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Shifts in rotifer life history in response to stable isotope
enrichment: testing theories of isotope effects on
organismal growth
Elena Gorokhova
Article citation details
R. Soc. open sci. 4: 160810.
http://dx.doi.org/10.1098/rsos.160810
Review timeline
Original submission:
Revised submission:
Final acceptance:
18 October 2016
28 February 2017
1 March 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-160810.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?
Yes
Do you have any ethical concerns with this paper?
No
Have you any concerns about statistical analyses in 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
Recommendation?
Accept with minor revision (please list in comments)
Comments to the Author(s)
This is a very interesting and potentially important paper in which rotifer Brachionus plicatilis
was grown on 15N-enriched algae at the 15N concentration range from 0.37% (natural
abundance) to 5% (25-times enriched). There are two main findings: first, that the ≤5%
enrichment causes significant biological effects, including doubling of longevity; and second, that
there is a sharp contrast between the general trend and the 3.5% 15N datapoint, in agreement
with the isotopic resonance hypothesis. These results highlight the importance of understanding
the effects of stable isotope concentrations on the biology of complex multicellular organisms.
The paper is eloquently and competently written, and only minor comments can be made.
1.
The abstract asks for more specificity. Example: “…at the enrichment levels commonly
used in ecological studies”. This is too vague. Why not just say – up to 5% 15N? Also,
“…theoretically predicted effects of heavy isotope enrichment..” – by what theory?.. Why not
mention the isotopic resonance hypothesis?
2.
The first paragraph contains many nontrivial statements (Example: “Stable isotopes are
safe” – something the paper goes on to refute), but no references.
3.
P.3. “As light isotopes engage more easily in chemical reactions, the relative abundance
of heavy stable isotopes increases, leading to progressively slower reactions.” This is only true in
respect to one isotopically labeled compound undergoing chemical transformation, while in a
system as a whole the isotopic abundances are constant. Also, a reference to a specific study
demonstrating this effect would be appreciated.
4.
P.5. “understanding these effects is crucial for developing better theories” – what is the
difference between understanding and theory?
5.
“5% (heavily enriched)”. Most people would call >50% heavily enriched, not 5%.
Review form: Reviewer 2
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?
Yes
Do you have any ethical concerns with this paper?
No
Have you any concerns about statistical analyses in this paper?
No
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3
Recommendation?
Accept with minor revision (please list in comments)
Comments to the Author(s)
The author shows that feeding the rotifer Brachionus plicatilis 15N-enriched algae substantially
changes the life history traits of these animals. This contradicts assumptions made in studies
employing stable isotope analysis, which is widely used in ecological studies. This study is
important and timely because it demonstrates how stable isotope labeling can strongly affect
rotifer lifespan, reproduction and population growth.
Pg 5, Line 59 – State the source of the experimental strain of B. plicatilis used in this study. Since
this species is known to be a complex of at least 15 sibling species, describe the procedures
deployed to ensure proper classification of this strain.
Pg 6, lines 8-13 – “Under these conditions, the rotifers have a pre-reproductive
(neonate) stage of 1–2 days, reach maximum reproductive output at day 4–5, produce 16-20 eggs
and live 10–12 days...” Describe how these observations were performed. The methods only
describe how populations are maintained in stock cultures, not how the life histories of
individual females were characterized.
Pg 6, line 59 – Describe where these resting eggs were obtained.
Pg 7, line 48 – Why is this a different algae concentration than in line 10?
Pg 7, line 55 – What is the typical standard deviation for these measurements and the minimum
difference among treatments required for detection?
Pg 9, line 25 – Author could do a better job describing lifespan results – list mean lifespans and %
increase for each treatment.
Pg 9, lines 33-55 – Is there anything about Isochysis galbana that could have produced the effect
of 15N on its food value for rotifer reproduction? Confirmation of this enhancement effect in
other algae would be useful.
Pg 10, line 3 – Could the decrease of fecundity with increasing 15N supplementation be due to
caloric restriction, a widely recognized factor extending lifespan? This interpretation is
mentioned on pg 11, line 40 and could be emphasized more. Even though feeding rate was not
different among treatments, perhaps nutritive value of labeled algae was reduced.
Pg 10, line 50 – I would not characterize your results as “reproductive toxicity”. It seems to me
that you are observing a change in the nutritional quality of the Isochrysis rather than toxicity.
