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Expression of a symbiosis-specific gene in Symbiodinium
type A1 associated with coral, nudibranch and giant clam
larvae
M. Mies, C. R. Voolstra, C. B. Castro, D. O. Pires, E. N. Calderon and P. Y. G. Sumida
Article citation details
R. Soc. open sci. 4: 170253.
http://dx.doi.org/10.1098/rsos.170253
Review timeline
Original submission:
Revised submission:
Final acceptance:
22 March 2017
24 April 2017
27 April 2017
Note: Reports are unedited and appear as
submitted by the referee. The review history
appears in chronological order.
Note: This manuscript was transferred from another Royal Society journal without peer review.
Review History
RSOS-170253.R0 (Original submission)
Review form: Reviewer 1
Is the manuscript scientifically sound in its present form?
Yes
Are the interpretations and conclusions justified by the results?
Yes
Is the language acceptable?
Yes
Is it clear how to access all supporting data?
Not Applicable
Do you have any ethical concerns with this paper?
No
© 2017 The Authors. Published by the Royal Society under the terms of the Creative Commons
Attribution License http://creativecommons.org/licenses/by/4.0/, which permits unrestricted use,
provided the original author and source are credited
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2
Have you any concerns about statistical analyses in this paper?
No
Recommendation?
Accept with minor revision (please list in comments)
Comments to the Author(s)
Hello authors:
I very much enjoyed reading your paper about symbiont acquisition in corals, clams and slugs. I
think you addressed an important question about the genes involved in the establishment of
symbiotic relationships. The many RNA-seq experiments will benefit from more of this type
experiment!
Just a couple of suggestions and comments:
First of all, I am concerned that you did not do a second negative control. You sampled adult host
tissue with symbionts (positive control) and free living Symbiodinium cultures (negative control).
But you neglected to do the second negative control: to sample the larval tissue without
symbionts. I know you cannot go back and collect these samples. And even without these data, I
think your results are interesting. But it is always better to have both negative controls when
working with symbiosis. Maybe a sentence or two about why you didn't do this would help close
the circle.
Second, if you are so sure that the slugs were digesting Symbiodinium, what evidence can you
show? We see them clearly sequestered in the cerata in Fig 1B. On line 197 you indicate that you
had visual evidence of digestion. Why is this evidence not shown in Fig 1E? (also the letters for
the images in the figures are confusing with the genera initials) Can you at the very least describe
what was the visual evidence you are referring to? similarly the secondary evidence against
symbiosis (lines 249-257) is reasonable from an algal perspective. And yet these slugs are always
seen with living algal cells inside their bodies. Can you reword this to either make your point
more strongly or to back off the strength of your statement. The sentence in the abstract could be
adjusted to read something like, "evidence from this experiment did not support a mutualistic
relationship..."
Good luck with your paper and with your future work. A few more small comments by line
number below.
Line 29: A lot is known about establishment. But maybe not about the molecular biology or the
signals?
Line 58-62: Is Symbiodinium diversity relevant to this study? You might remove this to make it
more concise.
Line 79: I think you need at least one more sentence justifying this. Describe the studies you are
leaning on. Why are you so sure that this proton pump is the symbiosis red flag indicator?
Line 145-6: After you describe your samples, you did not mention freezing or buffers? How did
you preserve your RNA? Could your signal be spotty because of degradation?
Line 236: Only 1/3 coral replicates showed the expected results. Why is this? You included
multiple individual larvae in each sample. What does this mean for your results. How sensitive
was your assay? I think you need to discuss the inconsistent results in light of the methods a bit
more clearly. You were collecting along a developmental time series. Why did you top out at 72
hours? Was there literature or a pilot study that suggested these time points?
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3
Lines 244-257: please review the grammar in this paragraph
Line 266: this is a bit shaky? You write "intense changes in the circadian rhythm" but you do not
explain why this would generate inconsistent expression. Please explain further.
Review form: Reviewer 2
Is the manuscript scientifically sound in its present form?
Yes
Are the interpretations and conclusions justified by the results?
No
Is the language acceptable?
Yes
Is it clear how to access all supporting data?
Yes
Do you have any ethical concerns with this paper?
No
Have you any concerns about statistical analyses in this paper?
No
Recommendation?
Accept with minor revision (please list in comments)
Comments to the Author(s)
In this paper the authors investigated the expression of a symbiosis-specific gene, H+-ATPase as
a proxy of symbiosis establishment. The authors chose three different species, a coral species, a
nudibranch and a giant clam, known to incorporate Symbiodinium A1 through horizontal
(external) uptake, and measure the expression of H+-ATPase gene during larvae development.
