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Keywords:

  • compound-specific stable isotope analysis;
  • feeding experiment;
  • lipids;
  • trophic fractionation

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Material and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

1. Fatty acid–specific stable isotope analysis (FA-SIA) is expected to encompass most of the limits encountered when using more classical trophic markers such as bulk tissue stable isotope or fatty acid analyses. However, an essential premise for using FA-SIA for trophic studies is that the individual FA δ13C values of the consumer reflect those of its diet. Field studies using FA-SIA have so far made this assumption, which is not necessarily supported by the rare experimental tests.

2. A feeding experiment was conducted on Daphnia to test whether the δ13C values of individual fatty acids in Daphnia were actually related to those of its food.

3. Only the stable isotope composition of polyunsaturated (PUFA) and branched fatty acids (BrFA) was globally transmitted from the diet to Daphnia lipids, with however a significant isotope fractionation that varied depending on the considered diet source.

4. A model was constructed to evaluate how such variability may affect the reliability of FA-SIA to track the dietary sources of consumers’ PUFA and BrFA in the field. Results suggest that provided the endmembers are substantially isotopically different, FA-SIA could provide valuable insights into the pathways conveying these particular FA to consumers in the field.


Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Material and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

Trophic markers, such as bulk tissue stable isotope analysis (SIA), or fatty acid trophic markers (FATM), have been widely employed to track the origins and document the pathways conveying nutrient or organic molecules sustaining the secondary production of food webs (Alfaro et al. 2006; Perga et al. 2006). Both methods are based on the two same premises: (i) potential basal sources have distinct signatures and (ii) the source signature is predictively transmitted from the food to the consumer. Both methods however encounter the same limits as these premises are not necessarily met. In aquatic food webs for instance, some fatty acids (FA) had been identified as specific of certain algal classes in laboratory experiments but further field studies tend to show that they can be shared by various distinct taxa, therefore limiting the source specificity of these FA as trophic biomarkers (Bec et al. 2010). There are also many examples of SIA in which the aquatic and terrestrial endmembers could not be discriminated from their δ13C values (France 1996). Another issue concerns the adequate measurements of the endmembers. Phytoplankton stable isotope signature has rarely been measured directly (Grey, Jones, & Sleep 2000) as phytoplankton cells are difficult to isolate from other particles (bacteria, heterotrophic protists and detritus of different origins) that compose the bulk particulate organic matter. In addition, in SIA studies, phytoplankton is usually tacitly considered as a single, isotopically homogenous dietary source but the isotopic heterogeneity within the phytoplankton community was recently shown to be a potential source of overestimation of terrestrial C contribution to lake food webs (Perga, Kainz, & Mazumder 2008).

Many physiological processes were also shown to limit the condition of the predictive transmission of the source signature during trophic transitions. For SIA, trophic fractionation factors can be highly variable between species or dietary modes (Post 2002; McCutchan et al. 2003). Similarly, bioconversion of some FA from other FA precursors, as well as de novo biosynthesis, can impede the success of FATM approaches (Bec et al. 2003; Brett, Müller-Navarra, & Persson 2009).

Some of the biases related to the source specificity of the signature might be solved resorting to fatty acid–specific stable isotope analyses (FA-SIA; Pel, Floris, & Hoogveld 2004; Pel, Hoogveld, & Floris 2003). Indeed, two endmembers might share some of their marker FA: for instance, sea ice algae and pelagic diatoms, the two end-members of the study by Budge et al. (2008), shared high levels of the diatom FA biomarkers, i.e. 16:4(n-1) and 20:5(n-3), but these FA were shown to exhibit differing δ13C values and then discriminating them. Therefore, compound-specific stable isotope analysis is expected to provide a greater specificity to biomarkers (Evershed et al. 2007) and to encompass the difficulty to isolate single members from the bulk particulate organic matter (Pel, Hoogveld, & Floris 2003).

However, the second essential premise for using FA-SIA as trophic biomarkers in the field is, as for classical SIA, that the isotope ‘signal’ is actually transmitted from the diet to the consumer, i.e. that the individual FA δ13C values of the consumer reflect those of its diet. Field studies using FA-SIA on zooplankton have so far made this assumption (Budge et al. 2008). However, experimental tests of this essential assumption are still rare and restricted to a limited number of arthropod taxa (Chamberlain et al. 2004; Pond, Leakey, & Fallick 2006; Lau, Leung, & Dudgeon 2009). In addition, these rare experiments did not necessarily support this premise (Chamberlain et al. 2004; Pond, Leakey, & Fallick 2006; Lau, Leung, & Dudgeon 2009).

