Introduction
The strong, positive relationships between the stable carbon isotopic
composition (expressed as δ13C values) of organisms and that of
their diet can allow the identification of the autotrophic sources of
organic matter at the base of a food web (DeNiro and Epstein, 1978; Vander
Zanden and Rasmussen, 1999; McCutchan et al., 2003). Likewise, stable
nitrogen isotope ratios (expressed as δ15N values) can be used
to estimate the trophic position of consumers in food webs (DeNiro and
Epstein, 1981; Minagawa and Wada, 1984), and stable oxygen isotope ratios
(expressed as δ18O values) have been found to reflect those of
the water in the environment that organisms live in (Hobson, 2008; Soto et al.,
2013).
Approaches are continuing to be developed that apply stable isotope ratio
analysis to chitinous remains of aquatic invertebrates preserved in lake
sediments (Heiri et al., 2012; Leng and Henderson, 2013). For example, the
δ13C values of the fossil head capsules of benthic larvae of
non-biting midges (Chironomidae) and δ13C values of the remains of water fleas of the
genus Daphnia (Cladocera) have been used to investigate past changes in carbon
cycling and energy pathways in lake food webs (Perga, 2011; Wooller et al.,
2012; van Hardenbroek et al., 2013; Belle et al., 2014; Frossard et al.,
2014). The δ15N values of chironomid head capsules and of
Daphnia resting eggs (ephippia) have also been examined to investigate changes in
nitrogen sources in an arctic lake (Griffiths et al., 2010). Past variations
in lake water δ18O values have been reconstructed by analyzing
the δ18O values of fossil chironomid head capsules (Wooller et
al., 2004; Verbruggen et al., 2010b), and a correspondence has been found
between δ18O values of lake water and of chironomid head
capsules and Daphnia ephippia buried in surface sediments (Verbruggen et al.,
2011).
Daphnia can occur in high abundances and often dominate the zooplankton community
in lakes (Lampert, 2011). Being first-order consumers of algae, bacteria and
detritus (Geller and Müller, 1981; Gophen and Geller, 1984; Kamjunke et
al., 1999; Lampert, 2011), they form an important link between primary
production and the higher orders of the pelagic food web. This makes
Daphnia particularly suited for ecological investigations of freshwater ecosystems
and food webs using stable isotopes. While Daphnia usually reproduce
parthenogenetically, they may also reproduce sexually. Environmental cues
such as food availability, photoperiod and population density (Kleiven et
al., 1992; Cáceres and Tessier, 2004) may trigger sexual reproduction,
upon which eggs are formed enclosed by rigid sheaths (ephippia). The
chitinous ephippia are found abundantly in a wide range of lake sediment
types and remain well preserved in sediments hundreds to thousands of years
old (Szeroczyńska and Samarja-Korjonen, 2007). Since the chemical
composition of chitinous invertebrate remains stays largely unchanged even
in fossils more than 10 000 years old (Miller et al., 1993; Verbruggen
et al., 2010a), they are believed to retain their isotopic composition after
deposition (Heiri et al., 2012). Therefore, ephippia may provide material
for reconstructing the past stable isotopic composition of Daphnia in lakes and,
consequently, for investigating past conditions in aquatic food webs (e.g.,
Wooller et al., 2012; van Hardenbroek et al., 2013, 2014; Schilder et al.,
2015).
The use of δ13C and δ15N values of organisms to
infer likely organic carbon and nitrogen sources relies heavily on
assumptions regarding the difference between δ13C and δ15N values of organisms and their diet (Δ13C, Δ15N). There is a need for more controlled laboratory studies
investigating Δ13C and Δ15N (Martínez del Rio
et al., 2009) as well as the relationships between the δ18O values of
organisms and those of environmental water (Rubenstein and Hobson, 2004).
Δ13C, which is generally assumed to be between 0 and +1 ‰
for a range of animals, including invertebrates
(DeNiro and Epstein, 1978; McCutchan et al., 2003), has been studied for
chironomids under controlled laboratory conditions (Goedkoop et al., 2006;
Wang et al., 2009; Heiri et al., 2012; Frossard et al., 2013) and ranges
from -0.8 to +1.2 ‰. For Daphnia magna, Δ13C values
range from +1.7 to +3.1 ‰ (Power et al., 2003).
Δ15N, which is usually assumed to be between +3 and +4 ‰
(DeNiro and Epstein, 1981; Minagawa and Wada, 1984)
ranges from -1.5 to +3.4 ‰ for chironomids (Goedkoop
et al., 2006; Wang et al., 2009; Heiri et al., 2012) and from +1 to
+6 ‰ for Daphnia (Adams and Sterner, 2000; Power et al., 2003;
Matthews and Mazumder, 2008). In terms of oxygen, the δ18O
values of lacustrine invertebrates are strongly and positively related to
the δ18O values of local precipitation and the water in which
the invertebrates live (Wang et al., 2009; Nielson and Bowen, 2010;
Verbruggen et al. 2011; van Hardenbroek et al., 2012; Soto et al., 2013),
although laboratory studies have shown that the oxygen isotopic composition
of the diet can also affect invertebrate δ18O values (Wang et
al., 2009; Nielson and Bowen, 2010).
There can be distinct offsets in isotopic composition between whole-body
tissue and chitinous structures of invertebrates. Culturing experiments
comparing cephalopod soft tissue and their chitinous mouthparts have shown
that their chitinous structures can have δ15N values 3 to 4 ‰ lower than soft body tissue (Hobson and Cherel, 2006).
