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Research
Cite this article: Gorokhova E. 2017 Shifts in
rotifer life history in response to stable isotope
enrichment: testing theories of isotope effects
on organismal growth. R. Soc. open sci.
4: 160810.
http://dx.doi.org/10.1098/rsos.160810
Shifts in rotifer life history in
response to stable isotope
enrichment: testing
theories of isotope effects
on organismal growth
Elena Gorokhova
Department of Environmental Science and Analytical Chemistry, Stockholm
University, Svante Arrhenius väg 8, 10691 Stockholm, Sweden
EG, 0000-0002-4192-6956
Received: 18 October 2016
Accepted: 1 March 2017
Subject Category:
Biology (whole organism)
Subject Areas:
ecology/developmental biology/physiology
Keywords:
stable isotopes, 15 N-labelling, growth and life
histories, longevity, isotope resonance theory,
kinetic isotope effect
In ecology, stable isotope labelling is commonly used for
tracing material transfer in trophic interactions, nutrient
budgets and biogeochemical processes. The main assumption
in this approach is that the enrichment with a heavy isotope
has no effect on the organism growth and metabolism.
This assumption is, however, challenged by theoretical
considerations and experimental studies on kinetic isotope
effects in vivo. Here, I demonstrate profound changes in life
histories of the rotifer Brachionus plicatilis fed 15 N-enriched
algae (0.4–5.0 at%); i.e. at the enrichment levels commonly
used in ecological studies. These findings support theoretically
predicted effects of heavy isotope enrichment on growth,
metabolism and ageing in biological systems and underline the
importance of accounting for such effects when using stable
isotope labelling in experimental studies.
1. Introduction
Author for correspondence:
Elena Gorokhova
e-mail: [email protected]
Electronic supplementary material is available
online at https://dx.doi.org/10.6084/m9.
figshare.c.3718381.
Stable isotopes are safe and non-radioactive, occurring naturally
in the environment. They do not decay, which makes them
attractive natural tracers in experimental studies [1,2]. Naturally
occurring differences in isotopic signatures (δ-values) between
the substrate and the product of the reaction are used in
ecology and biogeochemistry to understand flows and processes.
Another approach is enrichment studies that deploy compounds
enriched in particular isotopes to the system and follow their
fate. Such stable isotope labelling is commonly used for tracing
material transfer in trophic interactions, material flows and
biogeochemical processes. It may also indicate whether systems
in question can metabolize certain compounds. There is a variety
of commercially available compounds with the heavy isotope
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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2.1. Test organisms
Rotifers are particularly well suited for testing life-history traits because of their short lifespan,
parthenogenic reproduction and ease of culture [18]. The rotifers Brachionus plicatilis Müller (Nevada
strain, originally from SINTEF Center of Aquaculture Norway) were cultured in 1 l glass beakers at
15 ppt ASW (artificial seawater, Instant Ocean), constant illumination (30 µE cm−2 s−1 ) and temperature
................................................
2. Material and methods
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making up 99% of the tracer element. These compounds can be integrated into a nutrient medium for
primary producers and feed regimes of consumers when addressing, for example, nutrient uptake, prey
selectivity and fate of dietary nutrients [2]. One should keep in mind, however, that natural abundance
and enrichment studies are often subject to different assumptions and caveats, and many of them are not
sufficiently understood.
In biota, heavy isotopes of common elements (C, H, N and O) exist in much lower abundance than
their light counterparts (1.1% for 13 C, 0.015% for 2 H or D, 0.2% for 18 O and 0.37% for 15 N). Despite their
small contribution, heavy isotopes produce remarkable kinetic difference because the gain in weight to
charge increases the stability of heavy isotope-containing chemical bonds, which may substantially slow
their reactivity—this phenomenon is called the kinetic isotope effect (KIE). As light isotopes engage more
easily in chemical reactions, the relative abundance of heavy stable isotopes increases in the substrate,
leading to progressively slower reactions [3]. However, one of the main assumptions of the isotopeenrichment approach is that the increased levels of a heavy isotope have no effect on the organism
growth and metabolism. This assumption is challenged by both theoretical and empirical research. In
particular, alterations in growth and metabolic profiles induced by 13 C- and/or 15 N-enriched media
were reported for bacteria [4–6], algae [7] and fungi [8]. A hypothesis has been put forward that the
increased uptake of the heavy isotope in a biological system would affect the kinetics of biochemical
reactions, increase the stability of the biomolecules and organism longevity [9,10]. Supporting this view,
the relative abundance of heavy isotope-containing metabolites in yeast exhibited an ageing-associated
trend, with most abundant amino acids declining in their 13 C and 2 H content in senescent cultures [11].