Actually, the FA δ13C values of the consumer are expected to be related to those of its diet if consumer’s FA are from dietary origin, i.e. if (i) the dietary FA are integrated within the consumer’s lipids with no further modification of their C-chain (i.e. elongation or desaturation) and (ii) the consumer’s FA are not the result of any de novo synthesis from non-lipid components. Although this assumption might not be validated for every FA and in all types of consumers, Daphnia might be an ideal candidate as: (i) earlier studies suggested that up to 98% of Daphnia FA were from dietary origins, with very limited de novo synthesis (Goulden & Place 1990), and (ii) Daphnia has very limited desaturase activities and might not be able to synthesize de novo (n-3) and (n-6) PUFA such as 18:2(n-6), 18:3(n-3) and 20:5(n-3) (von Elert 2002). For such reasons, it has been suggested that the δ13C values of these essential FA should be transmitted unchanged from the diet to the consumer (Stott et al. 1997).

In this study, we conducted an experiment in which Daphnia sp. were fed three protist food sources: since two were pigmented protists (the diatom Cyclotella sp. and the flagellate Rhodomonas lacustris) and the other a heterotrophic protist (the ciliate Cyclidium glaucoma), these food sources exhibited very distinct FA and FA δ13C compositions. The overarching aim of this paper was to test whether the δ13C values of individual FA in Daphnia were actually related to those of its food and to critically consider the improvements and beneficial knowledge of FA-SIA for future food web studies.

Material and methods

  1. Top of page
  2. Summary
  3. Introduction
  4. Material and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

Nutrition experiment

The pigmented flagellate (cryptophyte) Rhodomonas lacustris (12 μm long) and the centric diatom Cyclotella sp. (12 μm) were batch-cultivated in a modified Synura® medium (Vera et al. 2001). The ciliate Cyclidium glaucoma (18 μm long) was grown in the same medium enriched with milk powder (0·8 g L−1).

Daphnia sp. was isolated from zooplankton samples collected in Lake Annecy (France), cultured in spring water, and fed every other day with a 50/50 mixture of freeze-dried fish foods (Tetramin® + Tetraphyll®) broken down into fine particles by ultrasound.

Each nutrition experiment was conducted in triplicates using the following protocol: 3 × 120 neonates of Daphnia sp. (<12 h old) were placed in three glass tanks filled with 800 ml of spring water that was renewed every other day. The glass tanks were placed in a temperature-controlled chamber (20 °C) with a 12·12 h light/dark cycle. For the nutrition experiments, ciliate cells were separated from their medium by repeated centrifugations and resuspensions in fresh medium. Subsequently, the ciliate suspension was slowly filtered through a 5-μm membrane filter without vacuum, and retained cells were immediately resuspended from the filter into spring water. Food suspensions of the flagellate Rhodomonas lacustris and diatom Cyclotella sp. were obtained by centrifugation and resuspension of the cultured cells in spring water. Subsamples of the protists stock cultures were taken to estimate the number of cells in the food suspensions using a Sedgwick-Rafter chamber. Carbon concentrations offered to daphnids were estimated using the carbon conversion factors proposed by Menden-Deuer & Lessard (2000). The four food suspensions, i.e. the diatom Cyclotella sp., the flagellate Rhodomonas lacustris (1 mg C L−1), the ciliate Cyclidium glaucoma (1 mg C L−1) and the mixed diet composed of the flagellate Rhodomonas lacustris (0·5 mg C L−1) and ciliate Cyclidium glaucoma (0·5 mg C L−1) were added daily within the 10 days lasting experiment. Previous tests had shown that ciliates from the strain used in this experiment did not feed on the flagellate R. lacustris (unpublished results), hence limiting substantial changes in the ciliate FA content when present within the mixed diet as a result of predation on the flagellate. At the end of the experiment, daphnids were individually pipetted and placed on precombusted GF/A filters after being rinsed with clean spring water. Protists stock solutions were filtered on precombusted GF/C filters and stored at −60 °C before lipid analysis.