Heiri et al. (2012) reported that offsets of up to 2 ‰
between chironomid body tissue and chitinous head capsule δ13C
and δ15N values are possible. For Daphnia, field studies suggest that
(non-ephippial) exoskeleton parts can have 0.8 ‰ lower
δ13C and 7.9 ‰ lower δ15N
values than whole Daphnia (Perga, 2010), while no clear differences in δ13C and δ15N values between Daphnia and ephippia have been
reported in the only available study which examined this offset for Daphnia and free
ephippia collected in a vertical net trawl in Lake Geneva, Switzerland
(Perga, 2011). For vertebrates, differences in stable C and N isotopic
composition between tissue types have been related to differences in
contents of specific compounds (e.g., relative abundance of lipids,
carbohydrates and protein or of different amino acids; e.g., DeNiro and
Epstein, 1978; Pinnegar and Polunin, 1999). Differences in biochemical
composition also provide a potential explanation for the observed
differences in δ13C and δ15N values between the whole-body tissue and chitinous structures of aquatic invertebrates. For oxygen
and hydrogen, studies examining the offsets between the stable isotopic
composition of the whole-body tissue of lacustrine invertebrates and their
chitinous structures are still lacking.
To date, no controlled experiments investigating the offset between δ13C, δ15N and δ18O values of whole-body
tissue and ephippia have been published for Daphnia. Similarly, no laboratory
experiments have been performed examining the relationship between δ18O values of environmental water and Daphnia or their ephippia. Quantifying
these offsets and relationships is essential for the further development of
palaeoecological approaches based on stable isotope analyses of Daphnia remains and
for interpreting results from the fossil record.
We present results from an experiment developed to examine the relationships
between the δ13C values of diet and the δ18O
values of environmental water, on the one hand, and the δ13C and δ18O values of Daphnia, on the other. The experiment was specifically designed to examine
whether offsets in δ13C, δ15N and δ18O values exist between Daphnia and their ephippia. Furthermore, we
investigated whether the stable isotopic compositions of Daphnia and their ephippia
are influenced by temperature by performing the experiment at two different
temperatures.
Methods
<italic>Daphnia</italic> cultivation
Three ex-ephippial Daphnia pulicaria clones (LC PUL 53, 99 and 101; Möst, 2013) from
Lower Lake Constance (Switzerland) that showed extensive ephippia production
in culture in pre-tests were selected for the experiment. For each clone, 20
neonate Daphnia (< 48 h old) were grown in 2.5 L batch cultures prior to the
experiment. From these batch cultures, seven to eight second to third clutch neonates
(< 48 h old) were transferred to 180 mL jars, containing 160 mL of
filtered lake water (natural abundance or labeled water, according to
treatment conditions described below). The lake water was filtered with 0.45 µm glass fiber filters (Sartorius Stedim AG, Switzerland). Initially,
Daphnia were fed three times per week with fresh algae, concentrated to an
equivalent of 1 mg C L-1. After day 21 of the experiment, the amount of
food was doubled because the number of Daphnia in most jars exceeded 30
individuals. Experimental water was exchanged once per week and ephippia (if
present) were retained in the cultures. Due to potentially higher
productivity and evaporation, the water was exchanged twice per week in
Treatment 4 (20 ∘C).
Food and water sources in the experiment
Three weeks before the experiment, two 1 L chemostats were started
simultaneously to produce the algae (Acutodesmus obliquus, Turpin) to be used as food for
Daphnia in the experiment. The algae were cultivated in a “WC”-medium (Guillard,
1975). For one of the chemostats, 45 % of the sodium bicarbonate in the
medium (5.67 of 12.6 mg L-1) was replaced by sodium
bicarbonate containing 99.9 % 12C (Sigma Aldrich, USA), lowering the
δ13C values of the algae from this chemostat by, on average, 1.8 ± 1.2 ‰ (one standard deviation (1 SD)) (see
results). Once per week, the chemostat-grown algae were harvested,
centrifuged (5000 rpm) to remove residual medium, stored at 9 ∘C
in the dark and used to feed the Daphnia during the following week. Seven days
before the start of the experiment, 250 L of lake water were collected from Greifensee (Switzerland) (pH 8.0, TP 0.04 mg L-1, TN 1.6 mg L-1; data provided by the Cantonal Bureau for Waste, Water, Energy and
Air (AWEL, Zürich; www.awel.zh.ch)). This water was stored in the dark
at 12 ∘C for the duration of the experiment. Of this water, 50 L
were stored in a separate container, and 0.9 mL of water containing 97 %
18O (Sigma Aldrich, USA) were added to increase the δ18O
value of the water by 5.6 ‰ relative to the unlabeled
water (see results). Before exchanging the water in Treatment 4, the water
was allowed to equilibrate with the ambient laboratory air temperature (20 ∘C).
Experimental design
The experiment consisted of four cultivation treatments: a control treatment
in which Daphnia were cultivated in untreated, filtered lake water at 12 ∘C
on a diet of fresh chemostat-grown algae (Treatment 1), and
treatments with conditions identical to Treatment 1, with the exception of
the algae in Treatment 2, which had 1.8 ± 1.2 (1 SD) ‰
lower δ13C values. The culturing water in
Treatment 3 had δ18O values that were 5.6 ‰
higher than in the other treatments and Treatment 4 had a temperature (20 ∘C) that was higher than the other treatments.
