffelke, B., Shedrawi, G., ... & Willis, B. L. (2014). Sediment and turbidity associated with offshore dredging increase coral disease prevalence on nearby reefs. PLOS one, 9(7), e102498.
Validity of the findings: Overall comments: The authors bring up an important concern of using methods not designed to adequately sample a population for disease. This allows the authors to make important points about the consequences of finding insignificant differences in a study that could have management concerns. The authors also make a valid recommendation that is to tailor the methods to the questions and species that are being studied.  This is an important recommendation as many field studies rely on established sampling protocols (such as transects or quadrats) and may not consider that these methods may not be the most appropriate for the given study. While these conclusions are well stated in the discussion, the authors make claims that overstep the boundaries of their study (detailed below)

Generalization of results: The authors claim that ecological survey methods are insufficient to estimate disease prevalence (Lines 266-267). I would err on the side that, given the data and results presented, this statement is an over-generalization of the results. The authors only simulated data and surveys for one species of coral with one level of disease. In lines 219-220, the authors report that different species of corals have different abundances, which would influence the sample size for a given transect length. To support these claims, I would suggest that the authors vary both abundance and disease prevalence to assess the effects that these variables have on how ecological sampling methods perform in relation to estimating disease prevalence, otherwise the statements made in the discussion and conclusion sections are not fully supported by the results of the simulations presented. Alternatively, the authors can refocus their discussion and conclusions to be focused on low abundance corals. In the current state, the manuscript fails to meet all of the criteria stated for the validity of findings, namely "Conclusion are well stated, linked to original research question & limited to supporting results".

Choice of coral species: As a key reef building coral species that has been recently affected by disease, the choice to simulate data based on Orbicella is understandable. This choice though would limit the scope of the paper to large corals of low abundance. Underlying this data though is naturally lower abundance and cover (Knowlton & Jackson 2008) of corals in the Atlantic Ocean. This choice is biased as the ecological methods would naturally sample fewer corals. I would suggest either that the authors either refocus the discussion to focus on large, low-abundance corals or expand their analyses to include varying densities and abundances of corals as stated above. Also, not all studies focus on single coral species or disease (eg Lamb et al 2018) and could thus circumvent some of the issues underlying using ecological methods to study coral disease. In these instances, all corals in the belt-transect are enumerated and health status determined which could have higher coral abundances than studies focusing on a single species. Surveying for complete coral communities also has the added cost of needing to understand the diversity of coral species at a site

Lamb, J. B., Willis, B. L., Fiorenza, E. A., Couch, C. S., Howard, R., Rader, D. N., ... & Harvell, C. D. (2018). Plastic waste associated with disease on coral reefs. Science, 359(6374), 460-462.

Knowlton, N., & Jackson, J. B. (2008). Shifting baselines, local impacts, and global change on coral reefs. PLoS biology, 6(2), e54.
Additional comments: Overall, the authors present a very important potential shortcoming of coral disease studies in a well organized and clear manner. The methods employed are rigorous for the parameters provided and provide a compelling case for the given study species. The main shortcoming of the study is that the parameters investigated have a small scope while the authors make claims that span beyond the scope of the results they obtained. The authors claim that in all cases, using ecological methods is a poor method for coral disease studies, though they did not examine cases beyond a coral with low abundance and a moderate amount of disease.


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: CORAL DISEASE PREVALENCE ESTIMATION AND SAMPLING DESIGN
Review round: 2
Reviewer: 1

Output:
Basic reporting: Overall Comments: The authors clearly make the point that current methods in disease surveys of key reef building species in the Caribbean may not be sufficient to accurately and reliably measure disease prevalence on a reef. This finding is important as reefs are under a huge amount of stress from anthropogenic sources. Understanding the methods required to reliably and accurately measure disease are then important and this manuscript works on moving the field to a statistically sound foundation.
Use of English: The authors use proficient English throughout the manuscript.
Use of references: The use of references throughout the paper is well done and sufficient to support the claims that they make.
Experimental design: Overall comments: The authors employ a simulation strategy that is used to assess the accuracy and precision of ecological methods in disease surveys on key reef building species. Simulation of data here is important to test these methods and the authors employed a sound step-wise strategy to best capture reality based on the data they had. While this doesn’t address the entire scope of corals and potential distributions, for generally large reef building species, the methods and results are likely to be valid.
Validity of the findings: Overall comments: The authors bring up and address an important concern of using methods not designed to monitor disease. Not only does the authors provide the background in the statistical theory that makes belt transects poor for monitoring disease, they prove the theory using simulation studies. This allows the authors to make important points about how disease surveys of key reef building species are potentially inaccurate which can have implications for management and understanding disease processes. The authors then recommend tailoring study design to the questions being investigated according to study species and general assumptions of the subsequent analyses that will be performed.
Additional comments: Overall, the authors present a very important potential shortcoming of coral disease for large reef building species. The authors adequately investigate the use of ecological methods in disease surveys and find that in some cases, this may not be sufficient.


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: INTESTINAL MICROBIOME AND ITS POTENTIAL FUNCTIONS IN BIGHEAD CARP (ARISTICHTHYS NOBILIS) UNDER DIFFERENT FEEDING STRATEGIES
Review round: 1
Reviewer: 1

Output:
Basic reporting: The paper is written in clear and unambiguous English, except in few places as it is pointed out in the comments to the author.