Lipid analysis

Lipids were extracted from triplicate samples in a mixture of chloroform/methanol (2:1, v/v; Folch, Les, & Stanley 1957). Fatty acid analyses were performed on total lipids (TL) extracted from protists and on neutral lipids (NL) and phospholipids (PL) from Daphnia sp. Lipid classes were separated by thin-layer chromatography on silica gel plates, with hexane/diethyl ether/methanol/acetic acid (90:20:3:2 v/v). Lipid classes were identified by comparison with commercial standards purchased from Sigma (Sigma-Aldrich, St Louis, Missouri, USA). Acylglycerols (mainly triacylglycerols and diacylglycerols) were grouped together with free fatty acids and sterols esters to constitute the neutral lipids. Fatty acid methyl esters (FAME) were prepared by hydrolysis and methylation. The lipid extract was maintained at 90 °C for 40 min in sealed tubes containing hexane and 2 N methanolic sodium hydroxide. The tubes were then cooled, and 2 N methanolic sulphuric acid was added. After 20 min at 90 °C and a short centrifugation, the supernatant was transferred to another tube and dried under nitrogen and the FAME stored at −40 °C in hexane. FAME were analysed on a Chrompack CP 9001 gas chromatograph connected to a recording integrator. The GC was equipped with a Chrompack CP7747 capillary column (25 m × 0·32 mm i.d., film thickness: 0·20 μm). The oven temperature was programmed to increase from 160 to 240 °C at a rate of 2·5 °C min−1. FAME were identified by comparison with known laboratory standards and commercial standards from Sigma (Sigma-Aldrich, St Louis, Missouri, USA) and Supelco (Bellefonte, Pennsylvania, USA). The concentration of total FA was estimated using a double internal standard (13:0 and 23:0), added as free fatty acids before derivatization. FA concentrations were expressed as per cent of total identified FA.

Fatty acid–specific stable isotope analyses

Isotopic analyses of individual FAME were carried out under a continuous helium flow using an HP 5890 gas chromatograph coupled with a CuO furnace (850 °C) and a cryogenic trap (−100 °C) coupled with a VG Optima mass spectrometer, monitoring continuously ion currents at m/z = 44, 45 and 46. The gas chromatograph was installed with a BPX70 column (60 m length, 0·32 mm internal diameter, 0·5-μm-film thickness). Oven temperature rose from 50 to 260 °C at 3 °C and held for 40 min. All δ13C values were calibrated against CO2 standards previously calibrated against Pee Dee Belemnite standard. A mix of 4 FAME (C12:0, C14:0, C16:0 and C18:0), purity of which had been previously checked by GC and individual δ13C measured by EA-IRMS, was used as reference material. Analytical precision, determined from consecutive runs of the reference material over the whole range of linearity of the isotopic source, was <0·3‰. Each sample was run three times, and values were averaged. FAME δ13C were corrected for the methyl group addition during methylation according to the formula (1)

  • image(eqn 1)

where δ13CFAME and δ13CMeOH are the δ13C values of the measured FAME and methanol used during methylation, respectively. δ13CFA represents the δ13C of the given FA prior to methylation, and n is the number of carbon atoms in the (non-methylated) FA. For this study, the δ13C of the methanol used for the preparation of FAME was −45·3‰.

Data analyses

Results for per cent composition of all detected FA are summarized in Appendix 1. In the scope of this paper though, we focused only on the results for the most abundant FA, for which quantities and δ13C values could be measured simultaneously in diets and Daphnia lipids. These FA were namely three-saturated FA (SAFA: C14:0, C16:0 and C18:0), two mono-unsaturated FA [MUFA: C16:1(n-7) and C18:1(n-7)], three polyunsaturated FA [PUFA: C18:3(n-3), C18:4(n-3) and C20:5(n-3)] and the sum of branched FA (BrFA). FA compositions of food sources or Daphnia lipids were compared using pairwise comparisons with Bonferroni-adjusted significance levels.