Each treatment consisted of 30 glass jars, which were sterilized using an
autoclave. Prior to the experiment, each glass jar was assigned to one of
three replicate groups (A, B, C). The neonate Daphnia were evenly distributed in the
jars to ensure that every experimental replicate group contained 10 jars,
with 3 to 4 jars per clone. All the jars for a given treatment were held in
one large tray, and the jars within each treatment were evenly distributed
within the trays. The trays were held in the dark in temperature-controlled
incubators.
The experiment was designed to assess the following: (a) the effect of a
change in algal δ13C values on those of Daphnia and their ephippia
(Treatment 2); (b) the effect of a change in environmental water δ18O values on those of Daphnia and their ephippia (Treatment 3); (c) the
effect of a difference in temperature (i.e., 12 and 20 ∘C) on
the δ13C, δ15N and δ18O values of Daphnia and their ephippia (Treatment 4); and (d) the offset
between Daphnia and ephippia in terms of their δ13C, δ15N
and δ18O values (Treatments 1–4). Statistical analyses were
performed with the PAST software package, version 1.97 (Hammer et al.,
2001), except for tests used to compare the algae from both chemostats. To
account for repeated measures, linear mixed effects models (LMEs) were
applied, fitting a random intercept for each probing date with the lme
function in the nlme package in the R statistical package (R Core team,
2013). Significance was analyzed using an F test. A Bonferroni correction
was applied to the multiple (six) comparisons of the stable isotopic
composition of Daphnia between the treatments (Tukey post hoc tests).
Sample collection
After the weekly harvest, a small portion of algae from each chemostat was
rinsed with deionized water and centrifuged five times to remove the
culturing medium. The concentrated algae were freeze-dried and a small
aliquot (150 to 200 µg) was loaded into tin cups (6 × 4 mm, Lüdi
Swiss, Switzerland) to measure the δ13C, δ15N and
δ18O values of the algae (δ13Calgae, δ15Nalgae and δ18Oalgae). In each
treatment, one jar was assigned to monitoring variation in δ18O
values of the water (δ18Owater). Once per week, before
discarding the water, 12 mL were transferred to a 12 mL glass vial with no
head space (Labco, UK) and stored in the dark at 7 ∘C. Every
second sample was analyzed for δ18Owater values. Every
third week a sample of the water in the storage barrels was collected,
stored and measured for δ18Owater values.
The experiment was terminated after 62 days. He and Wang (2006) have
demonstrated that the Daphnia carbon turnover rate is 11 to 36 % per day, which
suggests that after 62 days our Daphnia likely had achieved isotopic equilibrium
with the experimental diet and water. Daphnia and ephippia were harvested and
pooled according to treatment (1–4) and replicate group (A, B, C). Adult
Daphnia were hand-picked from a Bogorov sorting tray (Gannon, 1971) with fine
forceps under a binocular and freeze-dried, after which they were loaded
into tin cups (6 × 4 mm, Lüdi Swiss, Switzerland; ∼ 10 to
12 individuals per measurement) for analysis of δ13CDaphnia, δ15NDaphnia and δ18ODaphnia values. For each treatment replicate group, three
samples were prepared and measured, resulting in 36 measurements for each
chemical element. Ephippia were collected and treated in 10 % KOH for 2 h to remove any algal matter and egg yolk. Replicate measurements
(three each for C, N and O) of ephippia not treated with KOH were prepared to
assess any influence of this treatment on the isotopic compositions of
ephippia. The ephippia were loaded into pre-weighed tin cups (6 × 4 mm,
Lüdi Swiss, Switzerland): ∼ 10 to 15 for δ13Cephippia and δ15Nephippia analysis and 15
to 20 for δ18Oephippia analysis. Three samples were
prepared and measured for each treatment replicate group, except for
Treatment 4, which yielded only sufficient numbers of ephippia to measure
once per treatment replicate group.
Assessing the source of oxygen in <italic>Daphnia</italic>
Following Wang et al. (2009), our experimental setup was used to approximate
the proportional contribution of oxygen in the Daphnia stemming from the
environmental water relative to that from the diet, using the following
equation:
p=δ18ODaphnia(A)-δ18ODaphnia(B)δ18Owater(A)-δ18Owater(B),
where p is the proportion of oxygen in Daphnia stemming from the water, δ18ODaphnia(A) and δ18Owater(A)
would be the δ18O values of Daphnia and the water if Daphnia were cultivated in
non-manipulated, filtered lake water, and δ18ODaphnia(B) and δ18Owater(B) would be the
δ18O values of Daphnia and the water if Daphnia were cultivated in the
18O-enriched, filtered lake water.
Stable isotope mass spectrometry
The δ13C and δ15N values of the algae, Daphnia and
ephippia were measured on a Costech ESC 4010 elemental analyzer interfaced
via a ThermoConflo III with a Thermo Delta V isotope ratio mass spectrometer
(IRMS) at the Alaska Stable Isotope Facility (ASIF) at the University of
Alaska, Fairbanks. The analytical precisions for δ13C and
δ15N values are expressed as 1 SD from the mean based on the
results from multiple (n= 13) analyses of a laboratory standard
(peptone) and were ± 0.2 ‰ and ± 0.1 ‰,
respectively. The δ18O values of the
water samples were measured on an online pyrolysis thermochemical reactor
elemental analyzer (TCEA) (Finnigan ThermoQuest) coupled to a continuous
flow (Conflo III) IRMS (Finnigan MAT Delta V) at the ASIF. Analytical
precision is expressed as 1 SD from the mean based on the results from
multiple (n= 3) analyses of a laboratory standard (doubly labeled water;
±0.3 ‰). The δ18O values of the
algae, Daphnia and ephippia were measured using the same techniques and instruments
as used for the water samples. Analytical precision based on replicate (n= 12) laboratory standard measurements (benzoic acid, Fisher Scientific,
Lot No 947459) was ±0.4 ‰ . Stable isotopic
compositions are expressed in standard delta (δ) notation in
‰ relative to V-PDB (Vienna Pee Dee Belemnite) for δ13C values, AIR for δ15N values and V-SMOW (Vienna Standard Mean Ocean Water) for δ18O values.