The introduction is pretty poor and has to be improved substantially. The main drawback is insufficient background/context of the study.

Professional quality figures, tables provided.
Experimental design: The experimental design is simple and the experiment is well executed to answer the research question.

Some parts of the methods are pretty vague and not well described thus leading to confusion. Specific parts are pointed out in the comments to the author.
Validity of the findings: Though the dataset reveals interesting patterns in the microbial community composition based on the feeding type, the sample number used in the study is too few (n=4-6) compared to the contemporary standards. This might have had an effect on the conclusions.
Additional comments: The article describes the differences in the intestinal microbial communities of the big head carp fed naturally (with zooplankton) and the formulated diets. In addition, authors have also presented the biochemical properties of the blood in relation to the feeding regime. In conclusion, the authors claim that the formulated diet has an impact on the microbial populations as well as certain biochemical parameters of the blood.
The article is generally well written and easy to read, though I have the following concerns.

Major issues

1.	The feeding groups could be renamed based on the feeding type (as written in the title). It’s a bit confusing to a reader that the paper starts with the feeding regime experiment and then when it comes to naming the groups, they are named based on the pond fertilization strategy.
2.	The introduction is not cohesive and does not lead to the objective in question. To make it interesting for the reader I would strongly recommend that the introduction should be modified/rewritten by bringing in specific examples of diet affecting the microbiome (either from other fish/mammalian experiments).
3.	The amplicon generation protocol is not clear in either the manuscript or the provided reference, Gu et al 2016. In addition, the Gu et al 2016 has an error in their manuscript. They say that  primers “ 27F 5’-barcode- AGAGTTTGATCCTGGCTCAG-3’ and 533R 5’-AGAGTTTGATCCTGGCTCAG -3’”  was used for the amplification and in the next paragraph it is mentioned that “The PCR products were separated by 2% agarose gel electrophoresis and bands of the desired size (approximately 250 bp) were purified”. Theoretically, 27F and 533R primers do not produce amplicons with length 250. The amplicon generation protocol should be clearly elaborated (construction of the amplicon primers, single index, dual index details should be provided) or a proper reference should be included.
4.	Line 136, “(i) 200 bp reads”- Is it 200 bp or 250 bp. Sequencing length is 250 bp in the previous part of the manuscript. If the forward and reverse reads are trimmed to 200 bp then I do not think that they can be assembled.
5.	Figure 1 is a plot showing the rarefaction. The difference in the mean CHAO1 index and the statistical differences are not depicted in the plot. hence a plot like FIG2 should be made for chao1 index as well.
6.	Line 270 “Whether growth performance is related to intestinal microbial
diversity merits further investigations”. This is a key finding of the study. It has to be elaborated in relation to the other farmed animal studies/references that are raised using formulated diets. Perhaps less diversity of bacteria and better growth of the animal go hand in hand?

Minor issues

1.	Line 69, “rectangular enclosure ecosystem”- the word ecosystem should be deleted.
2.	Line 73, were the mentioned zooplankton species isolated and identified? It should be described in the methods.
3.	Line 74, What do you mean by 1/2 feeding? I assume it is feeding half of the recommended feeding rate?. If yes please mention it clearly by introducing a line of text in the methods.
4.	Line 99, the exact dose used for the killing should be clearly mentioned.
5.	Line 117, the  short forms must be written in full.
6.	Lines 153 and 156, the words treatments should be replaced by samples.
7.	There are some unwanted characters above all the scatter plots that should be removed.
8.	Table 1- the survival rates- Though not significantly different, what could be the reason for ~20-25% reduced survival in the group B, C, D?. This has to be put into context in the discussion.
9.	It is not clear what the authors mean by “main performance” in the table 1 header ?. Should be replaced with a more understandable word.
10.	Line 114, “Approval ID: CAFSCJ-2014-001” The document in the supplementary materials should be cited for this statement.


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: INTESTINAL MICROBIOME AND ITS POTENTIAL FUNCTIONS IN BIGHEAD CARP (ARISTICHTHYS NOBILIS) UNDER DIFFERENT FEEDING STRATEGIES
Review round: 1
Reviewer: 2

Output:
Basic reporting: The paper is well written and clear.  There are some language mistakes and some unclear sections that should be improved. More specifically, see here:

Line 40:  important to understand these things in what?  vertebrates? among diverse fish groups?
Line 48-49:  maybe say why knowing these things matter.
Line 63:  2) "and to determine the relationship" instead of "and what's"
Line 82:  Not clear what this means:  "This was done to get natural fish food organisms."
Line 91: add "to" so that it reads "randomly distributed to the ponds"
Line 97: should read "One hundred and eighty days after the start of the experiment,"
Line 127-133:  It is unclear of the authors used proper negative and/or kit controls for sequencing.  Especially important since they handle the samples so much, like running on 2% agarose gel and then gel-purifying the products etc.

Line 221-223:  I do not believe these are accurate names for genera.  It may be better to refer to these sequences as OTUs. For example, Gammaproteobacteria_unclassified is not a genus.