Pg 11, line 48 – “...elemental composition of the algae (at least, %C and %N)...” are too crude of
measures to detect changes in the nutritive value of Isochrysis for rotifer reproduction.
Decision letter (RSOS-160810)
21-Feb-2017
Dear Dr Gorokhova
On behalf of the Editors, I am pleased to inform you that your Manuscript RSOS-160810 entitled
"Shifts in rotifer life history in response to stable isotope enrichment: testing theories of isotope
effects on organismal growth" has been accepted for publication in Royal Society Open Science
subject to minor revision in accordance with the referee suggestions. Please find the referees'
comments at the end of this email.
The reviewers and handling editors have recommended publication, but also suggest some minor
revisions to your manuscript. Therefore, I invite you to respond to the comments and revise your
manuscript.
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4
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5
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6
Reviewer comments to Author:
Reviewer: 1
Comments to the Author(s)
This is a very interesting and potentially important paper in which rotifer Brachionus plicatilis
was grown on 15N-enriched algae at the 15N concentration range from 0.37% (natural
abundance) to 5% (25-times enriched). There are two main findings: first, that the ≤5%
enrichment causes significant biological effects, including doubling of longevity; and second, that
there is a sharp contrast between the general trend and the 3.5% 15N datapoint, in agreement
with the isotopic resonance hypothesis. These results highlight the importance of understanding
the effects of stable isotope concentrations on the biology of complex multicellular organisms.
The paper is eloquently and competently written, and only minor comments can be made.
1.
The abstract asks for more specificity. Example: “…at the enrichment levels commonly
used in ecological studies”. This is too vague. Why not just say – up to 5% 15N? Also,
“…theoretically predicted effects of heavy isotope enrichment..” – by what theory?.. Why not
mention the isotopic resonance hypothesis?
2.
The first paragraph contains many nontrivial statements (Example: “Stable isotopes are
safe” – something the paper goes on to refute), but no references.
3.
P.3. “As light isotopes engage more easily in chemical reactions, the relative abundance
of heavy stable isotopes increases, leading to progressively slower reactions.” This is only true in
respect to one isotopically labeled compound undergoing chemical transformation, while in a
system as a whole the isotopic abundances are constant. Also, a reference to a specific study
demonstrating this effect would be appreciated.
4.
P.5. “understanding these effects is crucial for developing better theories” – what is the
difference between understanding and theory?
5.
“5% (heavily enriched)”. Most people would call >50% heavily enriched, not 5%.
Reviewer: 2
Comments to the Author(s)
The author shows that feeding the rotifer Brachionus plicatilis 15N-enriched algae substantially
changes the life history traits of these animals. This contradicts assumptions made in studies
employing stable isotope analysis, which is widely used in ecological studies. This study is
important and timely because it demonstrates how stable isotope labeling can strongly affect
rotifer lifespan, reproduction and population growth.
Pg 5, Line 59 – State the source of the experimental strain of B. plicatilis used in this study. Since
this species is known to be a complex of at least 15 sibling species, describe the procedures
deployed to ensure proper classification of this strain.
Pg 6, lines 8-13 – “Under these conditions, the rotifers have a pre-reproductive
(neonate) stage of 1–2 days, reach maximum reproductive output at day 4–5, produce 16-20 eggs
and live 10–12 days...” Describe how these observations were performed. The methods only
describe how populations are maintained in stock cultures, not how the life histories of
individual females were characterized.
Pg 6, line 59 – Describe where these resting eggs were obtained.
Pg 7, line 48 – Why is this a different algae concentration than in line 10?
Pg 7, line 55 – What is the typical standard deviation for these measurements and the minimum
difference among treatments required for detection?
Pg 9, line 25 – Author could do a better job describing lifespan results – list mean lifespans and %
increase for each treatment.
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7
Pg 9, lines 33-55 – Is there anything about Isochysis galbana that could have produced the effect
of 15N on its food value for rotifer reproduction? Confirmation of this enhancement effect in
other algae would be useful.
Pg 10, line 3 – Could the decrease of fecundity with increasing 15N supplementation be due to
caloric restriction, a widely recognized factor extending lifespan? This interpretation is
mentioned on pg 11, line 40 and could be emphasized more. Even though feeding rate was not
different among treatments, perhaps nutritive value of labeled algae was reduced.