Results
Food and water
The δ13Calgae values from both chemostats showed some
variation with time (Fig. 1). On all sampling dates except the first, the
algae cultured on 13C-depleted medium had lower δ13Calgae values than the standard algae (Fig. 1). As a
consequence, the mean δ13Calgae value for the culture
grown using 13C-depleted medium (-20.6 ± 1.84 ‰)
was 1.8 ± 1.2 ‰ (n= 9)
lower than the mean δ13Calgae of the standard algae (-18.8 ± 2.4 ‰),
and this difference was statistically
significant (LME, F(1,8) 18.04, p < 0.005). There was no
statistically significant difference between the algae cultures in terms of
δ15N values (standard algae 2.5 ± 0.3 ‰,
13C-depleted algae 2.2 ± 0.3 ‰; F(1,8) 4.58, p > 0.05), δ18O values (standard algae 13.4 ± 1.0 ‰,
13C-depleted algae 14.6 ± 1.1 ‰; F(1,7)
5.43, p > 0.05) or atomic C : N ratios (standard
algae 6.4 ± 1.3, 13C-depleted algae 6.5 ± 1.3; F(1,8) 0.18,
p > 0.05) (Fig. 1).
δ13C, δ15N and δ18O
values and atomic C : N ratios of the algae harvested from both chemostats
during the experiment. Open circles with dashed line represent the standard
algae, and the closed circles with solid line represent the algae that were
cultured on a medium with the addition of 13C-depleted bicarbonate. The
data points and error bars on the right side of the plots indicate average
values and 1 SD, respectively.
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The addition of 18O-enriched water led to an increase in δ18Owater values in the storage barrels by 5.6 ‰
(δ18O value of -3.4 ± 0.1 ‰ , n= 3)
relative to the non-labeled water (δ18O value of -9.0 ± 0.1 ‰ n= 3) (Fig. 2). The δ18Owater values from the experimental jars in
Treatment 1, 2 and 4 were not significantly different (one-way ANOVA,
F(2,2) 30.1, p > 0.05) between the three treatments
throughout the experiment, and the mean was -8.2 ± 0.5 ‰ (n= 11). Water from experimental jars from
Treatment 3 had a mean δ18Owater value of -3.3 ± 0.6 ‰ (n= 4). The mean δ18Owater values
in the storage barrels and the mean δ18Owater values in
the experimental jars after 1 week were used to approximate the baseline
δ18Owater values during cultivation for resolving
Eq. (1) by taking the mean of the two values. This resulted in estimates
of -8.6 ‰ for the cultures in non-manipulated lake water
at 12 ∘C (Treatment 1 and 2) and -3.4 ‰ for
the cultures in Treatment 3 with 18O-enriched water.
δ18O values of the water in the storage barrels
for the standard water (open circles, dashed line) and the artificially
18O-enriched water (closed circles, solid line) sampled on day 0, 13
and 35; and the δ18O values of the water sampled from the
experimental jars before water was exchanged for Treatment 1 (open diamonds,
control), Treatment 2 (open triangles, 13C-depleted algae), and
Treatment 3 (closed diamonds,18O-enriched water) sampled on day 13,
27, 41 and 62; and Treatment 4 (open squares, 20 ∘C) sampled on
day 13, 27 and 41. The plus symbols (+) on the right side indicate the
mean of the mean experimental jar values and the mean storage barrel values
for the standard water and the 18O-enriched water, respectively.
![]()
δ13C, δ15N and δ18O
values of Daphnia body tissue (left, open circles) and ephippia (right, closed
circles) for Treatment 1 (control), 2 (13C-depleted algae), 3
(18O-enriched water) and 4 (elevated temperature). Each data point
represents one of the treatment replicate groups and consists of three
measurements, of which the standard deviation is indicated by the error bars
(only one measurement per replicate treatment group was available for
ephippia in Treatment 4). The black horizontal lines in the δ13C and δ15N plots represent the average value of the
algae used in that treatment.
![]()
<italic>Daphnia</italic> stable isotope ratios
Mean stable isotope values for Daphnia are based on 9 measurements (three
measurements for each of the three replicates per treatment). The mean
δ13CDaphnia value in Treatment 2 (where Daphnia were offered
13C-depleted algae) was lower (-20.2 ± 0.1 ‰) than in Treatment 1 (-18.7 ± 0.1 ‰) and 3
(-17.9 ± 0.1 ‰ ) (Fig. 3). For treatments at 12 ∘C (1–3), the mean δ13CDaphnia value was 0.5 ± 0.3 ‰
higher than the mean δ13Calgae value that Daphnia were cultured on. The mean δ13CDaphnia value in Treatment 4 (20 ∘C; -19.0 ± 0.1 ‰) was 0.2 ± 0.1 ‰ lower
than the mean δ13Calgae value. The results from all
treatments in terms of δ13CDaphnia values were
significantly different from each other (one-way ANOVA and Tukey post hoc
test; Table 1)
Results of the tests for statistical differences between the four
(1–4) treatments (one-way ANOVA) and between pairs of treatments (Tukey
test) for δ13CDaphnia, δ15NDaphnia and
δ18ODaphnia values. The results of the Tukey test are
presented below the F and p values for the one-way ANOVA, showing Q values
(lower left part of matrix) and p values after Bonferroni correction (upper
right).