I feel the argument of the paper is interesting, it is well-written and I suggest that it could be
accepted for publication if the following suggested revisions are clarified and/or complied with.
Specific comments:
Page 2, line 55: please update references. Current references are not specific to primary
production of Symbiodinium but rather Climate Change.
Page 3, lines 69-70: please replace “is very well researched” with “known”.
Page 5, line 143: please clarify why only 72 hours. In table 1 you mention that larval development
for Mussismilia hispida and Tridacna crocea go up to 12 and 17 days approximately. Your time
frame goes only until 3 days, a rather short duration concerning the larval development of these
species.
Page 8, lines 240-241: I cannot agree, your conclusions are overstated. If we take a look at Table 2
we can see that for the coral species, from 15 observations (5 time points x 3 larvae replicates) you
only have 1 positive expression of the gene H+-ATPase. You should be thoughtful since one
event represents quite preliminary results. You could have clearly benefited from a longer period
of time, in order to make sure we can still verify the expression after 96hrs, one week time.
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4
Decision letter (RSOS-170253)
20-Apr-2017
Dear Dr Mies,
The editors assigned to your paper ("Expression of a symbiosis-specific gene in Symbiodinium
type A1 associated with coral, nudibranch and giant clam larvae") have now received comments
from reviewers. We would like you to revise your paper in accordance with the referee and
Associate Editor suggestions which can be found below (not including confidential reports to the
Editor). Please note this decision does not guarantee eventual acceptance.
Please submit a copy of your revised paper within three weeks (i.e. by the 13-May-2017). If we do
not hear from you within this time then it will be assumed that the paper has been withdrawn. In
exceptional circumstances, extensions may be possible if agreed with the Editorial Office in
advance.We do not allow multiple rounds of revision so we urge you to make every effort to
fully address all of the comments at this stage. If deemed necessary by the Editors, your
manuscript will be sent back to one or more of the original reviewers for assessment. If the
original reviewers are not available we may invite new reviewers.
To revise your manuscript, log into http://mc.manuscriptcentral.com/rsos and enter your
Author Centre, where you will find your manuscript title listed under "Manuscripts with
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When submitting your revised manuscript, you must respond to the comments made by the
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In addition to addressing all of the reviewers' and editor's comments please also ensure that your
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If your study uses humans or animals please include details of the ethical approval received,
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5
• Competing interests
Please declare any financial or non-financial competing interests, or state that you have no
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• Authors’ contributions
All submissions, other than those with a single author, must include an Authors’ Contributions
section which individually lists the specific contribution of each author. The list of Authors
should meet all of the following criteria; 1) substantial contributions to conception and design, or
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critically for important intellectual content; and 3) final approval of the version to be published.
All contributors who do not meet all of these criteria should be included in the
acknowledgements.
We suggest the following format:
AB carried out the molecular lab work, participated in data analysis, carried out sequence
alignments, participated in the design of the study and drafted the manuscript; CD carried out
the statistical analyses; EF collected field data; GH conceived of the study, designed the study,
coordinated the study and helped draft the manuscript. All authors gave final approval for
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Once again, thank you for submitting your manuscript to Royal Society Open Science and I look
forward to receiving your revision. If you have any questions at all, please do not hesitate to get
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Yours sincerely,
Alice Power
Editorial Coordinator
Royal Society Open Science
on behalf of Kevin Padian
Subject Editor, Royal Society Open Science
[email protected]
Associate Editor's comments:
Comments to the Author:
I thank the authors for submitting, and the reviewers for assessing this manuscript. This
manuscript was transferred in from PRS-B (without review) and deals with an important yet
unresolved process; the establishment of symbiosis between dinoflagellates and three larval
hosts. All of us recognized the importance of the issue, thought it was timely, and clearly
conveyed. Nevertheless, all three of us were left a little disappointed in some aspects. Specifically,
there were key issues with the overall set up, and the interpretation and discussion of data.
Clearly, the study design has its limitations (e.g., lack of a negative control, duration of
observations), and the patterns in the data aren't are robust. Further, the introduction appears to
undercut what is already known about establishment of symbiosis in these systems. I urge the
authors to set up the study more generally, and build up to the lack of mechanistic/molecular
resolution which this study fills. I would also like to see more background/rationale for choosing
Downloaded from http://rsos.royalsocietypublishing.org/ on June 18, 2017
6
this specific candidate gene, and importantly, discuss clearly the limitations of focussing on only
one gene. In general, the authors need to critically discuss the data and minimize overselling it. It
is an interesting study, which has nothing seriously wrong. As such, a forward-looking setup and
discussion would be more useful than one that undermines what is already known, and oversells
the weak data trends observed. I look forward to receiving a thoroughly revised version. Again,
thanks to everyone!