Line 243:  add "the" so that it reads "the results of the present study"
Line 293:  change "indicating" to "suggests"
Line 301:  remove comma after "carp"
Line 305-6:  change "basing from" to "Based on"
Experimental design: Experimental design is clear and for the most part done okay.  However, all the arguments seem to be made at the phylum level, e.g., 241-256, yet the arthors are making predictions about function.  It would be very helpful if the authors made arguments based on family-level predictions, which is the level for which functional constraints or predictions are made. For example, Fig. 3, would be more meaningful if compared at the family level..  The authors try to discuss, quite a bit, of the genera involved, however, I think it would make more sense to describe families of relevant bacteria throughout and make arguments about function then.  The work on functional predictions would be substantially improved if they performed analysis such as PICRUSt or something similar.
Validity of the findings: Again, the findings would be significantly strengthened by also comparing familly-level predictions to modeled findings in PICRUSt
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: INTESTINAL MICROBIOME AND ITS POTENTIAL FUNCTIONS IN BIGHEAD CARP (ARISTICHTHYS NOBILIS) UNDER DIFFERENT FEEDING STRATEGIES
Review round: 2
Reviewer: 1

Output:
Basic reporting: Introduction improved based on the comments from last round of review
Experimental design: Experimental design has more clarity compared to the first version.
Validity of the findings: Better explained in the revised manuscript.
Additional comments: The manuscript has been improved considering the comments from the last round of review, although there are few things that needs to be addressed.

The authors wrote in the rebuttal that only the PCR recipe and the thermocycling conditions were used from the Gu et al. Hence I request the authors to write in detail the protocol that they used for the preparation of the libraries for the illumina sequencing.


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: INTESTINAL MICROBIOME AND ITS POTENTIAL FUNCTIONS IN BIGHEAD CARP (ARISTICHTHYS NOBILIS) UNDER DIFFERENT FEEDING STRATEGIES
Review round: 2
Reviewer: 2

Output:
Basic reporting: significantly improved
Experimental design: same; description is improved and clearer
Validity of the findings: no comment
Additional comments: The revised manuscript is a significant improvement.  The authors have addressed my concerns and made the recommended changes.


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: IN-SITU INCUBATION OF A CORAL PATCH FOR COMMUNITY-SCALE ASSESSMENT OF METABOLIC AND CHEMICAL PROCESSES ON A REEF SLOPE
Review round: 1
Reviewer: 1

Output:
Basic reporting: In this study, the authors placed a small, isolated patch reef and a section of bare sediment in large and small benthic incubation tents respectively to measure day and night benthic metabolic rates near the island of Saba. The authors incorporated small amounts of seawater exchange across the tent boundary throughout the experiment into mixing models in order to quantify reef metabolic rates within each tent. Making in situ metabolic measurements at depth, and especially at night!, is a difficult task and I commend the authors for the great amount of work involved in this study. This type of work is incredibly valuable for our understanding of coral reef metabolic rates and is critical for pushing scientific understanding of coral reef processes forward. However, I have a few concerns with this manuscript especially with respect to the leakage of the tents and method descriptions in this study that should be addressed prior to publication.

(1) The study introduction discusses investigating the carbonate chemistry of an offshore patch reef with much attention to NCC and NCP but does not include clearly testable hypotheses. Please clearly state the hypotheses being tested in this manuscript in the introduction and discuss whether the evidence supports or does not support the hypotheses tested in the discussion.
Experimental design: (1) My biggest concern with this manuscript is the leaking of the tents and how they were accounted for in the models as much of the text appears to contradict itself with respect to how well constrained these leaks were in the models. For example, line 242 implies that the mixing model was derived for only 20% of the time of the incubations. How do you know mixing rates for the time that measurements were not conducted both inside and outside of the chamber? The authors later state in line 395 that there are occasional large breach events that have not been accounted for in the model for O2? How does this not also affect the conclusions of the other parameter fluxes? In line 316, the authors state the O2 is an excellent marker for the seal of the tent but then state that only incubations 3-5 provide O2 data that allows asymptotic leak rates to be assumed? Is this for the asymptotic model only? If the assumptions for the asymptotic model are invalidated across 2 of the 5 incubations, then why is this model employed and described in the manuscript? Line 430 then states that depending on the method to account for leakage used, either net calcification or net CaCO3 dissolution was measured, which further makes me question whether there’s a strong understanding of what actually happened in the tents based on the modeling methods employed in this study. As stated in the section 1 of this review, some of this confusion could in part be due to the language used throughout the manuscript in addition to alternating between discussions of the large patch reef tent and the small sediment tent and discussions of two mixing models employed in this study. Nonetheless, the manuscript appears to contradict itself in these statements regarding the most central methodology of this manuscript. The authors recommend these tents as critical tools to be used elsewhere for metabolic measurements, but the contradicting statements in the manuscript suggest there may be critical flaws in the methods and their interpretation. These points and apparent contradictions need to be addressed before publication.

(2) I do not follow the logic in line 437. Does the “5-25 kg CaCO3 m-2 yr-1 calcifier surface” refer to calcification rates of individual coral taxa within the tent? As the paper is written, it’s not clear how the ReefBudget portion of the study was carried out and how to compare these rates to the chamber flux measurements in this study. Please include a table summarizing the %-cover by each taxa, the rates of CaCO3 production, and resulting taxa-level contribution to the ReefBudget numbers to show how these ReefBudget numbers were derived and include a more formal comparison between this ReefBudget calculation to the NCC in this study before publication.

