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I appreciate the time you have spent on revising your manuscript. It has resulted in an excellent study.
[# PeerJ Staff Note - this decision was reviewed and approved by Dezene Huber, a PeerJ Section Editor covering this Section #]
Overall, your manuscript is well-written, clear, and generally well received. The two reviewers raised a number of concerns, though they are relatively minor and do not require further experiments or wet lab work.
The paper is well-written.
Some additional background is needed in the introduction:
it is confusing that you start off reference florida when the study takes place in the cayman islands.
I would also mention why cervicornis is especially easy to grow like this.
A discussion of Gignoux-Wolfsohn et al. 2017 should be added as they found a strong effect of presumed genotype (referred to as colony in the paper becuase they did not actually genotype) on A. cervicornis microbiomes. Although, many of those corals were dominated by Endozoicomonas. This difference and implications would be good to discuss as well.
S. Gignoux-Wolfsohn, F. M. Aronson, S. V. Vollmer, Complex interactions between potentially pathogenic, opportunistic, and resident bacteria emerge during infection on a reef-building coral. FEMS microbiology ecology 93, (2017).
The sampling strategy and methods are well-described.
A few comments on methods:
104- should be and yellow
106- were these wild colonies?
159: Did you do one analysis with all 3 of your factors? or separate?
Findings are well-described.
Comments on the results:
Missing figure legends?
Figure 4: Would be nice if this were all rickettsiales asvs. Given that there are only 11 I dont think it would significantly detract from your point and would allow the reader to make more clear comparisons between figure 3 and figure 4
Its also unclear if the y axis on this figure refers to total abundance or abundance within the rickettsiales?
177-184: its confusing that you are discussing most abundant asvs but referencing a figure where everything is grouped by order.
228-230: Would you expect rerunning the pipeline to produce different results? how much variation was there?
The discussion needs a little more work to place your findings in the context of the previous literature.
281: Also, they sampled differently, there could be differences you aren't capturing using syringe sampling. This sampling strategy should be discussed.
303: But Gignoux-Wolfsohn 2020 used V6 and found high abundance of Rickettsiales.
288-304: There is a lot of discussion of A. palmata that seems out of place given that this was not the objective of this study.
It is also interesting that you are sampling them right next to each other removing the environmental variation that confounds natural studies of genotypes. It would be interesting to have more of a discussion of the effect of site as found in Chu and Vollmer and Gignoux-Wolfsohn and Vollmer 2014
The discussion needs another paragraph focused on your finding of genotype-specificity of the dominant strains of Rickettsia as seen in Figure 4, espeically the finding that all samples across colonies of the green genotype were dominated by the same ASV. This is an interesting finding! To me it suggests a closer relationship between these rickettsia and the coral host. You can delve into more of a discussion of microbial symbionts of corals (both beneficial and pathogenic). What does this tell you about when these corals are aquiring the rickettsia?
see M. J. Neave et al., Differential specificity between closely related corals and abundant Endozoicomonas endosymbionts across global scales. The ISME journal, (2016).
In this study, the Authors analyzed the microbial community composition of a keystone and endangered coral species, Acropora cervicornis, after sampling colonies that were reared in a Caribbean ocean nursery. Based on 16S rRNA gene metabarcoding, the Authors identified that bacterial communities did not display significant spatial heterogeneity across single colonies, but that they were influenced by the host genotype (with one bacterial genus dominated most samples). This work provides interesting information about the microbial communities associated with A. cervicornis and is a useful baseline for future studies.
Overall, the manuscript is clear, well written and structured, and the language is professional. Figures are relevant; raw data as well as scripts used for analysis are available.
The introduction flows well and is appropriately referenced, but could benefit from some additional information and minor revisions:
Lines 71-73: The allusion to coral microbiome manipulation comes a little bit abruptly and is not really the scope of the present study. While still useful to mention it, it would be interesting to expand on the roles of the coral microbiome and justify why it is important to characterize it (which would emphasize the value of this study).
Line 79: The Authors refer to coral “microbial communities”, which would actually encompass not only bacteria and archaea (assessed here), but also other microbes. As for example Symbiodiniaceae communities were not investigated, it would be more accurate to refer to “prokaryotic communities” or “bacterial communities” (when relevant) throughout the manuscript.