Comments to Author:
Reviewers' Comments to Author:
Reviewer: 1
Comments to the Author(s)
Hello authors:
I very much enjoyed reading your paper about symbiont acquisition in corals, clams and slugs. I
think you addressed an important question about the genes involved in the establishment of
symbiotic relationships. The many RNA-seq experiments will benefit from more of this type
experiment!
Just a couple of suggestions and comments:
First of all, I am concerned that you did not do a second negative control. You sampled adult host
tissue with symbionts (positive control) and free living Symbiodinium cultures (negative control).
But you neglected to do the second negative control: to sample the larval tissue without
symbionts. I know you cannot go back and collect these samples. And even without these data, I
think your results are interesting. But it is always better to have both negative controls when
working with symbiosis. Maybe a sentence or two about why you didn't do this would help close
the circle.
Second, if you are so sure that the slugs were digesting Symbiodinium, what evidence can you
show? We see them clearly sequestered in the cerata in Fig 1B. On line 197 you indicate that you
had visual evidence of digestion. Why is this evidence not shown in Fig 1E? (also the letters for
the images in the figures are confusing with the genera initials) Can you at the very least describe
what was the visual evidence you are referring to? similarly the secondary evidence against
symbiosis (lines 249-257) is reasonable from an algal perspective. And yet these slugs are always
seen with living algal cells inside their bodies. Can you reword this to either make your point
more strongly or to back off the strength of your statement. The sentence in the abstract could be
adjusted to read something like, "evidence from this experiment did not support a mutualistic
relationship..."
Good luck with your paper and with your future work. A few more small comments by line
number below.
Line 29: A lot is known about establishment. But maybe not about the molecular biology or the
signals?
Line 58-62: Is Symbiodinium diversity relevant to this study? You might remove this to make it
more concise.
Line 79: I think you need at least one more sentence justifying this. Describe the studies you are
leaning on. Why are you so sure that this proton pump is the symbiosis red flag indicator?
Line 145-6: After you describe your samples, you did not mention freezing or buffers? How did
you preserve your RNA? Could your signal be spotty because of degradation?
Downloaded from http://rsos.royalsocietypublishing.org/ on June 18, 2017
7
Line 236: Only 1/3 coral replicates showed the expected results. Why is this? You included
multiple individual larvae in each sample. What does this mean for your results. How sensitive
was your assay? I think you need to discuss the inconsistent results in light of the methods a bit
more clearly. You were collecting along a developmental time series. Why did you top out at 72
hours? Was there literature or a pilot study that suggested these time points?
Lines 244-257: please review the grammar in this paragraph
Line 266: this is a bit shaky? You write "intense changes in the circadian rhythm" but you do not
explain why this would generate inconsistent expression. Please explain further.
Reviewer: 2
Comments to the Author(s)
In this paper the authors investigated the expression of a symbiosis-specific gene, H+-ATPase as
a proxy of symbiosis establishment. The authors chose three different species, a coral species, a
nudibranch and a giant clam, known to incorporate Symbiodinium A1 through horizontal
(external) uptake, and measure the expression of H+-ATPase gene during larvae development.
I feel the argument of the paper is interesting, it is well-written and I suggest that it could be
accepted for publication if the following suggested revisions are clarified and/or complied with.
Specific comments:
Page 2, line 55: please update references. Current references are not specific to primary
production of Symbiodinium but rather Climate Change.
Page 3, lines 69-70: please replace “is very well researched” with “known”.
Page 5, line 143: please clarify why only 72 hours. In table 1 you mention that larval development
for Mussismilia hispida and Tridacna crocea go up to 12 and 17 days approximately. Your time
frame goes only until 3 days, a rather short duration concerning the larval development of these
species.
Page 8, lines 240-241: I cannot agree, your conclusions are overstated. If we take a look at Table 2
we can see that for the coral species, from 15 observations (5 time points x 3 larvae replicates) you
only have 1 positive expression of the gene H+-ATPase. You should be thoughtful since one
event represents quite preliminary results. You could have clearly benefited from a longer period
of time, in order to make sure we can still verify the expression after 96hrs, one week time.
