Experimental assessment of environmental influences on the stable isotopic composition of Daphnia pulicaria and their ephippia

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A higher water temperature (20 • C) resulted in lower δ 13 C values in Daphnia and ephippia than in the other treatments with the same food source and in a minor change in the difference between δ 13 C values of ephippia and Daphnia (to −1.3 ± 0.3 ‰).This may have been due to microbial processes or increased algal respiration rates in the experimental containers, which may not affect Daphnia in natural environments.There was no significant difference in the offset between δ 18 O and δ 15 N values of ephippia and Daphnia between the 12 • C and 20 • C treatments, but the δ 18 O values of Daphnia and ephippia were on average 1.2 ‰ lower at 20 • C compared with 12 • C. We conclude that the stable isotopic composition of Daphnia ephippia provides information on that of the parent Daphnia and of the food and water they were exposed to, with small offsets between Daphnia and ephippia relative to variations in Daphnia stable isotopic composition reported from downcore studies.However, our experiments also indicate

Introduction
The strong, positive relationships between the stable carbon isotopic composition (expressed as δ 13 C 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 δ 15 N 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 δ 18 O values) have been found to reflect those of the water in the environment 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 δ 13 C values of fossil head capsules of benthic larvae of non-biting midges (Chironomidae) and 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 δ 15 N 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 δ 18 O values have been reconstructed by analyzing the δ 18 O values of fossil chironomid head capsules (Wooller et al., 2004;Verbruggen et al., 2010b), and a correspondence has been found between δ 18 O values of lake water and of chironomid head capsules and Daphnia ephippia buried in surface sediments (Verbruggen et al., 2011).Figures

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Full 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 ten thousand 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., 2013van Hardenbroek et al., , 2014)).
The use of δ 13 C and δ 15 N values of organisms to infer likely organic carbon and nitrogen sources relies heavily on assumptions regarding the difference between δ 13 C and δ 15 N values of organisms and their diet (∆ 13 C, ∆ 15 N).There is a need for more controlled laboratory studies investigating ∆ 13 C and ∆ 15 N (Martínez del Rio et al., 2009), and the relationships between the δ 18 O values of organisms and those of environmental water (Rubenstein and Hobson, 2004).∆ 13 C, 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)  ∆ 13 C values range from +1.7 to +3.1 ‰ (Power et al., 2003).∆ 15 N, 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).Measurements of Daphnia and ephippia collected in the field have been used to infer that Daphnia exoskeletons have 0.8 ‰ lower δ 13 C and 7.9 ‰ lower δ 15 N values than whole Daphnia (Perga, 2010) whereas no clear differences in δ 13 C and δ 15 N values between Daphnia and ephippia were apparent (Perga, 2011).To date, no controlled experiments investigating the offset between whole body tissue and ephippia have been published for Daphnia.Quantifying this offset is essential for further development of palaeoecological approaches based on stable isotope analyses on Daphnia remains and for interpreting results from the fossil record.In terms of oxygen, the δ 18 O values of aquatic invertebrates is strongly and positively related to the δ 18 O values of local precipitation and the water in which the invertebrates live (Wang et al., 2009;Nielson and Bowen, 2010;van Hardenbroek et al., 2012;Soto et al., 2013).To our knowledge, no controlled experiments have been performed examining the relationship between δ 18 O values of environmental water and Daphnia, or their ephippia.
We present results from an experiment developed to examine the relationships between the δ 13 C values of diet and the δ 18 O values of environmental water, and the

Daphnia 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 7-8 secod 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 refreshed once per week and ephippia (if present) were retained in the cultures.Due to potentially higher productivity and evaporation, the water was refreshed 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 "WC"-medium (Guillard, 1975).For one of the chemostats, 45 % of the sodium bicarbonate in the medium (5.67 mg L −1 of 12.6 mg L −1 ) was replaced by sodium bicarbonate containing 99.9 % 12 C (Sigma Aldrich, USA), lowering the δ 13 C values of the algae from this chemostat by on average 1.8 ± 1.2 (one standard deviation (1 SD)) ‰ (see Sect. ing week.Seven days before the start of the experiment 250 L of lake water were collected from Lake 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.50 L of this water were stored in a separate container and 0.9 mL of water containing 97 % 18 O (Sigma Aldrich, USA) were added to increase the δ 18 O value of the water with 5.6 ‰ relative to the unlabeled water (see Sect. 3).

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 δ 13 C values.The culturing water in Treatment 3 had δ 18 O 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 3-4 jars per clone (Fig. 1).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 (Fig. 1).The trays were held in the dark, in temperature controlled incubators.
The experiment was designed to assess the following: 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 (LME) 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.