Changes in the δ13C value of a given FA between diet and Daphnia lipids (Δ13CFAi.j.) were calculated for each individual FAi, for each individual diet j, as the differences in the δ13C values of FAi between Daphnia13CFA.Daphniai.j) and its diet j13CFAi.j) (2):

  • image(eqn 2)

Results

  1. Top of page
  2. Summary
  3. Introduction
  4. Material and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

Fatty acid compositions

The three diet sources exhibited significant differences in their per cent composition of the nine selected FA (Fig. 1). Yet, the relationships in FA patterns between Daphnia and their dietary source depended on the considered FA. Daphnia lipid contents in 16:0 and 18:0 were rather independent from the contents of these FA in the dietary source. Indeed, the diatom food source had significantly higher amounts of 16:0 and 18:0 than the two other diet sources. Daphnia fed diatom had yet rather similar amounts of 16:0 and 18:0 as in the other treatments. For the seven other FA though, Daphnia FA composition exhibited a marked dietary influence. In addition, Daphnia fed on the mixed diet (flagellate + ciliate) showed a composition in those FA that was intermediate between those of Daphnia fed each of these individual food sources. The adequacy between diet and Daphnia FA composition was yet lower for MUFA and 14:0 than for BrFA and PUFA. Hence, Daphnia fed a food source in which MUFA or 14:0 was particularly abundant [16:1(n-7) and 14:0 in diatoms, 18:1(n-7) in ciliates] would exhibit higher abundances of this given FA in its NL or PL. However, Daphnia could still exhibit substantial amounts of these FA in their lipids even when supplied at very low amounts by the diet [for instance, 14:0, 16:1(n-7) and 18:1(n-7) in flagellates]. In contrast, BrFA were present in Daphnia lipids only when supplied by the diet (the ciliate C. glaucoma). The PUFA composition of the dietary source was also clearly reflected in that of Daphnia NL, but the adequacy of PUFA patterns between dietary and Daphnia PL was somewhat lower. For instance, although 20:5(n-3) was c. 15 times less abundant in ciliates than in flagellates and diatoms, amounts of this FA in Daphnia PL were similar in all the treatments.

image

Figure 1.  Error plots (average ± standard deviation) of some fatty acids (FA) contents for the food sources (Diatom: Cyclotella sp., Flagellate: Rhodomonas lacustris, Ciliate: Cyclidium glaucoma) and neutral lipids (NL) and phospholipids (PL) of Daphnia fed these food sources in the nutrition experiment [D-Diatom: Daphnia fed the diatom Cyclotella sp., D-Flagellate: Daphnia fed the flagellate R. lacustris; D-Ciliate: Daphnia fed the ciliate C. glaucoma; and D-mixed: Daphnia fed the mixed diet (50% Flagellate; 50% Ciliate)].

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Stable carbon isotope ratios of individual FA

Average δ13C value of FA in the flagellate R. lacustris was −32·9‰ (SD = 2·8‰), with a minimal value of −35·8‰ for 22:6(n-3) and a maximal value of −29·2‰ for 18:0 (Fig. 2a, Appendix 2). The average δ13C value of FA in the diatom Cyclotella sp. was relatively similar to that of the flagellate R. lacustris (−31·8‰; SD = 1·4‰), with a minimal value of −33·2‰ for 18:4(n-3) and a maximal value of −27·9‰ for 18:0. FA δ13C values for the ciliate C. glaucoma were much less depleted, with an average value of −22·3‰ (SD = 1·6‰) and values were ranging from −25·6‰ [18:2(n-6)] to −20·1‰ (14:0). The intermolecular variability in FA δ13C values within a single food source ranged from 4·8‰ (ciliate) up to 6·6‰ (flagellate).

image

Figure 2.  δ13C values (average ± standard deviation) for individual fatty acids (FA) for (a) the three diet sources, (b) neutral lipids (NL) and (c) phospholipids (PL) of Daphnia fed the three individual and the mixed diet sources.

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Consistently, FA of Daphnia fed ciliates were enriched in 13C by 6–7‰ compared with those of Daphnia fed the autotrophic protists (Fig. 2b,c). In addition, FA δ13C values of Daphnia fed the mixed diet ranged between those of Daphnia fed to two individual food sources (Fig. 2b,c). However, looking closer to the data, isotope differences for individual FA, Δ13CFAi.j, were highly variable between diets and FA (Fig. 3). For instance, Δ13C18:1(n-7) ranged from −5‰ (for PL of Daphnia fed ciliates) up to +1‰ (for PL of Daphnia fed diatoms). Δ13CFAi.j was also highly variable between FA of Daphnia fed a single food source. For instance, for Daphnia fed flagellates, Δ13CFAi.j could range from −4‰ (14:0, 16:0 and 18:0) to +1‰ [18:1(n-7)]. Δ13CFAi.j values were more similar between NL and PL of Daphnia fed a single diet source.

image

Figure 3.  Differences in fatty acids (FA) δ13C values between diet and Daphnia’s individual neutral lipids (NL) and phospholipids (PL) FA for the three diet sources.