Lines 79-86: Either here or in the discussion, the Authors could emphasize why it is important to investigate spatial heterogeneity of bacterial communities in corals. If the communities are homogeneous, it would enable to collect fewer samples across the colony and hence reduce the stress of sampling. If the community composition varies across the colony, then it would be important to collect a sufficient number of samples from different locations on the same host, otherwise results wouldn’t be representative and within-colony differences could bias interpretations.
Line 86: It could be relevant to mention that some studies have reported a strong association between the coral host genotype and its bacterial community composition (for example: Glasl et al. 2019 PeerJ; or Hester et al. 2016 ISME - a review in which the authors listed studies having found an effect of coral species or environment on coral-associated bacterial communities).
Research questions are well-defined and most methods clearly explained, however the points outlined below require further explanations. Importantly, the Authors haven’t specified how they dealt with potential bacterial contaminants (as they didn’t seem to have done DNA extraction controls). If that is the case, this shortcoming should be acknowledged. Also, it is not indicated whether read counts were normalized prior to statistical analyses, which could bias the results given that samples had varying sequencing depths.
Line 115: What was the purpose of the agitation with the syringe? Was it to clear off the tissue from potential debris, trigger mucus release, or else…?
Line 120: Seawater was excluded from the samples by decanting, however it is unlikely that microorganisms present in seawater were entirely removed from samples. It would have been interesting to sample the seawater as well, and assess which part of the bacterial microbiome was coral-specific vs derived from the environment. This could perhaps be commented on?
Line 124: Was an equal amount of each sample used for DNA extraction (if so, how much)?
Lines 133-134: Negative PCR controls were performed, however there is no mention of DNA extraction controls. This is an important procedure to identify laboratory/reagent contaminants and is becoming a critical inclusion in microbiome studies (see Slater et al. 2014 BMC Biology; de Goffau et al. 2018 Nature Microbiology; Davis et al. 2018 Microbiome). If the Authors did not perform blank DNA extractions (and sequenced them alongside samples) to identify and remove contaminants, this should be explicitly acknowledged.
Lines 151 and further: Was any variance stabilization method (such as with DESeq2 or rarefaction) applied on the data to account for the different read counts across samples? Not normalizing the read counts could greatly impact and bias the results (especially since samples fell within a large range of counts, as outlined on line 168).
Line 160: Please specify that ANCOM stands for “Analysis of Composition of Microbiomes”
Results are nicely reported and discussed, and the conclusion clearly summarizes the findings of the study. Could the Authors please address the following points?
Lines 174-175: Was a pairwise comparison performed to check whether the difference in microbiome composition between the Y and the G/R was statistically significant?
Lines 209 and further: Did the Authors try to conduct the same analysis pipelines by removing these very abundant Rickettsiales from the samples? Sometimes rare members of the microbiome play important roles. The Authors specified on lines 232-234 that differences between coral colonies of different genotypes were based on microbes with low relative abundance. Such an analysis (with Rickettsiales removed from the dataset) would therefore be relevant to enable patterns to emerge that are based on low-abundance bacteria.
Lines 272-273: As the Authors outlined on the previous lines, it is interesting that the microbiome seems relatively uniform within A. cervicornis colonies and if so, stress of sampling could be reduced in future studies by collecting fewer samples while still obtaining a representative microbial community. However, the conclusion needs to be tempered, as patterns observed in one or two species cannot be directly extrapolated to all acroporid corals. This study in particular considered nursery-reared corals, which were hung on a structure and not growing on the reef as natural colonies would be. This in itself might lead to different microbial signatures and it would be relevant to repeat a similar study with wild A. cervicornis corals to confirm the present results. Moreover, other acroporid species (some of which have different morphology/physiology, or individuals sampled from another location) might possess microbial structures that are not consistent with those observed in this study. Hence, lack of spatial heterogeneity should not be assumed for most acroporid corals, at least without further evidence.
Lines 284-285: Maybe the Authors could suggest that more studies on spatial heterogeneity of microbial communities need to be undertaken across numerous coral species to verify these conclusions. If colonies are large enough, taking more than 3 samples per location on the colony would be a more robust design.
It could be useful to suggest that future studies should investigate the microbial community composition of corals transferred to the reef and compare how they relate to nursery-reared communities. Perhaps the success of individual corals could be linked with the composition of their microbiome, or some detrimental shifts could be identified in colonies that perform less well?
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