Holo-2bRAD: A Hologenomic Method for High-Resolution Analysis of Coral Microbiomes During Bleaching

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

See attached

Comments for author File: Comments.pdf

Comments on the Quality of English Language

Needs reading by an English speaker

Author Response

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Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

At first glance, the article makes an extremely favorable impression. The authors propose the use of a new, relatively inexpensive approach to determining the health of coral reefs, including the composition of their microbiota. The coral species used by the authors, Galaxea fascicularis, has been repeatedly proposed as a model organism and, in principle, corresponds to the task set by the authors. However, upon reading the manuscript, a large number of questions arose that need to be resolved before making any conclusions about the possibility of publication.

Firstly, the Materials and Methods section is inadequately written. From section 2.1. Sampling and DNA Extraction, we learn that the results are based on three colonies, measuring 10-15 cubic centimeters, collected in Yalong Bay. There is no information on how the colonies were selected, why colonies of this size were chosen, and in what condition (healthy or somewhat bleached) they were selected. There is no information on which part of each colony was used to obtain the libraries. Did the authors use the dead part of the colony or only the living part, and was the skeletal part included? Since we are dealing with massive colonies, and adult specimens of this species reach a diameter of more than a meter, and the ability to reproduce sexually in this species occurs when colonies are larger than 5 cm and older than 8 years, could the use of small colonies 3-5 cm in diameter have influenced the results obtained?

Further on in the text (see Fig. 1, line 217, Table 2, line 317), it becomes clear that some additional material was used by the authors, and only in section 3.5 (results) do the authors mention for the first time that they conducted research on some other colonies (the number is not specified) with varying degrees of bleaching (unfortunately, the authors do not specify how exactly the bleaching was assessed).  I strongly recommend that the authors include a description of the material collected  in July 2024 in the Materials and Methods section and describe in detail the methodology for collecting, assessing, and studying all colonies used in this study.
Figure 1 raises many questions. It is unclear whether healthy and bleached colonies were analyzed separately. How does this figure relate to the three stages of bleaching? What do the authors mean by “genome download” in the context of this study?
The authors should explain all this in detail and accurately in the “Materials and Methods” section and, based on the rewritten section, redo Figure 1.
DESeq2 is not ideally suited for microbiome data due to the compositional nature of such datasets. Nevertheless it is not mentioned in the Materials and Methods as well. It would also be desirable for the authors to describe in the Materials and Methods section how the abundance data were generated.

Table 2 (Genotyping accuracy of coral and sea water)  does not stand up to criticism. The abbreviations used in the table are not explained anywhere, and the authors leave it up to the reader to guess what they mean. Same abbreviations (without explanation) arrive also at figures 5 and 6)

Without resolving these priority issues, the manuscript cannot be considered for publication.

I do not want to dwell on minor inconsistencies and inaccuracies, as they will undoubtedly be addressed when working on the structure of the article.

Author Response

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Author Response File: Author Response.docx

Reviewer 3 Report

Comments and Suggestions for Authors

The article provides valuable result regarding application of novel technique (2bRAD-M technology) for the analysis of coral-associated microbial populations. The work may be accepted after minor revisions.

Questions and commentaries.

1. All figures should be enlarged and their resolution should be increased as most of figures are not readable.
2. Table S1 may be included in the main text of the manuscript.
3. Line 252. the remaining genomes were excluded on the basis of lacking unique 2bRAD tags. Could you clarify – why these genomes may be lack of unique 2bRAD tags?
4. Line 343. The results of the analysis of the microbiota of coral and seawater samples are presented. Please, describe seawater sampling in subsection 2.1. Sampling.
5. Could you provide examples of the comparison of the results of microbial population analysis using 2bRAD-M technology and 16S rRNA gene fragment metabarcoding? Are you planning further works to compare these methods?

 

Author Response

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Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The authors have taken criticism of the original version of the article and have made many revisions.

As it stands the article is now publishable.

 

The authors have not taken my suggestion to delineate the basic structure of the coral symbiosis which is the specific endosymbiosis of a dinoflagellate in the endoderm of the coral animal (cnidarian). I think that this is a missed opportunity.

However, in their present definition of the coral holobiome, the authors sufficiently alert the reader to the difficulties of interpreting coral bleaching from these results without further work on the constituent parts of the holobiome, particularly the dinoflagellate algal endosymbiont (the zooxanthella).

In my humble opinion this could be made more explicit in the present iteration

Author Response

Response: We thank the reviewer for the positive evaluation of the revised manuscript and for this insightful suggestion. We agree that coral bleaching is fundamentally associated with the disruption of the endosymbiotic relationship between the coral host and dinoflagellates (Symbiodiniaceae). In the present manuscript, we have emphasized the complexity of the coral holobiont and noted that interpreting bleaching-related microbial changes requires considering the different components of this symbiotic system. We appreciate the reviewer’s constructive comment and believe it highlights an important direction for future research.

 

Reviewer 2 Report

Comments and Suggestions for Authors

I would like to thank the authors for their nice response.
Unfortunately, not for quality of the new version of the MS.
I have a strong impression that the version of the MS that was submitted after the review is not good one. or  perhaps authors decided not to change anything but only claim some actions? 

1. In the previous round of the review authors were asked to add recent literature such as 
   Peixoto, R. S., & Voolstra, C. R. (Eds.). (2025). Coral Reef Microbiome. Springer Nature Switzerland.
   Unfortunately there is no response or an action, and Introduction section arrived without any changes
2.  In the Response 2 authors claimed that they moved all collection data in the Material and Methods section.  Nevertheless, I see it at the same place - section 3.5. I do not see any information how the water samples were collected – this also has to be in material and Methods section as this is material – not results. And again  Table  3 with acronyms arrives BEFORE acronyms are introduced (because they suppoed to be introduced in Material and Methods section.
   
   Authors, Please, return to the previous round of the review and check carefully and make claimed changes one by one. The article requires significant work and must be checked before submission—the authors should submit a revised version, not a draft.

 Other remarks
1. It is unclear from Figure 5 and the accompanying text elsewhere whether the authors verified that all their specimens of the genus Galaxea belong to a single species—Galaxea fascicularis s.s. Please state this clearly.

2. lines 398–400. In the reference to Table 3, the authors write: "Holo-2bRAD libraries were constructed and sequenced; alignment to the custom hologenome database revealed a significant disparity in microbial tag proportions between extreme bleaching phenotypes (Mann-Whitney U test, p < 0.05; Table 3).” However, there is no such value in Table 3. Please use consistent terminology in the table and in the text referring to it.
3. lines 420–422 "Three unresolved bacterial lineages (g__QQUY01-Pyrinomonadaceae, g__Cutibacterium, o__UBA12135)... “—the names listed are missing from Figure 6D, to which the authors refer, and are left as ”unresolved" in Figures 6A and 6D. Authors, please ensure that the text and figures are consistent. Also, compare the colony designations (FA1–FC3) in the text, tables, and all figures, and standardize them (see, for example, Figures 6A and 6B). It is also unclear what the authors mean by “W” and “NW” in Figure 6B. Please provide an explanation in the text or in the figure caption. In the same figure, please replace the numerals (7 and 15) with the names of the corresponding taxa.

minor remarks
Line 26 “Galaxea fascicularis” after species name, please add author, year and taxonomic position (Order and Family) in parentheses (same in the first mention in Introduction)
line 29 Thermoanaerobacterium – authors determined only one species of the genus – Thermoanaerobacterium thermosaccharolyticum -  please use it as binomen throughout the MS

Author Response

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Author Response File: Author Response.docx