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Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Material and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

With the increased use of SIA on bulk material to document trophic interactions within terrestrial and aquatic food webs, a number of confounding factors that might complicate data interpretation, such as isotope routing and turnover, or isotope heterogeneity of the endmembers, have been highlighted. Recent advances in gas chromatography–combustion–isotope ratio monitoring mass spectrometry now allow the measurements of the stable isotope compositions of specific compounds such as fatty acids, and the increased availability of such measurements is expected to help encompassing most of the problems that could be encountered when working with bulk material (Pel, Hoogveld, & Floris 2003). However, experimental studies are still required to document which information compound-specific stable isotope analyses could be expected to provide.

FA origins in Daphnia lipids

Not much is known about lipid metabolism and pathways in Daphnia (Brett, Müller-Navarra, & Persson 2009). In their seminal study, Goulden & Place (1990) measured fatty acid accumulation and synthesis rates of Daphnia pulex and D. magna when fed isotopically labelled chlorophycea Ankistrodesmus falcatus. According to their results, in their experimental context, at least 98% of the total lipids, and virtually all of the storage lipids, were derived from the diet. They suggested that storage lipid accumulation rates in daphniids were strongly regulated by the availability of dietary lipids. Overall, our feeding experiment is in line with Goulden & Place’s (1990) conclusions as NL FA composition was shown to depend more on the diet than PL. Such results suggest that NL might be more suitable for FATM approaches in the field than PL or even total lipids.

Since Goulden & Place’s (1990) study however, only one similar labelling experiment has been performed (Bychek et al. 2005). Most of our knowledge on Daphnia lipids was obtained through supplementation experiments (von Elert 2002) or controlled feeding experiments (Bec et al. 2003; as we did herein), in which changes in Daphnia fatty acid compositions in relation to dietary fatty acids were monitored. Feeding experiment studies have shown that diet has a very strong impact on the FA composition of some zooplankton (and in particular Daphnia spp.; reviewed in Brett, Müller-Navarra, & Persson 2009). Supplementation experiments identified (n-3) PUFA as essential fatty acids as for most animals (von Elert 2002; Ravet, Brett, & Muller-Navarra 2003). Studies on Daphnia lipids have focused on the physiological role of these (n-3) PUFA on Daphnia growth and fecundity (Weers & Gulati 1997; Ravet, Brett, & Muller-Navarra 2003), on the impact of environmental factors on their accumulation in Daphnia, such as temperature (Masclaux et al. 2009) and, more generally, on (n-3) PUFA pathways within aquatic food webs (Perga, Bec, & Anneville 2009). The physiological roles of the other FA that can be synthesized de novo have been much less documented. In addition, so far, poor efforts have been dedicated to the study of synthesis and conversion pathways in Daphnia and zooplankton in general (Brett, Müller-Navarra, & Persson 2009).