(3) In Table 4, how were the dissolution fluxes attributed to the respective surfaces within the 4 m2 patch? Additionally, why are negative dissolution fluxes described instead of stating that positive calcification was observed? The authors state that rates for the incubations were 0.63±0.46 kg CaCO3 m-2 yr-1, but I do not see the respective NCC rate for each incubation, nor how it was normalized to biotic surface areas. The authors allude to some aspect of ReefBudget methodology but this is not clear. Please describe the benthic flux attributions, ReefBudget methodology, and more clearly state the NCC rates for each incubation before publication.

(4) In line 40 and throughout the manuscript, accretion does not equate to NCC. Accretion = NCC – Net Export as per the cited Kleypas et al. (1999) reference. Please correct this throughout the manuscript to avoid mixing NCC and accretion terms before publication.
Validity of the findings: (1) The authors present a great deal of information and cover an impressive range of information in this study; however, I find the lack of data visualization in this manuscript difficult for supporting the conclusions in this manuscript. For example, why weren’t internal and external TA, DIC, temp, sal, DO, and nutrients plotted vs. time for each incubation? This data is important in its own right showing the variability of biogeochemistry in the enclosure and on the reef in general, but is also critical to interpreting the validity of the mixing models proposed in this study. Please show timeseries of the actual data in the manuscript and/or supporting information so that the reader can see how variables changed instead of reading less-clear descriptions of the data throughout the text.

(2) In line 213, why are external O2 and nutrients assumed to be constant? External changes in T, S, TA, and DIC are all clearly discussed during the incubations so I would expect that there would likely also be associated changes in O2 and nutrients outside the incubation tent. In Line 258 after stating that nutrients and O2 are assumed to be constant, the authors state that the ambient concentrations were essentially invariant relative to the in-tent changes but I do not see support of this with the data presented. It seems as though this assumption was due to the lack of measured external O2 data, but this is not clear and I do not see evidence that this is a valid assumption in the context of the rest of the data discussed in this manuscript. Please address this point before publication with either data to support the claims or a statement that this is a speculative, assumption (as are the O2 flux uncertainties) due to the lack of recorded data.

(3) Additionally, much of the accessible data is in the proprietary ‘.mat’ MATLAB format. Please upload this in a more universally accessible format such as ‘.csv’ or similar to improve access for those lacking expensive MATLAB site licenses.
Additional comments: Please see these additional minor comments and suggestions, which I hope will improve the clarity and presentation of the final published manuscript:

Line 34 : “bleaching of reef” to “bleaching of reef corals”

Line 41: “the coral’s overall response to the sum of environmental changes” NCC reflects the community and not the individual coral therefore “coral reef” should be used in place of “coral”

Line 50: There is an expansive literature on quantitative changes in measured depth and accretion from coral cores that would improve the discussion here. For example see the following:

Yates KK, Zawada DG, Smiley NA, Tiling-Range G. Divergence of seafloor elevation and sea level rise in coral reef ecosystems. Biogeosciences. 2017 Mar 15;14(6):1739.

Hubbard DK, Miller AI, Scaturo D. Production and cycling of calcium carbonate in a shelf-edge reef system (St. Croix, US Virgin Islands): applications to the nature of reef systems in the fossil record. Journal of Sedimentary Research. 1990;60(3).

Line 74: What is meant by “calm environments”? Eddy covariance and gradient flux measurements have been successfully employed across a range of reef environments. Please see Takeshita et al. (2016) and references therein for a discussion of these methods:

Takeshita Y, McGillis W, Briggs EM, Carter AL, Donham EM, Martz TR, Price NN, Smith JE. Assessment of net community production and calcification of a coral reef using a boundary layer approach. Journal of Geophysical Research: Oceans. 2016 Aug 1;121(8):5655-71.

Line 77: The authors state “where no accumulation of chemical signals in the water column are expected”, but boundary layers may still allow measurements to be conducted on reef metabolism. Please see Long et al (2013):

Long MH, Berg P, de Beer D, Zieman JC. In situ coral reef oxygen metabolism: An eddy correlation study. Plos One. 2013 Mar 11;8(3):e58581.

Line 82, 83: Consecutive sentences say “Moreover”. Please revise to avoid repetition

Line 174: “two-hourly” is this once every two hours or twice an hour? Please clarify in the text.

Line 180: “immediate” to “immediately”

"CaCO3 dissolution" should be used wherever "dissolution" is written to clarify what is dissolving

Line 347: This implies net calcification, not accretion. Additionally, concentration rates appear to by higher for incubations 3-5, which is likely due to the lack of accounting for leakage in incubations 1 and 2 discussed in line 316.

Line 376: Please clarify what is meant by the following sentence: “The contribution of dissolution is always negative, corresponding to net accretion of CaCO3.”