Daphniaδ13C values
Daphniaδ15N values
Daphniaδ18O values
F(2.3) 303.8 p < 1 × 10-8
F(2.3) 52.1 p < 1 × 10-5
F(2.3) 255.3 p < 1 × 10-8
1
2
3
4
1
2
3
4
1
2
3
4
1
< 0.002
< 0.002
< 0.05
1
> 0.9
< 0.005
< 0.002
1
> 0.1
< 0.002
< 0.005
2
28.16
< 0.002
< 0.002
2
1.686
< 0.01
< 0.002
2
5.646
< 0.002
> 0.05
3
13.62
41.78
< 0.002
3
10.16
8.476
> 0.1
3
24.6
30.25
< 0.002
4
6.968
21.19
20.58
4
15.32
13.63
5.154
4
11.88
6.234
36.48
Mean δ15NDaphnia values at 12 ∘C were 5.5 ± 0.1 ‰ (Treatment 1),
5.7 ± 0.1 ‰ (Treatment 2) and 6.2 ± 0.1 ‰ (Treatment 3),
and they were 3.4 ± 0.3 ‰ higher than the mean δ15Nalgae value
(Fig. 3). At 20 ∘C (Treatment 4), the mean δ15NDaphnia value (6.5 ± 0.2 ‰) was 4.0 ± 0.2 ‰ higher than the mean δ15Nalgae value. All treatments, except for Treatments 1 and 2 and Treatments 3 and 4,
were significantly different from each other with regard to δ15NDaphnia values (one-way ANOVA and Tukey post hoc test; Table 1).
Treatments 1 and 2 were both performed at 12 ∘C and with similar
water in terms of δ18O values. The mean δ18ODaphnia values in these treatments were 11.7 ± 0.1 ‰ and 11.0 ± 0.2 ‰ (Fig. 3). In Treatment 3, where the mean δ18Owater value was 5.2 ‰ higher than in the
other treatments, the mean δ18ODaphnia value
was 14.6 ± 0.3 ‰ , which was 2.9 and 3.6 ‰ higher than in Treatment 1 and 2, respectively. In
Treatment 4, with δ18Owater as in Treatment 1 and 2 but
run at a higher temperature (20 ∘C), the mean δ18ODaphnia value (10.2 ± 0.2 ‰) was
1.5 and 0.8 ‰ lower than in Treatment 1 and 2,
respectively. A significant difference in δ18ODaphnia
values was found between all treatments, except for Treatments 1 and 2 and Treatments 2 and 4 (one-way ANOVA and Tukey post hoc
test; Table 1).
Ephippia stable isotope ratios
In all treatments ephippia production started between day 27 and day 34 of
the experiment. Until day 48 of the experiment, ephippia production was low
(on average 1 to 1.5 ephippia per jar per week), after which production
increased to 4.5 to 6 ephippia per jar per week in Treatments 1, 2 and 3,
whereas production in Treatment 4 remained low. Across the replicate
treatments (A–C), the production of ephippia was similar with, on average, 12
to 13 ephippia per jar at the end of the experiment. The majority of the
ephippia were produced by clone LC PUL 99 (55 %), whereas LC PUL 101 and
53 were responsible for 23 and 22 % of the ephippia production,
respectively.
The measurements that we performed on untreated ephippia did not reveal a
detectable effect of the KOH treatment on the δ13Cephippia, δ15Nephippia and δ18Oephippia values (t tests: δ13C t= 0.41,
p > 0.05; δ15N t= 2.20, p > 0.05; δ18O t= 0.03, p > 0.05). The mean δ13Cephippia value was, on average, 0.2 ± 0.8 ‰
lower than the mean δ13CDaphnia
value, but this difference was not statistically significant (paired
t test: t= 0.83, p >0.05; Fig. 4). However, this value was strongly
affected by the results from Treatment 4 (20 ∘C), which yielded
unexpected values that will be discussed below. In the three treatments at
12 ∘C, δ13Cephippia values were, on average, 0.2 ± 0.4 ‰ higher than δ13CDaphnia,
although this difference was again not significant (paired t test: t= 1.50,
p > 0.05). Over all four treatments, δ15Nephippia values were, on average, 1.6 ± 0.4 ‰
lower than δ15NDaphnia values
(paired t test: t= 14.01, p < 5 × 10-8), and δ18Oephippia values were, on average, 0.9 ± 0.4 ‰
lower than δ18ODaphnia values
(paired t test: t= 5.58, p < 5 × 10-5).
The difference in δ13C, δ15N and
δ18O values between ephippia and Daphnia for all four treatments
(closed circles). The open circle gives the offset for the three treatments
at 12 ∘C excluding Treatment 4 (20 ∘C), which yielded
unexpected results for δ13C (see text). Error bars indicate
standard deviations.
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Discussion
Statistically significant differences were found between nearly all
treatments for all investigated Daphnia stable isotope ratios, even in cases where
we expected no differences based on the manipulations. For example,
Treatment 1 and 3 were identical in terms of δ13C values of the
food source and temperature and only differed in the δ18O
values of the water, and Treatment 1, 2 and 3 were identical in terms of
δ15N values of the food source and temperature. However, the
unexpected differences between these treatments were generally small and of
the same order of magnitude as the analytical precisions associated with
each element (Fig. 3). They may represent the inherent variability
associated with stable isotope ratios in organisms (Schimmelmann, 2011).