Our results are actually consistent with previous studies (Weers, Siewertsen, & Gulati 1997; Brett et al. 2006), showing that Daphnia FA composition varied generally according to the diet they were fed, although amongst the nine FA on which we focused, not all were from dietary origins (i.e. FA directly transmitted from the food to the consumer’s lipids with no further changes in the C backbone). Typically, amounts of C16:0 and C18:0 in Daphnia NL varied between 15–20% and 3–6%, respectively, but such differences were not dependent on the amounts of these same FA in Daphnia diet sources. Furthermore, proportions of 16:0 and C18:0 were constant in Daphnia PL (20% and 7%, respectively), whatever were the amounts of these FA in the dietary source, confirming that their amounts in Daphnia tissues, and especially in cell membrane, are regulated by active physiological processes, consistently with Bychek et al.’s (2005) observations. 14:0 and the MUFA 16:1(n-7) and 18:1(n-7) were more likely at least partially from dietary origins as Daphnia that were fed with diets rich in these FA exhibited the highest proportions of these same FA in their own lipids. However, differences in the amounts of these FA were not as large in Daphnia as in their respective diet sources, implying that differential accumulation, de novo synthesis or bioconversion occur when these lipids are supplied in limited amounts by the diet (Sargent and Henderson 1986). For instance, the flagellate R. lacustris was rich in 16:1(n-7) but very depleted in 18:1(n-7) compared with the other food sources; however, amounts of 18:1(n-7) in Daphnia fed the flagellate were relatively high as this FA might arise from the elongation of the abundant dietary 16:1(n-7). At last, BrFA and PUFA were clearly of dietary origins because dietary differences in the amounts of these FA were clearly reflected in Daphnia PL and NL. BrFA could not be detected in Daphnia fed diet sources that had not such FA, clearly highlighting that Daphnia cannot synthesize de novo these FA typical for heterotrophic microbes (Ederington, McManus, & Harvey 1995). Differences in the contents of (n-3) PUFA in Daphnia lipids, and especially in NL, were related to those between diets. The ciliate diet source was distinct from the other diet sources because of its relative depletion (about 10 times lower) in FA from the (n-3) series. Nevertheless, lipids, and especially PL, of Daphnia fed ciliates exhibited amounts of 20:5(n-3) that were not that much lower than lipids of Daphnia fed the other diet sources. 18:3(n-3) has been documented as a potential precursor for 20:5(n-3) (von Elert 2002; Bec et al. 2003) but as the ciliate food source was also poor in this shorter PUFA, it is more likely that these relatively high amounts of 20:5(n-3) arise from preferential retention of this peculiar FA (Kainz et al. 2009).

Relationships of FA δ13C values between diet and Daphnia

Our estimates of changes in δ13C between dietary and Daphnia FA might be affected by some experimental biases related to FA turnover in Daphnia. Indeed, phytoplankton cultured in batch conditions might undergo significant and relatively quick changes in their δ13C and FA δ13C values, because chemical conditions in batch cultures, especially pH and nutrients, can change over the course of the experiment (van Dongen, Schouten, & Damste 2002; Pond, Leakey, & Fallick 2006). The FA δ13C values of the food sources, which were actually measured after 10 days, could differ from those at the beginning of the feeding experiment by several per mil. Depending on the turnover of FA in Daphnia, Daphnia FA δ13C values could reflect those of their diet over a longer-integrated time period. As a result, the apparent difference in the FA δ13C values between Daphnia and its diet could be, at least partly, the consequence of these differences in FA turnover rates between the protists and their consumers. However, Daphnia grown under experimental conditions with food supplied at optimal rates will increase its mass by a factor 10 in 6 days (Brett, Müller-Navarra, & Persson 2009), which, in our context, guarantees a full FA turnover over the duration of the experiment. In addition, earlier feeding trials using 14C-labelled diets showed that lipid, and especially FA, turnovers in Daphnia NL and PL are very quick and that Daphnia FA C-isotope values reach equilibrium with those of its food within 24H (Farkas, Kariko, & Csengeri 1981; Goulden & Place 1990; Bychek et al. 2005). Hence, such experimental biases are very likely to have only minor effects on our results.

Although highly variable between FA and diets, FA in Daphnia lipids were generally 13C-depleted compared with their counterpart in the corresponding diet, although previous results on Collembola and shrimps showed that these differences, usually large, can be either positive or negative (Chamberlain et al. 2004, 2006a,b; Lau, Leung, & Dudgeon 2009).

Large isotope differences between the diets’ and the consumers’ lipids are expected for FA that might be synthesized de novo or result from the elongation or desaturation of FA precursors. Indeed, de novo synthesis from non-lipid dietary constituents involves the enzymatic oxidation of pyruvate to acetyl-coenzyme A during which significant selection of the lighter pyruvate occurs. Such processes result in FA 13C-depleted compared with other non-lipid constituents (De Niro & Epstein 1977) and, potentially, with the diet FA. Because 14:0, 16:0, 18:0, 16:1(n-7) and 18:1(n-7) might arise at least partly from de novo synthesis, they could be expected as more 13C-depleted in Daphnia lipids when a higher proportion had to be synthesized de novo. However, no relationships between Δ13C and FA dietary concentrations were detected for these FA. Such results might suggest that de novo synthesis proceeds from different precursors depending on the dietary composition. As elongase and desaturase enzymes preferentially use the lighter precursor, FA derived from elongation and/or desaturation processes in the consumer lipids were also expected to exhibit lower δ13C values than the dietary FA precursors (Monson & Hayes 1982). According to the mass–balance rule, the remaining pool of precursor FA in the consumer should be enriched in 13C compared with the dietary ones. As mentioned previously, 18:1(n-7) in Daphnia fed Cyclotella sp. might result from the elongation of 16:1(n-7) that was abundant in this dietary source, but contrary to our expectations, 18:1(n-7) δ13C value was higher than that of 16:1(n-7) in both the diet and the consumer. Such results imply that FA synthesis and conversion pathways are complex and that further researches are deserved to interpret δ13C patterns in Daphnia non-essential FA.