The authors hypothesized that cells might gradually lose the ability to retain heavy metabolites with
ageing and, by introducing D2 O in the culture medium, they were able to slow down this metabolic
deterioration and ageing.
Further, the isotopic resonance theory advocates a non-monotonous dependence of the reaction rate
upon the enrichment [12]. The proposed mechanism relates to the overall reduction of the system’s
complexity, represented by a total number of distinct quantum mechanical states in the macromolecules
and analysed using a peptide mass distribution. This theory predicts that at certain abundances of the
stable isotopes, the rates of chemical and biochemical reactions accelerate, affecting biological growth.
For example, the resonance conditions are predicted to occur at 0.35% of 13 C, 3.5% of 15 N, 6.6% of
18 O and 0.03% of 2 H, and these predictions were confirmed using experiments with E. coli grown at
varying isotopic composition [6]. Although these resonance isotopic concentrations are too high to occur
naturally in any environment on Earth, they are within the range of concentrations applied in laboratory
and field experiments with isotope-enriched substrates (e.g. [13–17]). For studies using isotope-enriched
substrates, the occurrence of KIEs and resonance conditions imply that exposing producers or consumers
to nutrient medium or food with isotopic signatures greatly deviating from their natural levels can cause
alterations in organism growth and metabolism. If present, these alterations may affect the experimental
outcome and lead to flawed conclusions. Therefore, understanding KIEs on growth and metabolism
across different levels of biological organization would facilitate application of stable isotope approach
in ecological and metabolomic studies.
Here, we used parthenogenic rotifer Brachionus plicatilis grown on 15 N-enriched algae and assayed
feeding, fecundity, lifespan and quality of the offspring at the 15 N concentration range of 0.37 at%
(natural abundance; δ15 N = −2.7‰) to 5 at% (heavily enriched; δ15 N = 13 172‰). The study provides the
first empirical evidence that the chronic intake of 15 N-enriched food can cause various changes in animal
life history, including beneficial effects on lifespan and detrimental effects on reproduction. Moreover, at
theoretically predicted resonance (3.5 at% 15 N), the reproduction and offspring quality were elevated in
comparison with other enriched diets. Our findings support the existence of an array of growth-related
responses to 15 N concentration in the diet and highlight the importance of accounting for these effects
when designing experimental studies with 15 N-labelling.
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For the experimental diets, the algae were enriched with 15 N by replacing Na14 NO3 with Na15 NO3
(98 at% pure, Aldrich, St. Louis, MO, USA) in the medium; 10% of NaNO3 was enriched with 15 N.
The algae were grown under standard light and temperature conditions with constant shaking, and
the isotope-enriched solution was added 5 days before harvesting, to ensure sufficient uptake. This
labelling procedure yielded a stock solution of algae with δ15 N values of 16 190‰ (6 at% 15 N). The
δ15 N values as well as elemental concentrations in the labelled (C%: 40.2 ± 1.2% and N%: 6.2 ± 0.6%)
and unlabelled (C%: 41.1 ± 1.3% and N%: 6.8 ± 0.5%) algae with three replicate samples per group
were determined at the UC Davis Stable Isotope Facility, USA. There was no significant difference for
either C% (t-test: t4 = 0.85; p > 0.45) or N% (t4 = 1.33; p > 0.25) between the labelled and unlabelled algae.
Therefore, all experimental diets were assumed to be equally nutritious. To prepare the experimental
diets, the labelled and unlabelled algae were mixed to correspond to 0.4, 0.5, 1, 3.5 and 5 at% of 15 N; the
unlabelled algae (δ15 N: −2.7‰ corresponding to 0.37 at%) were used as a control. For each food batch,
the algal concentrations were determined using a laser particle counter Spectrex PC-2000 (Spectrex Corp.,
CA, USA).
2.3. Life tables: experimental set-up and measured endpoints
For each experiment, resting eggs were hatched for 20–22 h in 20 ml ASW into a physiologically uniform
cohort of neonates. The life table experiment was carried out in 24-well tissue culture microplates
containing 2 ml culture medium, with one newly hatched female B. plicatilis per well (24 wells per
treatment). The experiment consisted of six treatments corresponding to 15 N in the experimental diets.
