Combined responses of primary coral polyps and their algal endosymbionts to decreasing seawater pH

With coral reefs declining globally, resilience of these ecosystems hinges on successful coral recruitment. However, knowledge of the acclimatory and/or adaptive potential in response to environmental challenges such as ocean acidification (OA) in earliest life stages is limited. Our combination of physiological measurements, microscopy, computed tomography techniques and gene expression analysis allowed us to thoroughly elucidate the mechanisms underlying the response of early-life stages of corals, together with their algal partners, to the projected decline in oceanic pH. We observed extensive physiological, morphological and transcriptional changes in surviving recruits, and the transition to a less-skeleton/more-tissue phenotype. We found that decreased pH conditions stimulate photosynthesis and endosymbiont growth, and gene expression potentially linked to photosynthates translocation. Our unique holistic study discloses the previously unseen intricate net of interacting mechanisms that regulate the performance of these organisms in response to OA.

The work is reasonably well executed. The approach of combining physiological and molecular analyses, and the analyses being done for both host and symbionts, is a big plus. The data are properly collected and interpreted. It is a pity that the Methods sections have to be presented in such a brief manner, going to the supplementary material is cumbersome for readers. I really wish that some can be presented in the main paper.
I recommend acceptance after minor revision. I have only a few technical comments for consideration in the revision, which are shown below. What were the natural pH and other conditions from which the coral colonies were collected from?
With 9 day exposure to the acidification conditions, the responses observed were shortterm/acute responses. For those that died or could not settle (would eventually die) the effect is clear. For those that survived, despite the observed structural, physiological and molecular alterations, would you expect them to recover, partially or in full, to the "normal" status (i.e. as if in pH8.2) if they were to be in the acidified condition over the long run? Is there any reported data of such recovery?
Related to the above question, if the observed effect will carry through adulthood and impact reproduction? I think it is important to give some discussion on the question here and the above. Review form: Reviewer 2 (Ewelina Rubin)

Recommendation
Accept with minor revision (please list in comments)

Scientific importance: Is the manuscript an original and important contribution to its field? Excellent
General interest: Is the paper of sufficient general interest? Excellent Quality of the paper: Is the overall quality of the paper suitable? Excellent

Do you have any concerns about statistical analyses in this paper? If so, please specify them explicitly in your report. No
It is a condition of publication that authors make their supporting data, code and materials available -either as supplementary material or hosted in an external repository. Please rate, if applicable, the supporting data on the following criteria.

Do you have any ethical concerns with this paper? No
Comments to the Author I think you did fantastic work on the study and the paper. I think the whole coral research field can benefit from studies that combine to measure transcriptomic responses and phenotypic traits. We need more studies like that. Those actually do move the coral research forward. it was a true please to read your paper.
I only have a few comments/suggestions for corrections 1) to my knowledge you are not the first presenting both coral and algal symbiont response to OA (please check Lin, Z., Wang, L., Chen, M., and Chen, J. (2018). The acute transcriptomic response of coral-algae interactions to pH fluctuation. Mar Genomics 42, 32-40.
2) there are few times in the discussion when you are using abbreviations of genes/protein (e.g. CARP4 and STPC) that you never defined in the paper before; I had to go to our supplementary tables but even there the proteins are not defined so it forces a reader to seek out this information in the reference material 3) I would not call the genes/proteins in table S4 to be "known to be biomineralization related" --I would call them putative associated with the biomineralization process. Because sometimes the "evidence" comes only from transcriptomics studies, only and not direct functional genomics studies. In my opinion, we can not be sure that something that was regulated is involved in biomineralization. I see many proteins that are known to be associated with other biological processes. For example --vitellogenin --there is direct evidence that the protein is present in coral oocytes --see Shikina, S., Chen, C.J., Chung, Y.J., Shao, Z.F., Liou, J.Y., Tseng, H.P., Lee, Y.H., and Chang, C.F. (2013). Yolk formation in a stony coral Euphyllia ancora (Cnidaria, Anthozoa): insight into the evolution of vitellogenesis in nonbilaterian animals. Endocrinology 154, 3447-3459.