Significant differences in the isotope values between diet and Daphnia lipids were also observed for FA that are only from dietary origins, such as BrFA and PUFA. For such FA that are transmitted directly from the diet to the consumers, these isotopic differences can be considered as the result of trophic fractionation processes. The assumption that the metabolism of dietary FA, and especially essential FA, might not be associated with significant isotope fractionation is based on the results of a single feeding experiment that showed a close similarity of 18:2(n-6) δ13C values between the diet and bones of pigs (Stott et al. 1997). However, our results show that significant trophic fractionation may occur even for FA that are directly assimilated from the diet with no biosynthetic inputs from the consumer. Such a fractionation could result from assimilation, lighter compounds being preferentially assimilated (Chamberlain et al. 2006b). Fractionation processes could also occur during the FA integration within the consumer’s lipids. FA in organisms are usually present as esterified to a glycerol backbone under the form of triacylglycerol or phospholipids rather than under their free form. Although such FA from dietary origins do not undergo strong changes in their C-backbone structure, their integration within the consumer’s lipids involves several hydrolysis and esterification steps. Our results could suggest that the involved enzymes might catalyse these processes with significant isotope fractionation rates.

Although the analytical limits of the method did not allow to obtain δ13C values for PUFA for Daphnia fed all the dietary sources, results on 18:4(n-3) and 20:5(n-3) show that the isotope fractionation could vary over a 1–2‰ range even between two different food sources. Such variability in trophic fractionation factors for a single FA had been observed in previous experiments on Collembola (Chamberlain et al. 2004) and shrimps (Lau, Leung, & Dudgeon 2009). Similarly, FA δ13C values of zooplankton and fish could not be directly related to those of particulate organic matter in a marine field study conducted in the Pacific Ocean, off the West Coast of Vancouver Island (Veefkind 2003). It is difficult, in our experimental context, to provide any potential explanation for such patterns but it highlights the need for further experiments on that specific point. The variability in trophic fractionation factors might constitute a major fence to the use of FA-SIA to track FA origins in the field.

Applications to field studies

The use of FA-SIA in the field is expected to allow tracking the origins of dietary FA, i.e. estimating the contribution of different food sources to these FA in consumers’ tissues. For FA that were identified in this study from dietary origins (PUFA and BrFA), we backcalculated the contributions of each individual diet sources to these dietary FA in the lipids of Daphnia fed the mixed diet (50% flagellate, 50% ciliate). The contribution of the ciliate C. glaucoma (αFAiCiliate.Daphnia) and of the flagellate R. lacustris (1 − αFAiCiliate.Daphnia) to Daphnia dietary FAi was estimated resorting to a simple two-source mixing model. The isotope compositions of FAi in the individual dietary sources (δ13CFAiCiliate and δ13CFAiFlagellate) were used as endmembers. Trophic fractionation values were, for each FAi, averaged (±SD) between PL and NL and the diet sources (Δ13CFAi) as it is usually impossible to determine trophic fractionation values for all potential endmembers in the field (Table 1). Hence, from the δ13C values of FAi in Daphnia fed the mixed diet (δ13CFAiDaphnia), the contribution of the ciliate C. glaucomaCiliate.Daphnia) to FAi in Daphnia lipids was estimated as follows:

  • image(eqn 3)
Table 1.   Estimated trophic fractionation values of dietary FA (PUFA and BrFA) in Daphnia lipids, averaged over NL and PL and food sources
Dietary FAiΔ13CFAi (‰)95% confidence interval (‰)
15:0i−2·9[−3·3; −2·4]
15:0a−2·6[−3·2; −2·0]
18:3(n-3)−3·7[−5·3; −2·1]
18:4(n-3)−1·9[−2·8; −1·0]
20:5(n-3)−0·8[−2·3; 0·8]