Line 447: Why compare this 6% calcifier system to the reefs of Bonaire with 16.6% coral cover? Please see Perry et al. (2013) to place the results of this patch reef into a more comparable context:

Perry CT, Murphy GN, Kench PS, Smithers SG, Edinger EN, Steneck RS, Mumby PJ. Caribbean-wide decline in carbonate production threatens coral reef growth. Nature communications. 2013 Jan 29;4:1402.

Line 467: Please see CaCO3 dissolution rates for coral reef sediments around the world to better place these reef sediment dissolution rates in context:

Eyre BD, Cyronak T, Drupp P, De Carlo EH, Sachs JP, Andersson AJ. Coral reefs will transition to net dissolving before end of century. Science. 2018 Feb 23;359(6378):908-11.

Line 467: Middelburg et al. (2005) is not in the reference list. Please ensure that all references are properly documented in the references section.

Line 488: How can we know that 100 grams of fish in the tent is “generous” based on what has been presented in this manuscript?

Paragraph 512: This should additionally discuss the pioneering work by Pawlik and colleagues regarding the role of sponges in POC, DOC, and DIC on coral reefs.

McMurray SE, Stubler AD, Erwin PM, Finelli CM, Pawlik JR. A test of the sponge-loop hypothesis for emergent Caribbean reef sponges. Marine Ecology Progress Series. 2018 Feb 8;588:1-4.

Figure 4: Which incubation number does this figure represent? What do the data from the other incubations look like with respect to the incubation data presented here in figure 4?

The authors declare no funding for this work but surely there were some exceptional amount of costs for both the personnel time, ship time, and resources used in this project. Please state how this project was funded.


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: IN-SITU INCUBATION OF A CORAL PATCH FOR COMMUNITY-SCALE ASSESSMENT OF METABOLIC AND CHEMICAL PROCESSES ON A REEF SLOPE
Review round: 1
Reviewer: 2

Output:
Basic reporting: No comment
Experimental design: No comment
Validity of the findings: No comment
Additional comments: van Heuven, Webb, et al. use a tent enclosure to estimate metabolic rates of a coral patch at ~20 m depth off Saba, Dutch Caribbean. This is a nice study and would be a useful addition to the literature, particularly because there are very few estimates to date of metabolic rates on reef slopes and it is both surprising and interesting that this reef is net heterotrophic. However, there are a few areas that can be improved with revision, or which need correction.

Title: while this study does report in situ variation in the carbonate system, it seems incidental to the primary purpose of the study, which was to assess metabolic rates of the patch reef. I’d suggest modifying the title to, ‘In situ incubation of a coral patch for assessment of metabolic rates on a reef slope’, or something like that.

First couple sentences of the abstract and first two paragraphs of the introduction: the paper starts with an extensive discussion of anthropogenic stressors on reefs, but this seems out of place as the study really isn’t assessing these impacts. I would substantially reduce if not cut this discussion entirely. Instead, almost all previous studies have investigated reef metabolism on reef flats (typically using a Lagrangian approach) because of the logistical challenges of doing so on a reef slope. This is one of the only studies to measure community metabolism on a reef slope, and I would refocus the introduction in that direction.

Line 40, I would disagree that the accretion/erosion balance of reef communities reflects corals’ overall response to anthropogenic stressors—lots of natural factors impact coral growth rates, certainly not just anthropogenic impacts, and rates of reef accretion are only indirectly related to coral growth rates (for example, reef accretion may be negligible in a wave-swept environment in spite of rapidly growing corals due to periodic physical loss of carbonates).

Line 93, the tent and chemistry measurements were used to assess reef metabolic rates, not the impacts of reef metabolism on in situ carbonate chemistry dynamics.

Line 113, “healthy” as applied to natural systems is often contentious and is problematic as it is rarely defined clearly (what precisely do you mean when you call the reefs “healthy”?). I would avoid the usage entirely. In addition, I’m not sure I would agree that the reefs around Saba are “healthy” even in the context of the wider Caribbean or that the reference cited leads to that conclusion.

Line 186, it doesn’t make sense that insufficient flushing of the umbilical line would lead to lower precision with the one-step titration as compared to the VINDTA since the same procedure was used to collect bottle samples for both. Instead, these levels of precision are about what you’d expect for the two methods.

Line 199 (and throughout), it should be ‘carbonate dissolution’ or ‘dissolution of carbonates’ or something like that, not simply “dissolution”.

Line 266 (and throughout), it should be ‘rates of change of concentrations’ or something like that, not “concentration rates”. Concentrations don’t have rates and written as such it confuses your meaning.

Line 346, the wording is confusing here—you’re using the TA anomaly technique to infer net calcification rates, so TA should be dropping if the reef is net calcifying (or vice versa).

Line 437 (and throughout, especially in tables), I would also provide estimates of metabolic rates in units of mmol/m2/hr as this will allow for easier comparison to other, similar studies and because these values probably translate better to what was measured (i.e., measurements were made over a few hours, not over a year and scaling up short-term fluxes to annual estimates may not be appropriate).

Line 461, ameliorate is misused—I’d just leave it as ‘a complementary tool’.

Line 486-528, in my mind, the net heterotrophy shown by this patch reef (even including net heterotrophy during the daytime!) is perhaps the most surprising and interesting finding of the study. I would consider moving this section to the beginning of the discussion section (i.e., hook the readers with your most interesting result first). Also, how do these rates of net heterotrophy compare to Pnet rates on shallower reefs? This is a neat result and the discussion of this aspect could be expanded a bit to place it in the larger context.