17-May-2021
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Best wishes, Dr Daniel Costa mailto: proceedingsb@royalsociety.org Reviewer(s)' Comments to Author: Referee: 1 Comments to the Author(s) This study investigates the effect of acidification on the primary polyps of the stony coral Stylophora pistillata using pH 7.8 and 7.6 versus pH 8.2 as the control. A set of morphostructural, physiological, as well as transcriptomic parameters were measured. The major findings included 1) there was a greater response to the pH change from 8.2 to 7.6 than other changes; 2) skeleton calcium deposition was reduced but coral tissue growth was elevated under acidification; 3) genes related to calcification were downregulated but those related to environmental sensing and signaling were upregulated; 4) symbiont density increased, along with upregulation of genes involved in carbon fixation and photosynthate translocation. The authors conclude that the coral larvae were, at least some, more resilient to acidification and those survived expressed mechanisms to counteract the effect of lower calcification under the extreme low pH.
The work is reasonably well executed. The approach of combining physiological and molecular analyses, and the analyses being done for both host and symbionts, is a big plus. The data are properly collected and interpreted. It is a pity that the Methods sections have to be presented in such a brief manner, going to the supplementary material is cumbersome for readers. I really wish that some can be presented in the main paper.
I recommend acceptance after minor revision. I have only a few technical comments for consideration in the revision, which are shown below.
What were the natural pH and other conditions from which the coral colonies were collected from?
With 9 day exposure to the acidification conditions, the responses observed were shortterm/acute responses. For those that died or could not settle (would eventually die) the effect is clear. For those that survived, despite the observed structural, physiological and molecular alterations, would you expect them to recover, partially or in full, to the "normal" status (i.e. as if in pH8.2) if they were to be in the acidified condition over the long run? Is there any reported data of such recovery?
Related to the above question, if the observed effect will carry through adulthood and impact reproduction? I think it is important to give some discussion on the question here and the above. Referee: 2 Comments to the Author(s) I think you did fantastic work on the study and the paper. I think the whole coral research field can benefit from studies that combine to measure transcriptomic responses and phenotypic traits. We need more studies like that. Those actually do move the coral research forward. it was a true please to read your paper. 2) there are few times in the discussion when you are using abbreviations of genes/protein (e.g. CARP4 and STPC) that you never defined in the paper before; I had to go to our supplementary tables but even there the proteins are not defined so it forces a reader to seek out this information in the reference material 3) I would not call the genes/proteins in table S4 to be "known to be biomineralization related" --I would call them putative associated with the biomineralization process. Because sometimes the "evidence" comes only from transcriptomics studies, only and not direct functional genomics studies. In my opinion, we can not be sure that something that was regulated is involved in biomineralization. I see many proteins that are known to be associated with other biological processes. For example --vitellogenin --there is direct evidence that the protein is present in coral oocytes --see Shikina, S., Chen, C.J., Chung, Y.J., Shao, Z.F., Liou, J.Y., Tseng, H.P., Lee, Y.H., and Chang, C.F. Decision letter (RSPB-2021-0328.R1)

02-Jun-2021
Dear Mrs Scucchia I am pleased to inform you that your manuscript entitled "Combined responses of primary coral polyps and their algal endosymbionts to decreasing seawater pH" has been accepted for publication in Proceedings B.
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Response to reviewer's comments: Combined responses of primary coral polyps and their algal endosymbionts to decreasing seawater pH
Scucchia Federica, Malik Assaf, Zaslansky Paul, Putnam Hollie, Mass Tali ___________________________________________________________________________________________________________ Dear editor and reviewers, your constructive and valuable comments helped us in significantly improving the quality of the manuscript. We have addressed all the comments by the reviewers as explained below in this document. Further, the modifications we implemented are all marked in both the manuscript and the supporting information file, for ease of identification. Below, we reproduce the reviewers' comments and questions in normal font with our responses in blue. Text that we have changed in the manuscript and copied into our responses is italics.
My co-authors and I thank you very much for your time and support and hope you find this experimental work relevant for your readership.