Author's Response to Decision Letter for (RSOS-170253)
See Appendix A.
Decision letter (RSOS-170253.R1)
27-Apr-2017
Dear Dr Mies,
I am pleased to inform you that your manuscript entitled "Expression of a symbiosis-specific gene
in Symbiodinium type A1 associated with coral, nudibranch and giant clam larvae" is now
accepted for publication in Royal Society Open Science.
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8
You can expect to receive a proof of your article in the near future. Please contact the editorial
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Editorial Coordinator
Royal Society Open Science
[email protected]
http://rsos.royalsocietypublishing.org/
Associate Editor Comments to Author:
Comments to the Author:
Thank you for addressing the concerns raised in the previous round thoroughly, and promptly! I
think this paper is much improved, and ready for print.
Appendix A
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Dear editor, thank you for your overall support and for thoroughly reading and reviewing our
manuscript. We also thank the reviewers for their thoughtful and helpful input, which is greatly
appreciated.
We have addressed all of the comments made by the editor and reviewers in a comprehensive way (see
reply below). We have also toned down the conclusions as requested and addressed why we used only
one gene and the inherent limitations as well as the relations associated with the duration of the
experiment.
Thank you very much,
REVIEWER #1
I very much enjoyed reading your paper about symbiont acquisition in corals, clams and slugs. I think
you addressed an important question about the genes involved in the establishment of symbiotic
relationships. The many RNA-seq experiments will benefit from more of this type experiment!
Just a couple of suggestions and comments:
First of all, I am concerned that you did not do a second negative control. You sampled adult host tissue
with symbionts (positive control) and free living Symbiodinium cultures (negative control). But you
neglected to do the second negative control: to sample the larval tissue without symbionts. I know you
cannot go back and collect these samples. And even without these data, I think your results are
interesting. But it is always better to have both negative controls when working with symbiosis. Maybe a
sentence or two about why you didn't do this would help close the circle.
Reply: Firstly, thank you for the overall support of our manuscript. The experimental design for this
manuscript is highly similar to an article of our group that has just been published (Mies et al. 2017,
Symbiosis, see literature cited). That work, while only now published, was performed many years ago
with another giant clam species (Tridacna maxima) and the H+-ATPase and RuBisCO were targeted
along with an actin gene (belonging to the host larvae). In that experiment, the isolated larvae (without
Symbiodinium) did express the actin gene and not the H+-ATPase or RuBisCO. However, during peerreview, we were a bit criticized as one of the reviewers claimed that the actin gene was out of context as
we were investigating gene expression in Symbiodinium and not in the host. That influenced us a bit and
we chose not to use host genes in the experiment depicted in this manuscript. Still, no expression was
found in the 0h samples - which was immediately after symbiont offering, meaning that most of the
larvae had not had the time to acquire symbionts. In the revised manuscript, we have added a brief
explanatory sentence.
Second, if you are so sure that the slugs were digesting Symbiodinium, what evidence can you show?
We see them clearly sequestered in the cerata in Fig 1B. On line 197 you indicate that you had visual
evidence of digestion. Why is this evidence not shown in Fig 1E? (also the letters for the images in the
figures are confusing with the genera initials) Can you at the very least describe what was the visual
evidence you are referring to? similarly the secondary evidence against symbiosis (lines 249-257) is
reasonable from an algal perspective. And yet these slugs are always seen with living algal cells inside
their bodies. Can you reword this to either make your point more strongly or to back off the strength of
your statement. The sentence in the abstract could be adjusted to read something like, "evidence from
this experiment did not support a mutualistic relationship..."
Reply: The process of digestion seems to take place only during larval development – the adult
individuals do not digest the symbionts, but discard them as a whole in the substrate. We did not take a
picture of the digested Symbiodinium cells, which would indeed have been useful. To address your
comment, we have now described the visual evidence for digestion and revised the mentioned parts in
the abstract and discussion.
Good luck with your paper and with your future work. A few more small comments by line number
below.
Downloaded from http://rsos.royalsocietypublishing.org/ on June 18, 2017
Line 29: A lot is known about establishment. But maybe not about the molecular biology or the signals?
Reply: corrected.
Line 58-62: Is Symbiodinium diversity relevant to this study? You might remove this to make it more
concise.
Reply: We actually feel it is important, as we address in the discussion that the success of Symbiodinium
A1 associated with coral and giant clam larvae may be specific and related to symbiont identity and
homology.