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 (150-200 µg) was loaded into tin cups (6 × 4 mm, Lüdi Swiss, Switzerland) to measure the δ 13 C, δ 15 N and δ 18 O values of the algae (δ 13 C algae , δ 15 N algae and δ 18 O algae ).In each treatment, one jar was assigned to monitoring variation in δ 18 O values of the water (δ 18 O water ).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 δ 18 O water values.Every third week a sample of the water in the storage barrels was collected, stored and measured for δ 18 O water values.
The experiment was terminated after 62 days.He and Wang (2006) have demonstrated that Daphnia carbon turnover rate is 11-36 % 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 a 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 δ 13 C Daphnia , δ 15 N Daphnia and δ 18 O Daphnia values.For each treatment replicate group, three samples were prepared and measured, resulting in 36 measurements for each chemical element.Ephippia were collected and Introduction

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Assessing the source of oxygen in Daphnia
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:

Food and water
The δ 13 C algae values from both chemostats showed some variation with time (Fig. 2).
On all sampling dates except the first, the algae cultured on 13 C-depleted medium had lower δ 13 C algae values than the standard algae (Fig. 2).As a consequence, the mean

Daphnia stable isotope ratios
The mean δ 13 C Daphnia value in Treatment 2 (where Daphnia were offered 13 C-depleted algae) was lower (−20.2± 0.1 ‰) than in Treatment 1 (−18.7 ± 0.1 ‰) and 3 (−17.9± 0.1 ‰) (Fig. 4).For treatments at 12 • C (1-3), the mean δ 13 C Daphnia value was 0.5 ± 0.3 ‰ higher than the mean δ 13 C algae value Daphnia were cultured on.The mean 4.0 ± 0.2 ‰ higher than the mean δ 15 N algae value.All treatments, except for Treatment 1 and 2, were significantly different from each other with regards to δ 15 N Daphnia values (One-way ANOVA and Tukey post-hoc test; Table 1).Treatment 1 and 2 were both performed at 12 • C and with similar water in terms of δ 18 O values.The mean δ 18 O Daphnia values in these treatments were 11.7 ± 0.1 ‰ and 11.0±0.2‰, respectively (Fig. 4).In Treatment 3, where the mean δ 18 O water value was 5.2 ‰ higher than in the other treatments, the mean δ 18 O Daphnia 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 δ 18 O water as in Treatment 1 and 2, but run at higher temperature (20 • C), the mean δ 18 O Daphnia value (10.2 ± 0.2 ‰) was 1.5 and 0.8 ‰ lower than in Treatment 1 and 2, respectively.A significant difference in δ 18 O Daphnia values was found between all treatments (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 1-1.5 ephippia per jar per week), after which production increased to 4.5-6 ephippia per jar per week in Treatment 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 12-13 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 we performed on untreated ephippia did not reveal a detectable effect of the KOH treatment on the δ 13 C ephippia , δ 15 N ephippia and δ 18 O ephippia values (Fig. 5  Fig. 6).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

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 δ 13 C values of the food source and temperature and only differed in the δ 18 O values of the water.Treatment 1, 2 and 3 were identical in terms of δ 15 N values of the food source and temperature.Treatment 1 and 2 were similar in terms of δ 18 O values of the water Daphnia were cultivated in 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. 4).They may represent the inherent variability associated with stable isotope ratios in organisms (Schimmelmann, 2011).In previous experiments δ 13 C Daphnia and δ 15 N Daphnia values have been found to differ as much as 1 ‰ between identical treatments (Power et al., 2003).Moreover, the stable isotope ratios of the algae showed some variability over the course of the experiment (Fig. 2).Therefore, a slight difference in timing in the buildup of biomass may have led to small differences in Daphnia stable isotope ratios.The differences in Daphnia stable isotope ratios were much larger when Introduction

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The food experiment: changing δ 13 C algae
Offering Daphnia algae with on average 1.8 ‰ lower δ 13 C algae values resulted in 1.5 to 2.1 ‰ lower δ 13 C Daphnia values.Since the δ 13 C algae values were variable over time, we cannot reconstruct the exact δ 13 C value of the carbon that Daphnia in our different treatments assimilated, and therefore cannot calculate a precise estimate of ∆ 13 C.
Based on the mean δ 13 C algae value over the duration of the experiment, however, ∆ 13 C between Daphnia and algae is estimated to be +0.5 ± 0.3 ‰ at 12 • C.This is in agreement with commonly found ∆ 13 C 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 ∆ 13 C 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 δ 13 C values based on C : N ratios following a model by McConnaughey and McRoy (1979).This leads to relatively higher δ 13 C 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 δ 13 C values they measured prior to lipid correction.at 12 • C δ 13 C ephippia was 0.7 ± 0.2 ‰ higher than the mean δ 13 C algae .The absence of a clear offset in δ 13 C 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 chironomid body tissue and chironomid head capsules (∼ 1 ‰; Heiri et al., 2012;Frossard et al., 2013).