Pg 10, line 50 – I would not characterize your results as “reproductive toxicity”. It seems to me
that you are observing a change in the nutritional quality of the Isochrysis rather than toxicity.
Pg 11, line 48 – “...elemental composition of the algae (at least, %C and %N)...” are too crude of
measures to detect changes in the nutritive value of Isochrysis for rotifer reproduction.
Author's Response to Decision Letter for (RSOS-160810)
See Appendix A.
Decision letter (RSOS-160810.R1)
01-Mar-2017
Dear Dr Gorokhova,
I am pleased to inform you that your manuscript entitled "Shifts in rotifer life history in response
to stable isotope enrichment: testing theories of isotope effects on organismal growth" is now
accepted for publication in Royal Society Open Science.
You can expect to receive a proof of your article in the near future. Please contact the editorial
office ([email protected] and [email protected]) to let us know if
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Best wishes,
Alice Power
Editorial Coordinator
Royal Society Open Science
[email protected]
Appendix A
Dear Editor, Dear Reviewers,
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I appreciate all these excellent questions and comments, they were very helpful in revising the
manuscript. Below are our answers and explanations, hopefully they are sufficient, but I can always
elaborate if necessary. The replies are numbered consequently to facilitate cross-referencing. In
addition to the changes prompted by the comments, the revision included text editing and including
a new Supplementary file with primary data.
Sincerely,
Elena Gorokhova
Reviewer: 1
The abstract asks for more specificity. Example: “…at the enrichment levels commonly used in
ecological studies”. This is too vague. Why not just say – up to 5% 15N?
Reply 1: Specified in the abstract that effects were observed in the concentration range 0.45 at%.
Also, “…theoretically predicted effects of heavy isotope enrichment..” – by what theory?.. Why not
mention the isotopic resonance hypothesis?
Reply 2: It is not only the IsoRes hypothesis, but also the theoretical considerations behind
KIE. To keep abstract short, I would prefer to retain the original sentence.
2.
The first paragraph contains many nontrivial statements (Example: “Stable isotopes are safe” –
something the paper goes on to refute), but no references.
Reply 3: Two references (Hagler and Jackson 2001; Hood-Nowotny and Knols 2007; both are
reviews) providing support for these statements are added.
3.
P.3. “As light isotopes engage more easily in chemical reactions, the relative abundance of
Downloaded from http://rsos.royalsocietypublishing.org/ on June 17, 2017
heavy stable isotopes increases, leading to progressively slower reactions.” This is only true in
respect to one isotopically labeled compound undergoing chemical transformation, while in a system
as a whole the isotopic abundances are constant. Also, a reference to a specific study demonstrating
this effect would be appreciated.
Reply 4: True, rephrased to avoid ambiguity. A reference (Hoefs, 2015) is added as
requested.
4.
P.5. “understanding these effects is crucial for developing better theories” – what is the
difference between understanding and theory?
Reply 5: Rephrased to keep it simple.
5.
“5% (heavily enriched)”. Most people would call >50% heavily enriched, not 5%.
Reply 6: Atomic percentage of 5 is a very heavy enrichment for 15N. The corresponding δ15N
value is 13172‰ is very high compared to the δ15N of -2.7‰ in the treatment with ambient
15
N levels (i.e., 0.37 at%). This information is not added to the Introduction to help a reader
with understanding the differences.
Reviewer: 2
Pg 5, Line 59 – State the source of the experimental strain of B. plicatilis used in this study. Since this
species is known to be a complex of at least 15 sibling species, describe the procedures deployed to
ensure proper classification of this strain.
Reply 7: The rotifers were Nevada strain obtained from SINTEF Center of Aquaculture
(Norway). This information is now provided in the M&M. No molecular verification has been
performed to confirm strain identity.
Pg 6, lines 8-13 – “Under these conditions, the rotifers have a pre-reproductive (neonate) stage of 1–
Downloaded from http://rsos.royalsocietypublishing.org/ on June 17, 2017
2 days, reach maximum reproductive output at day 4–5, produce 16-20 eggs and live 10–12 days...”
Describe how these observations were performed. The methods only describe how populations are
maintained in stock cultures, not how the life histories of individual females were characterized.