These estimates were compared with the contribution of the ciliate diet source to the same dietary FAi in the mixed diet (αFAiCiliate.mixed diet), based on the content of the two individual food source in FAi (αFAiCiliate and αFAiFlagellate) (4)

  • image(eqn 4)

Under the assumption that, as non-selective filter-feeders, Daphnia grazed and assimilated both protists in the mixed diet with identical efficiencies (Burns 1968), the contribution of ciliates to FAi to the mixed diet and to Daphnia lipids could be considered as similar.

In the mixed diet, the ciliate C. glaucoma provided 100% of the BrFA C15:0i and C15:0a, but contributed only poorly to the PUFA of the mixed diet [9%, 14% and 7% of 18:3(n-3), 18:4(n-3) and 20:5(n-3), respectively, Fig. 4]. Consistently, the two-source mixing model detected that the ciliate C. glaucoma provided BrFA but contributed only poorly to the (n-3) PUFA in Daphnia’s lipids, which were rather provided by the flagellate food source.

image

Figure 4.  Contribution of the ciliate Cyclidium glaucoma to the fatty acids (FA) content of Daphnia fed the mixed diet (50% flagellate and 50% ciliate); closed bars, calculated from the diet sources FA contents; open bars, backcalculated from the two-source mixing model (average ± confidence interval).

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Our results suggest that the stable isotope composition of PUFA and BrFA is globally transmitted from the diet to Daphnia lipids, with however a significant isotope fractionation (−4 to −1‰). Isotope fractionation factors for PUFA and BrFA varied depending on the considered diet source. Future studies should investigate which factors are driving such variability, to increase the predictability of trophic fractionation factors. More interestingly, it should be investigated whether isotopic fractionation operates at a molecular level during FA hydrolysis and esterification steps and – if it actually occurs – whether it depends on the physiological importance of the given FA for the consumer.

However, the variability in isotope fractionation observed at the individual FA level is finally quite comparable to that observed for discrimination factors at the bulk level between Daphnia and its diet (Power, Guiguer, & Barton 2003). Hence, in spite of such a variability observed for PUFA and BrFA trophic fractionation factors, FA-SIA might still be useful in the field, provided the potential dietary sources exhibit high differences in their isotope composition (c. 10‰ in our study). This is under such a premise that Chamberlain et al. (2006a) studied Collembola trophic preferences, using C3- and C4-derived organic matter as endmembers. Provided the endmembers are substantially isotopically different, FA-SIA could provide valuable insights into the pathways conveying certain FA, and especially essential FA, to consumers. Information potentially provided by FA-SIA are however not equivalent but complementary to those provided by SIA. While SIA would allow assessing the contribution of a dietary source to the consumer’s C biomass (i.e. secondary production), FA-SIA would decipher which of the dietary sources are the major providers of some FA (and especially essential FA). Hence, the combined use of SIA and FA-SIA would be useful to document an uncoupling between essential compounds and major organic matter transfers, as it was shown in heterogeneous environments (Koussoroplis et al. 2010).

Acknowledgements

  1. Top of page
  2. Summary
  3. Introduction
  4. Material and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

We thank both reviewers, such as Gabe Bowen, associate editor, for their useful comments on this work. Funding for this study was provided for by the ‘IXème Contrat Plan Etat Région’ from the ‘Région Rhône Alpes’ and the French ministry of Research. Special thanks to Ruben Veefkind, especially for interesting discussions on fatty acids’ isotopes ratios in aquatic food webs.

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  3. Introduction
  4. Material and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information
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Supporting Information

  1. Top of page
  2. Summary
  3. Introduction
  4. Material and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

Appendix 1. Percent composition in their main FA of the three individual diet sources and of the NL and PL of Daphnia fed the individual and mixed diets. Br = Branched FA; ND = not detected.

Appendix 2. FA δ13C values (in ‰, average ± standard deviation) of the three individual diet sources and of the NL and PL of Daphnia fed the individual and mixed diets. N.D. stands for not detected. For some FA, amounts were too limited to provide reliable δ13C values (B.L. stands for below detection limits).

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