Alternatively, since the stable isotope ratios of the algae showed some
variability over the course of the experiment (Fig. 1), a slight
difference in timing in the buildup of biomass may have led to small
differences in Daphnia stable isotope ratios. In previous experiments, δ13CDaphnia and δ15NDaphnia values have been found to differ as much as 1 ‰
between identical treatments (Power et al., 2003). The differences in
Daphnia stable isotope ratios were much larger when comparing treatments with
manipulated δ13Calgae and δ18Owater
values to those with non-manipulated algae and water.
The food experiment: changing
<inline-formula><mml:math display="inline"><mml:mrow><mml:msup><mml:mi mathvariant="italic">δ</mml:mi><mml:mn>13</mml:mn></mml:msup></mml:mrow></mml:math></inline-formula>C<inline-formula><mml:math display="inline"><mml:msub><mml:mi/><mml:mi mathvariant="normal">algae</mml:mi></mml:msub></mml:math></inline-formula>
Offering Daphnia algae with, on average, 1.8 ‰ lower δ13Calgae values resulted in 1.5 to 2.1 ‰
lower δ13CDaphnia values. Since the δ13Calgae values were variable over time, we cannot reconstruct
the exact δ13C value of the carbon that Daphnia in our different
treatments assimilated, and therefore we cannot calculate a precise estimate of
Δ13C. Based on the mean δ13Calgae value over
the duration of the experiment, however, Δ13C between Daphnia and
algae is estimated to be +0.5 ± 0.3 ‰ at 12 ∘C.
This is in agreement with commonly found Δ13C
values of 0 to +1 ‰ for a range of animals, including
invertebrates (DeNiro and Epstein, 1978; McCutchan et al., 2003). D. magna has been
reported to have a Δ13C value of +1.7 ‰
at 12 ∘C on a diet of aquarium food (Power et al., 2003).
However, in this study a lipid correction was applied to infer δ13C values based on C : N ratios following a model by McConnaughey and
McRoy (1979). This leads to relatively higher δ13C values, and
the procedure has been criticized, since it potentially provides biased
estimates when comparing isotopic ratios of different organisms and tissues
(Mintenbeck et al., 2008). Power et al. (2003) did not report the C : N of the
food and Daphnia, so we cannot back-calculate the δ13C values they
measured prior to lipid correction.
δ13Cephippia values also reflected the difference in δ13Calgae values between the treatments. At 12 ∘C, they
were not significantly different from the δ13CDaphnia
values (although they were consistently lower at 20 ∘C; see
below). This is in line with the findings by Perga (2011), who found that
the δ13C value of ephippia collected in the field was slightly,
but not significantly, higher than the δ13C value of Daphnia
collected in the same net trawls. This suggests that δ13Cephippia values are a reliable indicator of changes in
δ13CDaphnia values, and consequently of variations in
δ13C values of Daphnia diet: at 12 ∘C, δ13Cephippia was 0.7 ± 0.2 ‰ higher than
the mean δ13Calgae. The absence of a clear offset in
δ13C values between whole Daphnia and Daphnia ephippia at 12 ∘C is
in contrast to the difference found between whole Daphnia and Daphnia exoskeletons (0.8 ‰; Perga, 2010) and between chironomid body tissue and
chironomid head capsules (∼ 1 ‰; Heiri et
al., 2012; Frossard et al., 2013).
<inline-formula><mml:math display="inline"><mml:mrow><mml:msup><mml:mi mathvariant="italic">δ</mml:mi><mml:mn>15</mml:mn></mml:msup></mml:mrow></mml:math></inline-formula> N values of <italic>Daphnia</italic> and ephippia
At 12 ∘C, the observed Δ15N was +3.4 ± 0.3 ‰,
which agrees well with Δ15N values
referred to in the literature (+3 to +4 ‰; DeNiro
and Epstein, 1981; Minagawa and Wada, 1984). A range of Δ15N
values for Daphnia have been reported. D. pulicaria reared on a diet of frozen algae pellets
had a Δ15N of +1.4 ‰ (Matthews and
Mazumder, 2008). This is lower than the Δ15N we found.
According to Matthews and Mazumder (2008), the low Δ15N they
observed may be explained by the observation that a diet consisting of
detritus (dead algae) is associated with considerably (∼ 2.5 ‰)
lower Δ15N values than one consisting
of living plant matter (Vanderklift and Ponsard, 2003). Our observed Δ15N for D. pulicaria is within the range of reported D. magna Δ15N values
(+1 to +6 ‰; Adams and Sterner, 2000; Power et al.,
2003).
δ15Nephippia values were lower (1.6 ± 0.4 ‰)
than δ15NDaphnia values. In
contrast, Perga (2011) found δ15Nephippia values to be
slightly, but not significantly, lower than δ15NDaphnia
values in the field. Together with the results of Perga (2011), our data provide
an indication that δ15Nephippia values are indicative of
δ15N values of Daphnia and their diet, with only relatively minor
offsets between food, Daphnia and ephippia. For chironomids, differences of similar
magnitude between whole-body δ15N values and head capsule
δ15N values (-1 to +1 ‰) were observed
over a large range of δ15N values (2.5 to 15 ‰;
Heiri et al., 2012). Therefore, it seems likely that
differences between Daphnia and ephippia δ15N values may also be
similar across this δ15N range.