Detailed response to reviewers
Reviewer 1  The work is reasonably well executed. The approach of combining physiological and molecular analyses, and the analyses being done for both host and symbionts, is a big plus. The data are properly collected and interpreted. It is a pity that the Methods sections have to be presented in such a brief manner, going to the supplementary material is cumbersome for readers. I really wish that some can be presented in the main paper.
Thank you for the positive comments. We agree with the reviewer and acknowledge that placing the Methods sections in the supplementary could be unwieldy for readers. However, to be able to obey to the strict word limit requirement of the journal, we had to take the entire Methods section out of the main manuscript which, in the current form, is already very close to the world limit.
 What were the natural pH and other conditions from which the coral colonies were collected from?
The natural water conditions of the site from which coral larvae were collected are available through the Israel National Monitoring Program of the Gulf of Eilat  et al., 2021)) on a 12-hour/12-hour photoperiod was provided".
 With 9 day exposure to the acidification conditions, the responses observed were short-term/acute responses. For those that died or could not settle (would eventually die) the effect is clear. For those that survived, despite the observed structural, physiological and molecular alterations, would you expect them to recover, partially or in full, to the "normal" status (i.e. as if in pH 8.2) if they were to be in the acidified condition over the long run? Is there any reported data of such recovery?
We are not aware of any study that assessed the degree of recovery in subsequent coral developmental stages under acidification conditions. However, a study conducted on adult corals reported a successful recovery from decalcification after a long-term exposure to OA (12 months), when corals were transferred back to ambient pH conditions (Fine & Tchernov, 2007), showing that corals indeed possess a remarkable ability to adjust and survive to future acidification conditions. With our work, we thus hope to stimulate more research focused on the degree of modifications occurring in subsequent coral life stages exposed to long-term acidification conditions, to allow us to make more accurate predictions on the vulnerability of corals to future OA scenarios.
We have now added a brief discussion on this question in the manuscript:  Related to the above question, if the observed effect will carry through adulthood and impact reproduction? I think it is important to give some discussion on the question here and the above.
To the best of our knowledge, there are no studies investigating carry-over effects of the changes occurring in coral recruits due to acidification that have an impact on reproduction. There is however evidence of corals that had lived all their postsettlement lives at naturally low pH conditions (volcanic CO2 seeps) that showed no difference in fecundity as compared to corals living in non-acidic waters (Fabricius et al., 2017). We also would like to point out that such corals at naturally low pH conditions also showed improved adult performance as compared to corals living in non-acidic waters, to potentially compensate for their low recruitment rates (Fabricius et al., 2017).
Moreover, it appears that the shift to a less-skeleton/more-tissue phenotype (which is what we observed in the recruits) would not negatively impact the achievement of sexual maturity and gametogenesis, which appeared to develop similarly in both reduced-skeleton adult corals at acidified conditions and corals at ambient pH (Fine & Tchernov, 2007).
We have now added a brief discussion on this question in the manuscript: -lines 252-253 "Moreover, improved adult performance could compensate for low recruitment rates, as observed for corals living around CO₂ seeps (Fabricius et al., 2017)"; -lines 289-292 "The shift to a less-skeleton/more-tissue phenotype would not negatively impact the achievement of sexual maturity and gametogenesis, which appeared to develop similarly in both reduced-skeleton corals at acidified conditions and corals at ambient pH (Fine & Tchernov, 2007)".
 Fig. 3B: GO terms clustered based on the similarity of gene content: gene numbers or gene functions, or degree of enrichment of the pathway by DEGs? Can the dot size be variable to reflect the degree of enrichment? What about the significance level of the enrichment?
GO terms were clustered based on the portion of shared genes (i.e. tightly clustered terms share a higher number of identical genes). We have now better clarified this point in the figure description: -line 571 "Dendrogram on the left showing enriched GO terms and KEGG pathways clustered according to the portion of identical genes shared". We have modified the figure and changed the squares size based on significance level. We have also added the total number of genes within each GO term or KEGG pathway, to provide a clearer indication of the degree of enrichment: -line 574 "Red numbers indicate the total number of genes within each per GO term or KEGG pathway".
 Fig. 4: The same questions as for 3B.
We have improved the figure by adding the same changes as in Fig. 3B  We thank you the reviewer for the comment, and modified the text in the manuscript as follows: -lines 355-360 "Earlier works on adult corals investigating the response to OA of the algal endosymbiont focused either on transcriptomic (Lin et al., 2018) or physiological changes (Baghdasarian et al., 2017;Kaniewska et al., 2012), and reported negative or no physiological responses in the algal symbionts with the exposure to acidic seawater. However, it must be noticed that broad physiological differences exist among different species across Symbiodiniaceae (Díaz-Almeyda et al., 2017), and that gene expression changes of the endosymbiotic algae greatly vary among coral populations in response to acidification (Kenkel et al., 2018)".
 there are few times in the discussion when you are using abbreviations of genes/protein (e.g. CARP4 and STPC) that you never defined in the paper before; I had to go to our supplementary tables but even there the proteins are not defined so it forces a reader to seek out this information in the reference material.
 I would not call the genes/proteins in table S4 to be "known to be biomineralization related" --I would call them putative associated with the biomineralization process. Because sometimes the "evidence" comes only from transcriptomics studies, only and not direct functional genomics studies. In my opinion, we can not be sure that something that was regulated is involved in biomineralization. I see many proteins that are known to be associated with other biological processes. For example --vitellogenin --there is direct evidence that the protein is present in coral oocytes --see Shikina, S., Chen, C.J., Chung, Y.J., Shao, Z.F., Liou, J.Y., Tseng, H.P., Lee, Y.H., and Chang, C.F. (2013). Yolk formation in a stony coral Euphyllia ancora (Cnidaria, Anthozoa): insight into the evolution of vitellogenesis in nonbilaterian animals. Endocrinology 154, 3447-3459.