Fig 3, 4 (and throughout) a small thing, but the units for alkalinity should be ueq/kg (microequivalents per kilogram), not umol/kg, and temperature should have units indicated (deg C).

Fig S1, something is way off with the PAR values shown here. Per Table 1, your max values should be 120 umol photons/m2/s during incubation 1, which are sensible values, but here the max values shown are only 2-3 umol photons/m2/s.


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: IN-SITU INCUBATION OF A CORAL PATCH FOR COMMUNITY-SCALE ASSESSMENT OF METABOLIC AND CHEMICAL PROCESSES ON A REEF SLOPE
Review round: 3
Reviewer: 1

Output:
Basic reporting: Overall, the authors have done an excellent job revising the manuscript and responding to this reviewer’s previous comments. My biggest previous concern was regarding the discussion of leak rates and the models constructed. The rebuttal and revisions have largely cleared up these concerns and I believe the resulting manuscript much clearer and easier to follow. I commend the authors again for tackling the difficult problem or addressing leaks in this underwater tent to make such challenging benthic metabolism measurements and in utilizing models to overcome the tent leaks with the stated assumptions, but I also have some further concerns regarding conflicting verbiage in the text in regard to the leaks and the table of ReefBudget data that I believe can be addressed to further improve this manuscript. Please see them below in addition to a few minor comments.

Leaks:
In line 384, they state that this method offers a 1% leak rate—but this would be variable for every attempted seal and this should be stated in the manuscript. The authors state in line 366 they “do not know…that the tent did indeed leak”, but the manuscript clearly states that 1% leak rates were measured in the experiments and that they assume this to be the case for all incubations. The authors later state in their rebuttal that the tent was excessively leaky during two occurrences referencing lines 358. These lines collectively appear to contradict each other…
I commend the authors for maintaining transparency as best as they can about what they know and what they do not know, but these contrasting statements are challenging the core findings of this manuscript and I believe can be revised given a consistent clarifying text to the effect of: (1) 1% leak rates were inferred from 2 of the incubations and assumed to be constant throughout the study (2) 2 of the incubations did appear to have large leaks so those incubation leak rates may not fit the assumed constant 1% leak rate (3) these constant leak rate assumptions and inferred ambient environmental data were necessary given sparse ambient environmental data but future studies can ameliorate these shortcomings by continuous biogeochemical monitoring both within and outside the incubation tents.

Line 217: For the other three incubations, the “(known) variable environment” is actually an assumed variable environment because no external measurements were made. This should be clearly stated in the text.

ReefBudget:
Table S2 is confusing and unclear—I’m assuming that each individual colony is listed separately? I think overall percent cover of each species would improve clarity here. Furthermore, the “% of full planar 4.4 m^2 area” column matches the ReefBudget calcification rates (but please double check units/values!) for most of the coral species so I’m assuming this is in error... The authors state in the methods that there is a total of 4.2% coral cover in the enclosure so perhaps this column is supposed to be growth rates, but then M. faveolata has the growth rate listed for M. franksi (please also note that Montastrea faveolata/franksi/annularis complex is now Orbicella following recent genetic work). This table should list complete genus names for further clarity. The authors state that they followed ReefBudget protocols for this section but the number of errors in this table and lack of described methods makes me weary of the ReefBudget portion of this study. How was ReefBudget conducted? How were % cover and sizes of coral colonies quantified so precisely? Addressing these questions in addition to revising Table S2 will further improve the clarity of this section.

Where is the ambient O2 data reported? Are the authors referring to the 2016 Pelagia O2 vs salinity measurements? But these were collected a full year after the incubations were done and “<5 km away” so some mention of the assumptions associated with this data in the absence of the incubation-contemporaneous ambient measurements should be more clearly stated.

Figure 4e-f: Is the bold red line the complete model and the thinner red line the asymptotic model? This is not clear from the figure labels or caption.

Line 299: The authors state that ambient nutrients were invariable during the incubations, but I do not see actual ambient nutrient data supporting this claim for the duration of the incubations. Where is this coming from?

Please include figure captions and/or labels as appropriate for all of the supplemental materials as I do not see them in the downloaded version.
Experimental design: no further comment
Validity of the findings: no further comment
Additional comments: no further 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: IN-SITU INCUBATION OF A CORAL PATCH FOR COMMUNITY-SCALE ASSESSMENT OF METABOLIC AND CHEMICAL PROCESSES ON A REEF SLOPE
Review round: 3
Reviewer: 2

Output:
Basic reporting: No comment.
Experimental design: No comment.
Validity of the findings: No comment.
Additional comments: The authors have done a very nice job of responding to reviewer comments in their revision and I believe that this version of the manuscript is much stronger thanks to their thoughtful editing. This study provides a useful addition to the literature and I have only a few minor comments which I feel will improve the paper.

Line 18, “Here, we investigate diurnal trends of alkalinity and dissolved organic carbon, oxygen and nutrients…” but also dissolved inorganic chemistry. I’d mention that DIC was monitored as well here in the abstract, for clarity, or perhaps lump alkalinity and DIC together and say “carbonate chemistry, dissolved organic carbon, oxygen, and nutrients”.