The water experiment: changing <inline-formula><mml:math display="inline"><mml:mrow><mml:msup><mml:mi mathvariant="italic">δ</mml:mi><mml:mn>18</mml:mn></mml:msup></mml:mrow></mml:math></inline-formula>O<inline-formula><mml:math display="inline"><mml:msub><mml:mi/><mml:mi mathvariant="normal">water</mml:mi></mml:msub></mml:math></inline-formula> values
δ18Owater values were 5.2 ‰ higher in
Treatment 3 than in Treatment 1 and 2, and the mean δ18ODaphnia values in Treatment 3 were 2.9 ‰
higher than in Treatment 1 and 3.6 ‰ higher than in
Treatment 2. This implies that, as expected, differences in δ18ODaphnia values reflect differences in δ18Owater, yet that, as in other invertebrates, only part of the
oxygen incorporated by the Daphnia originated from the water. Wang et al. (2009)
reported that 69 % of the oxygen in chironomid larvae stemmed from the
water in their environment. Soto et al. (2013) estimated that 84 % of the
oxygen in protein isolated from chironomids came from the water in their
environment, and Nielson and Bowen (2010) reported that 69 % of the
oxygen in chitin from brine shrimp came from water in their environment.
Based on Eq. (1), we estimate that in our experiment 56 to 69 % of
the oxygen in Daphnia came from the water, based on Treatment 1 and 2,
respectively. These estimates are similar to the values reported by Wang et al. (2009) and Nielson and Bowen (2010).
δ18Oephippia values closely reflected differences in δ18ODaphnia: they were, on average, 0.9 ± 0.4 ‰ lower than δ18ODaphnia values. This
suggests that δ18Oephippia may be used as an indicator of
δ18ODaphnia, which in turn can be expected to be related
to lake water δ18O values. This is in agreement with the
correlation between surface sediment δ18Oephippia values
and lake water δ18O values found in a field survey of a number
of European lakes (Verbruggen et al., 2011).
The temperature experiment
Power et al. (2003) reported an increase of 0.1 ‰ in
Δ13C values for D. magna with a temperature increase from 12 to 20 ∘C (and +1.4 ‰ when
temperature increased from 12 to 26 ∘C). Therefore,
we expected Δ13C values for Daphnia in Treatment 4 (20 ∘C)
to be similar to or slightly higher than in the other treatments (12 ∘C).
Δ13C values were clearly lower, however, in
Treatment 4 (-0.2 ± 0.1 ‰) than in the other
treatments (+0.5 ± 0.3 ‰). While we cannot
exclude a negative relation between temperature and Δ13C values
for Daphnia, we choose to treat this result with caution due to the discrepancy
with the positive Δ13C values as reported in other studies
(DeNiro and Epstein, 1978; McCutchan et al., 2003; Power et al., 2003). A
higher lipid content of Daphnia may potentially lead to lower δ13CDaphnia values (McCutchan et al., 2003). However, the C : N
ratios of Daphnia in Treatment 4 were slightly lower (but not significantly
different; t test: t= 1.18 p > 0.05) than those of Daphnia in Treatment 1,
which does not agree with a higher lipid content in Daphnia from Treatment 4
(Smyntek et al., 2007). Alternatively, 13C depletion of algal biomass
during dark respiration may have affected the δ13Calgae in
Treatment 4 disproportionately due to the higher temperature.
Degens et al. (1968) found that δ13C values of the alga Dunaliella tertiolecta were 4 ‰ lower after 3 days in darkness. The rate of
respiration by algae depends on temperature and can be 2 to 4 times higher
at 20 ∘C than at 12 ∘C (e.g., Vona et al., 2004).
Microbial activity in the experimental jars could have been affected by
temperature and could have also influenced our results. Additionally, if
Daphnia in Treatment 4 had a different timing of growth compared to Treatment 1, as
might be expected, they may have been assimilating carbon from algae with
different δ13Calgae values during the main phase of their
growth compared to the other treatments, since δ13Calgae
values were relatively low in the beginning and at the end of the experiment
(Fig. 1). δ13Cephippia values were also lower in
Treatment 4, and 1.3 ± 0.3 ‰ lower than δ13CDaphnia values. For the same reasons as outlined above,
it remains unclear whether this observation is the consequence of a
fundamental change in the offset between δ13CDaphnia and
δ13Cephippia with temperature or whether it is affected by
variations in δ13Calgae and algal respiration rates or
differences in Daphnia growth rates between our treatments. Controlled experiments
over a range of temperature values analyzing not only δ13CDaphnia and δ13Cephippia values, but also
δ13C values of respired CO2 and microbial biomass
would be desirable to further explore this issue. Although the results of
Treatment 4 indicate that the difference between δ13Cephippia and δ13CDaphnia values may be more
variable than suggested by the cultivations at 12 ∘C, the offset
is still relatively small compared to the variation in δ13Cephippia values in lake sediment records (up to 10 ‰; e.g., Wooller et al., 2012).
Δ15N between Daphnia and algae was +4.0 ± 0.2 ‰
at 20 ∘C, 0.6 ‰ higher
than at 12 ∘C. A small increase (0.4 ‰) in
Δ15N in this temperature range has also been reported for D. magna
(Power et al., 2003). Power et al. (2003) found a decrease of 2.7 ‰
in Δ15N values for D. magna between 20 and 26 ∘C, however, and Barnes et al. (2007) found
a decrease of 0.6 ‰ in Δ15N values for sea
bass with a temperature increase from 11 to 16 ∘C.