Reply 8: These semi-quantitative estimates for life history traits were observed during pilot
studies that were needed to adjust feeding levels and other parameters of the experimental
setup; explained. They have no bearing on the results of the experiment.
Pg 6, line 59 – Describe where these resting eggs were obtained.
Reply 9: See Reply 7.
Pg 7, line 48 – Why is this a different algae concentration than in line 10?
Reply 10: Both numbers are correct. In the Life table experiment, the food was renewed
every second day, which means that algal concentrations gradually declined during the 48 h
incubation. The average food concentration during the incubation time between the media
change was calculated and used in the short-term feeding experiment (6 h); this is
explained now in the M&M, “Feeding experiment”. The feeding rate obtained was assumed
to represent average feeding rate during the 48-h incubation. This is the same approach as
we used when studying feeding rates of invertebrates in static systems (Hansson et al.
2001. Ratio-dependent functional responses - tests with the zooplanktivore Mysis mixta.
Marine Ecology Progress Series 216: 181-189).
Pg 7, line 55 – What is the typical standard deviation for these measurements and the minimum
difference among treatments required for detection?
Reply 11: Great point. The analytical standard deviation for cell density measurements is
usually <1% of the mean (at the densities used in this experiment). In the experiment, the
CV% varied 17 – 20% (mean values were around 19.6 – 20.7 and SD varied 3.2 – 4.6 across
the treatments). If we want to detect a difference of let’s say 20% between the control and
a treatment and use 80-90% power, 2-sided test and α = 0.05, the signal to noise ratio
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would be close to 1 (i.e., 0.20*20/4=1) and the number of replicates needed to detect this
difference is 18-20. Therefore, the experimental design (18 replicates) was capable
detecting differences ≥20% in the feeding rate between the groups, whereas the
differences of lower magnitude would not be detected. This information is now added to
the Discussion. Nevertheless, the mean values and distributions were nearly identical
among the treatments and, therefore, it is highly unlikely that a difference in feeding rate
were missed due to the experimental setup.
Pg 9, line 25 – Author could do a better job describing lifespan results – list mean lifespans and %
increase for each treatment.
Reply 12: Good point. The fold increase for each treatment is now presented in Table 1,
which is referenced in the result description. These values are perceived more clearly when
they are in the column format and not listed in the text.
Pg 9, lines 33-55 – Is there anything about Isochysis galbana that could have produced the effect of
15N on its food value for rotifer reproduction? Confirmation of this enhancement effect in other
algae would be useful.
Reply 13: This question is unclear: what “enhancement effect” is meant? The age at first
reproduction was delayed – the higher values for this parameter indicate compromised
maturation and reproductive effort. Haptophyte Isochrysis was used as one of the standard
food species for these rotifers. As far as I know, there are no published data on response of
this alga to 15N enrichment (or any other isotopic enrichment).
Pg 10, line 3 – Could the decrease of fecundity with increasing 15N supplementation be due to caloric
restriction, a widely recognized factor extending lifespan? This interpretation is mentioned on pg 11,
line 40 and could be emphasized more. Even though feeding rate was not different among
treatments, perhaps nutritive value of labeled algae was reduced.
Reply 14: The changes in elemental or nutritional content of any algal species growth in
Downloaded from http://rsos.royalsocietypublishing.org/ on June 17, 2017
isotopically enriched media have never been evaluated (as far as I know). However, a recent
publication from our lab has demonstrated a growth response of green algae to 15N
enrichment, with lower production at >0.5 at% 15N (Andriukonis & Gorokhova 2017. Kinetic
15
N-isotope effects on algal growth. Scientific Reports). Although lower production does not
necessarily imply lower nutritive value, this information is now included in the Discussion.
Pg 10, line 50 – I would not characterize your results as “reproductive toxicity”. It seems to me that
you are observing a change in the nutritional quality of the Isochrysis rather than toxicity.
Reply 15: True. Revised.
Pg 11, line 48 – “...elemental composition of the algae (at least, %C and %N)...” are too crude of
measures to detect changes in the nutritive value of Isochrysis for rotifer reproduction.
Reply 16: Also true. However, these are the only relevant values I have. A note has been
made to point out that elemental composition does not provide sufficient evaluation of the
dietary quality.