The algae were provided at 2 × 106 cell ml−1 , and the maternal females were transferred to a new medium
every second day. Plates were checked daily until the last animal died, mortality was recorded, and
the offspring were counted and removed. Body growth in this species follows the Bertalanffy function,
reaching 85–90% of maximum size between the first and second day of life [21]. Therefore, the neonate
(130–170 µm) can easily be distinguished from the maternal female (200–250 µm) during the first 24 h
after hatching. The offspring originated from the same female were pooled and preserved in RNA later
for biochemical measurements. All plates were kept in the dark at 26°C in climate-control chambers.
2.4. Feeding rate
To examine whether the 15 N-labelling affects individual food intake, a feeding experiment was
conducted using 24-well microplates with 10 individuals per well, 18 replicate wells with rotifers and
six control wells (algae only) per treatment. To prevent the hatching of amictic eggs and thus keep a
constant number of individuals per well, rotifers were also treated with 20 µM 5-fluoro-2-deoxyuridine
(FDU) [22]. The treatments were represented by the same diets as in the life table experiment, 1 ml per
well, 5 × 105 cell ml−1 . This concentration represented average food concentration occurring in the life
table experiment between the media renewals; these conditions were used to ensure saturating food
levels, a constant number of rotifers per well, and, hence, constant intake rate during the incubation [23].
The plates were incubated in darkness for 6 h and the filtering rate was calculated from the cell densities
measured with the particle counter before and after the incubation.
2.5. Biochemical assays
We measured RNA:protein ratio and individual protein content as indices of growth and condition
in the offspring using the neonates collected during the life table experiment. The animals from each
sample were divided between the two assays: 5–15 individuals were used in RiboGreen assay measuring
RNA quantity [24] and 5–10 individuals in NanoOrange assay measuring protein quantity [25]. Using
RiboGreen® Quantitation Kit (Invitrogen, Molecular Probes™), the RNA was quantified after extraction
................................................
2.2. Experimental diets
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(26 ± 2°C). The animals were fed with marine microalga Isochrysis galbana Parke 1949 grown in F
medium [19]; the food was provided daily at a density of 5 × 106 cell ml−1 . 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 (personal observation), which is in the range of variation
for this species [20]. All animals used in the study reproduced asexually; mictic females or males were
not observed.
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The life-history parameters used for treatment effect analysis were: age-specific fecundity, reproductive
period (the duration between the first and last offspring production), lifetime fecundity (total number
of offspring produced by a female) and lifespan calculated from the survival curve. The time of death
was determined as the midpoint between the last observation before death and the first after that.
Kaplan–Meier survival functions and daily reproduction curves were used to analyse the differences
between the treatments using Mantel–Cox log-rank tests as implemented in Prism v. 6.0 (GraphPad).
For other endpoints, D’Agostino & Pearson omnibus normality test was performed to confirm Gaussian
distribution and, subsequently, either Brown–Forsythe ANOVA with Dunnett’s multiple comparisons
test for normally distributed endpoints (filtering rate, length of the reproductive period, lifetime
fecundity, individual protein content and RNA:protein ratio) or Kruskal–Wallis (KW) with Dunn’s
multiple comparisons test for those significantly deviating from the normal distribution (age at first
reproduction) were used to evaluate treatment effects. The significance level was set at 5% (p < 0.05).
3. Results
3.1. Lifespan
The 15 N-enriched diets affected the shape of the survival curves (figure 1), and median lifespan
significantly increased in rotifers exposed to 15 N-enriched food compared with the control group (table 1
and figure 2). The greatest change corresponded to approximately 40% increase in the mean lifespan,
with maximal longevity values differing approximately twofold between the animals receiving the 5 at%
15 N diet and the controls (12 versus 24 d).
3.2. The timing of reproduction
Rotifers receiving 1 and 5 at% 15 N-enriched diets had significantly delayed reproduction (KW
statistic = 38.85, p < 0.0001). Moreover, the age at first reproduction increased with increasing 15 N
concentration, with the first reproduction occurring on average 1 day later than in the controls. However,
the 3.5 at% 15 N treatment deviated from this trend and was not significantly different from the control
(figure 2b; see also the electronic supplementary material, table S1). Moreover, the reproductive period
in the rotifers receiving 15 N-enriched diets was significantly longer (ANOVA: F5,138 = 3.189; p < 0.008;
Brown–Forsythe test: F5,138 = 10.79; p < 0.0001; figure 2c), and in the most affected treatment (5 at% 15 N),
it was more than 30% longer compared with the control. Again, the 3.5 at% 15 N treatment deviated
from this trend, being non-significantly different from the control (figure 2c; see also the electronic
supplementary material, table S2).