δ 15 N values of Daphnia and ephippia
At 12 • C, the observed ∆ 15 N was +3.4 ± 0.3 ‰, which agrees well with ∆ 15 N values referred to in the literature (+3 to +4 ‰, DeNiro and Epstein, 1981;Minagawa and Wada, 1984).A range of ∆ 15 N values for Daphnia have been reported.D. pulicaria reared on a diet of frozen algae pellets had a ∆ 15 N of +1.4 ‰ (Matthews and Mazumder, 2008).This is lower than the ∆ 15 N we found.According to Matthews and Mazumder (2008), the low ∆ 15 N they observed may be explained by the observation that a diet consisting of detritus (dead algae) is associated with considerably (∼ 2.5 ‰) lower ∆ 15 N values than one consisting of living plant matter (Vanderklift and Ponsard, 2003).Our observed ∆ 15 N for D. pulicaria is within the range of reported D. magna ∆ 15 N values (+1 to +6 ‰ ; Adams and Sterner, 2000;Power et al., 2003).δ 15 N ephippia values were lower (1.6 ± 0.4 ‰) than δ 15 N Daphnia values.In contrast, Perga (2011) found δ 15 N ephippia values to be slightly, but not significantly lower than δ 18 O water values were 5.2 ‰ higher in Treatment 3 than in Treatment 1 and 2, and the mean δ 18 O Daphnia 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 δ 18 O Daphnia values reflect differences in δ 18 O water , 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 56-69 % 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).δ 18 O ephippia values closely reflected differences in δ 18 O Daphnia : they were on average 0.9 ± 0.4 ‰ lower than δ 18 O Daphnia values.This suggests that δ 18 O ephippia may be used as an indicator of δ 18 O Daphnia , which in turn can be expected to be related to lake water δ 18 O values.This is in agreement with the correspondence between surface sediment δ 18 O ephippia values and lake water δ 18 O values found in a field survey of a number of European lakes (Verbruggen et al., 2011).(+0.5 ± 0.3 ‰).While we cannot exclude a negative relation between temperature and ∆ 13 C values for Daphnia, we choose to treat this result with caution due to the discrepancy with the positive ∆ 13 C 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 δ 13 C Daphnia 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, 13 C-depletion of algal biomass during dark respiration may have affected the δ 13 C algae in Treatment 4 disproportionally due to the higher temperature.Degens et al. (1968) found that δ 13 C values of the alga Dunaliella teriolecta were 4 ‰ lower after three days in darkness.The rate of respiration by algae depends on temperature and can be 2 to 4 times higher at 20

The temperature experiment
• C than at 12 • C (e.g.Vona et al., 2004).Microbial   2011) reported air temperature, and differences in air temperature at lakes 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. 6), and the ephippia stable isotope ratios Figures responded to the manipulations in δ 13 C algae and δ 18 O water we performed.Studies investigating the δ 13 C and δ 15 N values of fossil Daphnia ephippia have recorded shifts up to 5-10 ‰ in δ 13 C values (Wooller et al., 2012;Frossard et al., 2014) and 3 ‰ in δ 15 N values (Griffiths et al., 2010).Shifts of 2-3 ‰ in δ 18 O values have been reported for fossil chironomid head capsules (Wooller et al., 2004;Verbruggen et al., 2010b).
In our experiment, the SD 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 δ 13 C, δ 15 N and δ 18 O (±0.8 ‰ for δ 13 C when including Treatment 4 at 20 Discussion Paper | Discussion Paper | Discussion Paper | and ranges from −0.8 to +1.2 ‰.For Daphnia magna, Discussion Paper | Discussion Paper | Discussion Paper |

δ
13 C and δ 18 O values of Daphnia.The experiment was specifically designed to examine whether offsets in δ 13 C, δ 15 N and δ 18 O 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.
3).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 follow-Discussion Paper | Discussion Paper | Discussion Paper | (a) the effect of a change in algal δ 13 C values on those of Daphnia and their ephippia (Treatment 2), (b) the effect of a change in environmental water δ 18 O 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 δ 13 C, δ 15 N and δ 18 O values of Daphnia and their ephippia (Treatment 4), and (d) the offset between Daphnia and ephippia in terms of their δ 13 C, δ 15 N and δ 18 Discussion Paper | Discussion Paper | Discussion Paper | Screen / Esc Printer-friendly Version Interactive Discussion Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper |

δ
18 O water(A) − δ 18 O water(B)(1)    where p is the proportion of oxygen in Daphnia stemming from the water, δ18 O Daphnia(A) and δ 18 O water(A) are the δ 18 O values of Daphnia and the water if Daphnia were cultivated in non-manipulated, filtered lake water, and δ 18 O Daphnia(B) and δ 18 O water(B) the δ 18 O values of Daphnia and the water if Daphnia were cultivated in the 18 O-enriched, filtered lake water.2.6 Stable isotope mass spectrometry The δ 13 C and δ 15 N values of the algae, Daphnia and ephippia were measured on a Costech ESC 4010 elemental analyzer interfaced via a ThermoConflo III to a Thermo Delta V isotope ratio mass spectrometer (IRMS) at the Alaska Stable Isotope Facility Introduction Discussion Paper | Discussion Paper | Discussion Paper | (ASIF) at the University of Alaska, Fairbanks.The analytical precisions for δ 13 C and δ 15 N 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 δ 18 O values of the water samples were measured on an on-line 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 δ 18 O 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 for δ 13 C values, AIR for δ 15 N values and V-SMOW for δ 18 O values.
δ 13 C ephippia values also reflected the difference in δ 13 C algae values between the treatments.At 12 • C, they were not significantly different from the δ 13 C Daphnia 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 δ 13 C value of ephippia deposited in sediment traps was slightly, but not significantly higher than the δ 13 C value of Daphnia collected in the same lake.This suggests that δ 13 C ephippia values are a reliable indicator for changes in δ 13 C Daphnia values, and consequently for variations in δ 13 C values of Daphnia diet: Discussion Paper | Discussion Paper | Discussion Paper |

δ 15 N
Daphnia values in the field.Together withPerga's (2011) results, our data provide an indication that δ 15 N ephippia values are indicative of δ 15 N 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 δ 15 N values and head capsule δ 15 N values (−1 to +1 ‰) were observed over a large range of δ 15 N values(2.5-15‰;Heiri et al., 2012).Therefore, it seems likely that differences between Daphnia and ephippia δ 15 N values may also be similar across this δ 15 N range.
Power et al. (2003) reported an increase of 0.1 ‰ in ∆ 13 C 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 ∆ 13 C values for Daphnia in Treatment 4 (20 • C) to be similar to or slightly higher than in the other treatments (12 • C). ∆ 13 C values were clearly lower, however, in Treatment 4 (−0.2 ± 0.1 ‰) than in the other treatments Discussion Paper | Discussion Paper | Discussion Paper | 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 can be expected, they may have been assimilating carbon from algae with different δ 13 C algae values during the main phase of their growth compared to the other treatments, since δ 13 C algae values were relatively low in the beginning and at the end of the experiment (Fig. 2).δ 13 C ephippia values were also lower in Treatment 4, and 1.3 ± 0.3 ‰ lower than δ 13 C Daphnia 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 δ 13 C Daphnia and δ 13 C ephippia with temperature or whether it is affected by variations in δ 13 C algae and algal respiration rates or differences in Daphnia growth rates between our treatments.Controlled experiments over a range of temperature values analyzing not only δ 13 C Daphnia and δ 13 C ephippia values, but also δ 13 C values of respired CO 2 and microbial biomass would be desirable to further explore this issue.Although the results of Treatment 4 indicate that the difference between δ 13 C ephippia and δ 13 C Daphnia values may be more variable Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper |

Figure 1 .
Figure 1.Schematic representation of the experimental design used for culturing Daphnia.The top panel shows the four treatments and the bottom shows the setup of each treatment exemplified for Treatment 1.For each treatment, three replicate groups (A, B and C) and Daphnia pulicaria clones were distributed evenly among 30 experimental glass jars in a large tray.This was done so that each treatment replicate consisted of 10 jars with 3-4 jars for each clone.

Figure 6 .
Figure 6.The difference in δ 13 C, δ 15 N and δ 18 O 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 δ 13 C (see text).Error bars indicate SDs.
treated in 10 % KOH for 2 h to remove any algal matter and egg yolk.Replicate measurements (3 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): • C δ 13 C ephippia values were on average 0.2 ± 0.4 ‰ higher than δ 13 C Daphnia , although this difference was again not significant (paired t test, t 1.50, 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, δ18 O Daphnia values most likely primarily reflect environmental water δ 18 O values.The offset between δ 18 O ephippia and δ 18 O Daphnia in Treatment 4 (20 • C) • C, whereas δ 18 O ephippia 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 0.8-1.5 ‰ lower δ 18 O Daphnia values we found with an 8• C temperature rise.The data ofVerbruggen et al. (2011)and our experimental data would therefore be in agreement with a slight temperature effect on the fractionation of 18 O between lake water and Daphnia biomass.However, other mechanisms, such as a change in timing of Daphnia ephippia production with temperature and variations in δ 18 O values of food across the examined temperature gradient could also explain varying offsets between δ 18 O water and δ 18O Daphnia at different temperatures in the study ofVerbruggen et al. (2011).Moreover,Verbruggen et al. ( • 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 δ 18 O of the water they lived in.Although we only cultured Daphnia at two different temperatures, we found indications that temperature may have affected ∆