Line 27, I’d strongly suggest expressing calcification rates here and in several other places in units of mmol/m2/hr as this makes it easier to compared to NCP fluxes (i.e., same units used) as well as most of the similar data in the literature. It makes sense to scale up to kg/m2/yr when comparing these values to the ReefBudget approach, but in most other cases I think it’s more useful to use the hourly values. In effect, this means that the data will be expressed in both sets of units, depending on the context.

Line 48, NCC reflects responses of the entire community, not just the corals, and this is an important distinction. For example, many corals are not that sensitive to ocean acidification, and some species seem to be totally resistant, whereas dissolution of reef sediment and pavement appears to be much more sensitive to OA. Hence, NCC reflects the overall response of the community, not the responses of the corals (which are themselves only a component of the larger community).

Line 220, 267, 327, “dissolution” should be ‘carbonate dissolution’, ‘CaCO3 dissolution’, or something like that. Be sure to explicit about what is dissolving.

Line 325, I would clarify for the reader that respiration decreases TA slightly *due to the release of nutrients* (though respiration of only carbohydrates/lipids doesn’t impact nutrients or TA).

Line 333-335, “…although exterior AT and CT increased by as much as 60 μmol kg-1 in the latter half of the incubation inside this tent, interior AT and CT during those 4 hours did not increase at rates higher than 2.5 μmol kg-1 hr-1.” The wording is confusing here—you mean that the concentrations in the exterior increased by up to 60 umol/kg during the 4 hr incubation, but concentrations in the interior increased at a max of 2.5 umol/kg/hr?

Line 436-438, “This may be explained by varying community composition such as the absence of macro-bioeroders within our tent, especially not taking into account of mechanical bioerosion caused by parrot fish for instance.” But the chemical technique applied here does not and cannot assess mechanical bioerosion, only net carbonate precipitation and dissolution. This is an important distinction between the techniques—mechanical bioerosion is important to the process of reef accretion, and can be estimated with the ReefBudget approach, but mechanical erosion *cannot* be estimated with a chemical approach, only net carbonate precipitation/dissolution.

Line 439-454, well-written and nice comparison of the pros and cons of these techniques.


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

Input:
Title: CHARACTERIZATION OF BACTERIOPLANKTON COMMUNITIES AND QUANTIFICATION OF ORGANIC CARBON POOLS OFF THE GALAPAGOS ARCHIPELAGO UNDER CONTRASTING ENVIRONMENTAL CONDITIONS
Review round: 1
Reviewer: 1

Output:
Basic reporting: Review Guevara Campoverde et al.

Reference. 28742

Characterization of bacterioplankton communities and quantification of organic carbon pools off the Galapagos Archipelago under contrasting environmental conditions

Summary

The manuscript “Characterization of bacterioplankton communities and quantification of organic carbon pools off the Galapagos Archipelago under contrasting environmental conditions” describes the measurements of physico-chemical data, carbon pools, and microbial community composition at three sites in the Galapagos Islands, which have the potential to become test sites for ocean warming and ocean acidification due to their characteristics. These characteristics were not as prominent as in other years, probably due to an interference by a weak La Niña event. Therefore, no clear sites for the investigation of OW and OA effects were found. Furthermore, the paper describes the abundance of DOC and the formation of POC from waters of this station and finds a microbial community dominated by Gammaproteobacteria (Alteromonas), Roseobacter, and members of the SAR111 clade. The paper determines DOC availability as the driving force for differences in the microbial community, even though temperature is discussed as well as pH.
Experimental design: OK.
Validity of the findings: OK.
Additional comments: General comments

The manuscript is well written and contains several interesting findings. However, the overall structure of the manuscript makes it hard to follow the main hypothesis. It is not clear if the main task was to demonstrate the suitability of the sampling sites to investigate OW and OA or if the microbial community composition of different sites within the Galapagos Islands should be investigated or if the formation of aggregates in response to changing physico-chemical parameters is most important. A connection of the three different topics to form a coherent story is missing. In addition, streamlining all parts would help to increase the quality of the manuscript. Both are specifically true for the discussion.

Furthermore, the difference in temperature seems not to be significant. The max. value of CoI is 22.13 °C, while the min. value of RoR is 22.14°C. Therefore, temperature dependent oxygen solubility is questionable, especially since BoC and CoI show similar DO values, but should be furthest apart. Some papers cited for the argumentation on the dependency of DOC and aggregate formation on temperature state differences of 2-6°C, not 0.8°C as found in this study. Therefore, the argumentation on temperature needs to be more elaborated or the importance of temperature as a factor needs to be reduced. Especially, since Chl a/ phytoplanktom bloom occurrence is later discussed as factors that may have a higher influence than temperature (s. line 647 ff). Considering then the high abundance of DOC loving Gammaproteobacteria, temperature seems not to be the most likely factor shaping the communities. This discussion needs to be more clear and congruent.

Detailed comments

Abstract

Line 40: replace for with to thrive in or abundant in
Line 41: …taxa, such as members of the SAR11 clade, …

Introduction

Line 54: Comma before as well as
Line 97: Comma after nutrients – Consider splitting sentence

Material and Methods

Line 170: Change thought to through
Line 174: Sentence mentioning Germany is obsolete
Line 224: Accession number correct? ENA doesn’t find the accession number.