3.3. Reproductive output and offspring quality
The dynamics of the egg-laying in the rotifers receiving 15 N-enriched diets differed from the control
(figure 1). As lifespan increased, both the average age-specific fecundity and lifetime fecundity decreased
(Pearson r: R2 = 0.77, p < 0.02 and R2 = 0.57, p < 0.09, respectively). The latter differed significantly among
the treatments (ANOVA: F5,138 = 2.834; p < 0.02; Brown–Forsythe test: F5,138 = 4.278; p < 0.002), reaching
in the most affected treatments only 60–70% of the control values. A deviating response was observed in
the rotifers exposed to 3.5 at% 15 N that showed lifetime fecundity similar to that in the control (figure 2d;
electronic supplementary material, table S3). Moreover, offspring size measured as individual protein
content (ANOVA: F5,138 = 2.528; p < 0.04; figure 2e; electronic supplementary material, table S4) and
RNA:protein ratio (ANOVA: F5,138 = 2.883; p < 0.02; figure 2f ) showed a similar pattern, with significant
................................................
2.6. Statistics and data analysis
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with N-laurylsarcosine followed by RNase digestion [26]. Fluorescence measurements were done in
triplicate for each sample, standard, and negative control using FLUOstar Optima reader (BMG Labtech,
Germany) with 485 nm for excitation and 520 nm for emission and black solid flat-bottom microplates
(Greiner Bio-One GmbH). For protein measurements with NanoOrange® Protein Quantitation Kit
(Invitrogen, Molecular Probes™), Bovine serum albumin (BSA, Molecular Probes) was used as a protein
standard. The samples were analysed according to the manufacturer guidelines using the same type of
microplates, plate reader and wavelengths as for the RNA quantification.
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4
0.4% 15N
control
2
40
1
20
0
0
0.5% 15N
6
1% 15N
per cent surviving
80
4
60
40
2
20
0
0
100
6
5% 15N
3.5% 15N
80
per cent surviving
fecundity (offspring female–1)
100
4
60
40
2
20
fecundity (offspring female–1)
per cent surviving
60
0
0
0
5
10
15
age
20
25 0
5
10
15
20
25
age
Figure 1. Survival (solid line) and age-specific fecundity (dotted line, mean and standard deviation) of the rotifer Brachionus plicatilis
fed algal diets with varying 15 N-enrichment (0.4 to 5%); non-enriched algae were used as the control diet. See electronic supplementary
material, tables S1–S5 for statistical comparisons and electronic supplementary material, table S6 for primary data.
decreases for both growth proxies in all treatments but 3.5 at% 15 N (electronic supplementary material,
table S5).
3.4. Feeding activity
There were no significant differences in filtering rate between the treatments (ANOVA: F5,102 = 0.11,
p > 0.05) and no apparent trend related to 15 N concentration in the food (electronic supplementary
material, figure S1). However, the between-replicate variability was relatively high (CV% 15–17%) and
power analysis indicated that this experimental design was sufficient for detecting between-treatment
differences that exceed approximately 20%. The food depletion was similar between the treatments being
in the range of 13–18%, and no mortality occurred during the feeding trial.
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80
5
fecundity (offspring female–1)
100
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fold-change
1.09
χ2
4.069
0.5 at%
1.22
11.40
0.0007
p-values
0.044
.........................................................................................................................................................................................................................
.........................................................................................................................................................................................................................
1.0 at%
1.38
16.66
<0.0001
3.5 at%
1.18
11.40
0.0007
5.0 at%
1.42
15.92
<0.0001
26.07
<0.0001
.........................................................................................................................................................................................................................
.........................................................................................................................................................................................................................
.........................................................................................................................................................................................................................
log-rank test for trend
.........................................................................................................................................................................................................................
4. Discussion
We found significant effects of the dietary 15 N-enrichment on the rotifer life-history traits under
controlled conditions and invariant food intake. In particular, the extended lifespan and reproductive
toxicity were observed in the animals receiving 15 N-enriched food. Moreover, these changes were
associated with decreased protein allocation to the offspring as well as their growth potential, thus
potentially affecting the next generation. In addition, the effect of the 15 N concentration was not
monotonous as evidenced by a differential pattern of the responses in the rotifers from 3.5 at% 15 N
treatment compared with all other treatments with 15 N-enriched diet. More specifically, the lifetime
fecundity, timing of reproduction and offspring quality were similar between the rotifers fed 3.5 at% 15 N
diet and the control, albeit the lifespan in the former group was approximately 18% longer. These findings
support the existence of kinetic isotope effects in the biochemical reactions occurring in multicellular
consumers exposed to isotopically enriched diets. They also provide evidence for the occurrence of
enhanced growth at 3.5 at% 15 N as predicted by the isotopic resonance hypothesis [12]; notably, with
no penalty for the lifespan.