Previously observed Δ15N values in field studies of aquatic
food webs (Vander Zanden and Rasmussen, 2001), and specifically in
experimental studies of Daphnia (Adams and Sterner, 2000; Matthews and Mazumder,
2008), are, in some cases, lower than +3 to +4 ‰. A
potential effect of temperature on Δ15N values for Daphnia which,
based on presently available observations, may amount to 2.7 ‰
at temperatures above 20 ∘C (Power et al.,
2003) therefore deserves future attention. The offset between δ15NDaphnia and δ15Nephippia in our experiment
was, however, not significantly different (t test: t= 0.26 p > 0.05)
between Treatment 1 (control, 12 ∘C) and 4 (20 ∘C).
The effect of temperature on oxygen isotope fractionation during the
formation of chitin by aquatic organisms has not been examined previously in
experimental studies. Schimmelmann and DeNiro (1986) analyzed the δ18O values of the chitin of marine crustaceans collected along a
temperature gradient of 10 ∘C and van Hardenbroek et al. (2012)
studied the δ18O values of aquatic beetles in museum specimens
selected to represent a temperature gradient across North America. Both
studies concluded that the temperature effect on oxygen isotope
fractionation during chitin formation (if any) was smaller than the
variability due to minor differences in local environmental conditions. In
this study we kept close control over the environmental conditions and source
water δ18O values, and we found that δ18ODaphnia was slightly (0.8 to 1.5 ‰) lower
with an increase of temperature by 8 ∘C but otherwise similar
conditions. This may indicate an effect of temperature on oxygen isotope
fractionation by Daphnia. We do note, however, that the potential temperature
effect on oxygen isotope fractionation by Daphnia observed in our experiment was
relatively small and resulted from a large difference in temperature.
Therefore, δ18ODaphniavalues most likely primarily reflect
environmental water δ18O values. The offset between δ18Oephippia and δ18ODaphnia in Treatment 4 (20 ∘C)
was not significantly different, however (t test: t= 0.09,
p > 0.05), from that in Treatment 1 (control, 12 ∘C).
This suggests that, in contrast to the difference between δ18Owater and δ18ODaphnia, this offset is not
affected by temperature in the investigated temperature range (12
to 20 ∘C). Verbruggen et al. (2011) measured the
δ18O values of recently deposited ephippia from surface
sediments in lakes along a geographical gradient in Europe. They found a
strong correlation between δ18Oephippia values and lake
water δ18O values. In their data set, the δ18O
values of lake water increased by ∼ 4.8 ‰
with a temperature increase of 8 ∘C, whereas δ18Oephippia values increased by only ∼ 3 ‰ over this temperature gradient, a difference of
∼ 1.8 ‰. This difference is of a similar
order of magnitude as the 0.8 to 1.5 ‰ lower δ18ODaphnia values we found with an 8 ∘C temperature
rise. The data of Verbruggen et al. (2011) and our experimental data would
therefore be in agreement with a slight temperature effect on the
fractionation of 18O between lake water and Daphnia biomass. However, other
mechanisms, such as a change in timing of Daphnia ephippia production with
temperature and variations in δ18O values of food across the
examined temperature gradient could also explain varying offsets between
δ18Owater and δ18ODaphnia at different
temperatures in the study of Verbruggen et al. (2011). Moreover, differences in air temperature at lakes, which Verbruggen
et al. (2011) reported, do not necessarily lead to similar differences in lake water
temperatures.
Implications for palaeoecological studies
In general, we found that the stable isotopic composition of ephippia
closely reflected the stable isotopic composition of Daphnia. The offsets were
consistent within treatments and between most treatments (Fig. 4), and the
ephippia stable isotope ratios responded to the manipulations in δ13Calgae and δ18Owater that we performed. Studies
investigating the δ13C and δ15N values of fossil
Daphnia ephippia have recorded shifts of up to 5 to 10 ‰ in
δ13C values (Wooller et al., 2012; Frossard et al., 2014) and
3 ‰ in δ15N values (Griffiths et al., 2010).
Shifts of 2 to 3 ‰ in δ18O values have been
reported for fossil chironomid head capsules (Wooller et al., 2004;
Verbruggen et al., 2010b). In our experiment, the standard deviation of the
offset between Daphnia and ephippia stable isotope ratios was much smaller than the
reported shifts in stable isotope ratios of fossil remains: ±0.4 ‰
for δ13C, δ15N and δ18O (±0.8 ‰ for δ13C when
including Treatment 4 at 20 ∘C). If our findings are
representative of the offset in stable isotope ratios between Daphnia and their
ephippia in nature, they indicate that reported shifts in stable isotope
ratios of fossil ephippia can reliably be interpreted as indicating past
variations in Daphnia stable isotope ratios. These in turn can be expected to
reflect past changes in isotopic composition of Daphnia diet and/or the δ18O of the water they lived in. While experiments offer the
possibility to closely control the food sources and growth conditions for
Daphnia, they cannot cover the full range of environments and interactions found in
nature. Further studies in the field, in the fossil record and in an
experimental setting are therefore needed to confirm the findings that we present
here and to improve our understanding of the relationship between the stable
isotopic composition of food, ambient water and chitinous fossilizing
structures produced by Daphnia and other invertebrates. Although we only cultured
Daphnia at two different temperatures, we found indications that temperature may
have affected Δ13C and Δ15N on the one hand and the relationship
between δ18Owater and δ18ODaphnia
values on the other in an experimental setting. Future efforts focused on constraining the
effect of temperature on these offsets and relationships are therefore
particularly necessary.