Line 79: I think you need at least one more sentence justifying this. Describe the studies you are leaning
on. Why are you so sure that this proton pump is the symbiosis red flag indicator?
Reply: We have revised this passage to make it clearer; we heavily rely on the findings of Bertucci et al.
(2010) and Mies et al. (2017). The former characterized and comprehensively showed that this gene is
expressed only by Symbiodinium engaged in a mutualistic relationship; the latter validated the former’s
findings.
Line 145-6: After you describe your samples, you did not mention freezing or buffers? How did you
preserve your RNA? Could your signal be spotty because of degradation?
Reply: We have now added more detail. Briefly, collected samples were snap-frozen until RNA
extraction.
Line 236: Only 1/3 coral replicates showed the expected results. Why is this? You included multiple
individual larvae in each sample. What does this mean for your results. How sensitive was your assay? I
think you need to discuss the inconsistent results in light of the methods a bit more clearly. You were
collecting along a developmental time series. Why did you top out at 72 hours? Was there literature or a
pilot study that suggested these time points?
Reply: We think that more than one of the coral replicates would have expressed the H+-ATPase if the
experiment window lasted longer. However, we decided that 72 h would be adequate based on Mies et
al. (2017) and the facts that: i) while we had good broodstock individuals and experience in the culture
of these organisms, producing millions of larvae in captivity is very difficult, and ii) both giant clam and
coral larvae mortality are high (and poses an increasing problem the later time points are chosen),
which could leave us with little material for RNA extraction. Indeed, we used all the giant clam and coral
larvae that we produced for the 72 h experiments. Arguably we could have added one more sampling at
96 h for the nudibranch, but not further. Therefore, the obstacles in culturing these organisms kept us
from producing more larvae and having a longer experiment duration. We have addressed this more
comprehensively in the second paragraph of the discussion.
Lines 244-257: please review the grammar in this paragraph
Reply: corrected.
Line 266: this is a bit shaky? You write "intense changes in the circadian rhythm" but you do not explain
why this would generate inconsistent expression. Please explain further.
Reply: corrected.
REVIEWER #2
In this paper the authors investigated the expression of a symbiosis-specific gene, H+-ATPase as a
proxy of symbiosis establishment. The authors chose three different species, a coral species, a
nudibranch and a giant clam, known to incorporate Symbiodinium A1 through horizontal (external)
uptake, and measure the expression of H+-ATPase gene during larvae development.
I feel the argument of the paper is interesting, it is well-written and I suggest that it could be accepted
for publication if the following suggested revisions are clarified and/or complied with.
Specific comments:
Downloaded from http://rsos.royalsocietypublishing.org/ on June 18, 2017
Page 2, line 55: please update references. Current references are not specific to primary production of
Symbiodinium but rather Climate Change.
Reply: Firstly, thank you for the overall support of our manuscript. Indeed, the references were not
specific and we have now corrected this.
Page 3, lines 69-70: please replace “is very well researched” with “known”.
Reply: corrected
Page 5, line 143: please clarify why only 72 hours. In table 1 you mention that larval development for
Mussismilia hispida and Tridacna crocea go up to 12 and 17 days approximately. Your time frame goes
only until 3 days, a rather short duration concerning the larval development of these species.
Reply: see reply to next comment.
Page 8, lines 240-241: I cannot agree, your conclusions are overstated. If we take a look at Table 2 we
can see that for the coral species, from 15 observations (5 time points x 3 larvae replicates) you only
have 1 positive expression of the gene H+-ATPase. You should be thoughtful since one event represents
quite preliminary results. You could have clearly benefited from a longer period of time, in order to make
sure we can still verify the expression after 96hrs, one week time.
Reply: We believe that more than one of the coral replicates could have expressed the H+-ATPase if the
experiment window lasted longer. However, we decided that 72 h would be adequate based on Mies et
al. (2017) and the facts that: i) while we had good broodstock individuals and experience in the culture
of these organisms, producing millions of larvae in captivity is very difficult, and ii) both giant clam and
coral larvae mortality are significantly high, which could leave us with little material for RNA extraction.
And indeed, we used all the giant clam and coral larvae that we produced for the 72 h experiments.
Perhaps we could have added one more sampling at 96 h for the nudibranch, but not further. Therefore,
the obstacles in culturing these organisms kept us from producing more larvae and having a longer
experiment duration. We have addressed this in the second paragraph of the discussion and toned down
our overall conclusions, stating that, given that the H+-ATPase was once expressed, Symbiodinium and
coral larvae may engage in mutualistic symbiosis.