Heavy isotopes incorporated into biological molecules, such as lipids and amino acids, have been
proposed to confer cell resistance to reactive oxygen species (ROS) or reactive nitrogen species (RNS),
metabolic products which cause chemical damage [10]. For example, replacement of the bis-allylic
hydrogen atoms with deuterium atoms arrests PUFA autoxidation due to the isotope effect, rendering
cells more resistant to oxidative damage [27]. According to the free-radical theory of ageing, the enhanced
resistance to oxidative stress would result in an increased lifespan (e.g. [28]), as was observed in the
rotifers receiving 15 N-enriched food (figure 1). The animals receiving diets containing 0.4–5 at% 15 N had
lifespan extended up to 40% with concomitant changes in the timing of reproduction and decreased
lifetime fecundity by 30–40%. The decline in the reproductive output in combination with increased
longevity is typically observed in rotifers on low-calorie diets [29] and under exposure to some chemical
compounds [30]. However, in this case, both food intake (electronic supplementary material, figure S1)
and elemental composition of the algae (at least, %C and %N) were virtually invariable among the
treatments. Therefore, the observed changes in the life histories can only be attributed to the differential
metabolic processing of macromolecules with varying proportion of 15 N, the stability of these molecules,
and subsequent change in reproductive output and longevity. The elemental composition is not sufficient
to comprehensively characterize food quality, and it is also possible that nutritional value of the algae
grown in the 15 N-enriched media was lower compared with the non-enriched algae, which resulted in
the compromised diet. Recently, profound changes in growth dynamics of green algae grown at similar
levels of 15 N-enrichment (0.5–5 at%) were demonstrated, with the overall response being indicative of
stress [7]. Therefore, it is probable that biochemical composition of the algae exposed to 15 N-labelling
was also affected resulting in suboptimal nutritional quality for the rotifers.
In addition to increased stability, the isotope labelling of yeast has been reported to affect the
molecular architecture of proteins [31], raising questions about possible differences in functionality
of the isotope-labelled proteins. In bacteria grown in 15 N-enriched medium, multiple effects on the
key biosynthetic and metabolic pathways, such as citric acid cycle (a metabolic pathway that unifies
carbohydrate, fat and protein metabolism), pyruvate production and glycolysis were observed in concert
with growth inhibition [5]. As these pathways are highly evolutionarily conserved, it is not particularly
surprising to observe alterations in energy allocation and growth of the rotifers fed a 15 N-enriched food.
................................................
treatment
0.4 at%
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Table 1. Fold-change and log-rank (Mantel–Cox) analysis of the lifespan in Brachionus plicatilis exposed to diets with varying 15 Nenrichment in comparison to the control (0.37 at% 15 N concentration); see figures 1 and 2 for details and the electronic supplementary
material, table S6, for the primary data.
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3.5%
0.5%
5%
15
days
20
***
***
**
7
(d)
15
**
*
10
5
*
*
10
5
0
0
lifespan
lifetime fecundity
(b)
(e)
3
***
***
100
days
ng protein ind.–1
2
1
0
*
*
*
50
0
offspring size
age at first reproduction
(c) 12
(f)
**
0.4
*
ng RNA ng protein–1
days
9
6
3
0.3
*
0.2
**
**
0.1
0
0
reproductive period
RNA: protein
Figure 2. Variation in life-history traits (mean and standard deviation) of Brachionus plicatilis fed algal diets with varying 15 N-enrichment
(control and 0.4 to 5% 15 N). Lifespan (a), age at first reproduction (b), duration of the reproductive period (c), lifetime fecundity (d),
offspring size measured as protein content (e) and RNA:protein ratio in neonates (f ); asterisks indicate significant differences from the
control (*: p < 0.05, **: p < 0.01, ***: p < 0.001). See electronic supplementary material, tables S1–S5 for details on the statistical
comparisons.
................................................
1%
0.4%
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20
control
offspring female–1
(a)
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assistance.
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................................................
Data accessibility. All data are provided as an Excel table in the electronic supplementary material, table S6.
Competing interests. There are no competing interests to be declared.
Funding. No specific funding was received for this study.
Acknowledgements. I thank Lisa Mattsson and Karolina Eriksson-Gonzales (Stockholm University) for laboratory
8
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The observed increase in the age at first reproduction (figure 2b) and the lower protein content of the
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Isotope labelling should not be applied without understanding implications of KIEs in our study
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