Results

Line 398: Candidatus Pelagibacter ubique

Discussion

Line 405: Delete extensive
Line 447: the abbreviation for BoC was introduced already
Lines 460-461: The events are named El Niño and La Niña, check throughout the manuscript
Lines 558-570: It seems odd to start and spend 12 lines on a single taxon abundant only on one site, especially when the taxon cannot be correlated to any measured factor. Even though it is a Gammaproteobacterium, I suggest to reduce this two a sentence and discuss it with the other Gammaproteobacteria.
Line 570: Unclear switch to pH.
Line 606: Members of the SAR11-clade
Line 609: SAR11-clade
Line 609-614: Redundant since protorhodopsin was not measured
Line 616: Replace overgrown
Lines 638-640: Why were these methods not used then?
Line 647 ff: This sentence basically undermines all arguments made for temperature before.


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

Input:
Title: CHARACTERIZATION OF BACTERIOPLANKTON COMMUNITIES AND QUANTIFICATION OF ORGANIC CARBON POOLS OFF THE GALAPAGOS ARCHIPELAGO UNDER CONTRASTING ENVIRONMENTAL CONDITIONS
Review round: 1
Reviewer: 2

Output:
Basic reporting: no comment
Experimental design: Although the research question is well defined, considering the results and the data interpretation, it might not be the most relevant. I propose some improvements in the "General comments for the author" section.

The rest of the experimental design is fine, within the aims and scope of the journal with rigorous investigation performed and methods described with sufficient details.
Validity of the findings: Conclusions are well stated but may not be appropriate to the results of this study. Please see in the "General comments for the author" where I propose some improvements.
Additional comments: Summary:
The authors investigated patterns of the bacterial community at three sites in the Galapagos Archipelago used as proxies of three different environmental scenarios: a current scenario (reference site: Cowley Islet), an ocean acidification scenario (Roca Redonda), and an ocean warming scenario (Bolivar Channel). Contextual parameters were measured and metabarcoding was used to study the bacterial community from the free-living and the particle-attached fractions at each sites. In parallel, aggregate experiments were conducted to evaluate the volume/number of organic aggregates formed at each site and further estimate their contribution to patterns of the bacterial community.

Major comments:
Overall, I have mixed feelings about this work. The sampling, the original approach, the experimental procedure and analyses are very sound. However, I feel that the manuscript should be written differently to better highlight the findings and the hard work put in that study. I would advise the authors to refocus the hypotheses of their study without testing the OA and OW effects (even though it was the starting hypothesis). For instance, findings could be better highlighted with hypotheses comparing 3 sites with distinct environmental conditions in a tropical zone with a special focus on the impact of the organic carbon pools. In this example, it would be like testing the effect of three distinct ecological niches on the microbial community. In this case, OA and OW may then be discussed later on in the manuscript as perspectives. Also, this new focus fits better the current title.

Also, the authors may either be more positive in the interpretation of their results (by trimming some of the many “may”, “seem”, “likely”) or just remove assumptions where they are too unsure about them (e.g. L656 “may seem the more likely”).

Specific comments:
Please correct through the manuscript: replace “*on the* class level” by “*at the* class level”.

1. Introduction
L86: There is a typing mistake: subject*ed*“”

2. Materials and Methods
L170: There is a typing mistake: “th*r*ough”
L253-256: Could you explain why the data set was reduced following this approach?

3. Results
L318-320: The sentence is a bit hard to follow, please rephrase.
L343: There is a typing mistake: “most *to* the”
L345: Either specify which samples or the number of samples.
L361: SI Fig 2. Is not an RDA, it is the fig. regarding aggregate sizes.
L394: Instead of “likewise of the Rhodobacteraceae”, the authors may write “as for the Rhodobacteraceae”.

4. Discussion
L461: “[…] did not resemble the extremes recorded during strong El Nino or La Nina events.” Do you have a reference for this statement?
L478: What do you mean by “moderately positive correlation”? Is it a non-significant positive correlation?
L517 & Fig. S4: Where do these phytoplankton data come from?
L523: There is a typing mistake: “within *in*”, *in* to remove.
L555-556: Please rephrase the sentence.
L558: What do you mean by “preferentially found only”?

Paragraph starting L577: This part is a bit confusing as the authors still use the OW and OA hypotheses to discuss cyanobacterial patterns although they were falsified in the 4.1. and 4.2. sections.

L634: Why specifying the class for Polaribacter and not for Sulfitobacter and the other Rhodobacteraceae?

To further understand the community dynamics and their correlations with the different environmental conditions, the authors may also propose a functional approach in their perspectives. This would make sense as they hypothesize several times that despite some community turn over, the community may present some functional redundancy.

Figures
Fig. 2 : This is minor but the reading on the PCA may be nicer if the names of the parameters and samples were not overlapping.
Fig. 4: Please correct: “Taxonomic composition of the bacterial communities *at the* class level”

Regarding the supplementary figures, the legends were not easy to find, the authors may add a file document with the legends.

Also, I could not find where the Procrustes figure (SI Fig. 3) was discussed in the text.