Freshwater Biology (2012) 57, 696–703
doi:10.1111/j.1365-2427.2012.02735.x
Accumulation of polyunsaturated fatty acids by cladocerans: effects of taxonomy, temperature and food H E´ L E` N E M A S C L A U X * , †, A L E X A N D R E B E C * , †, M A R T I N J . K A I N Z ‡, F A N N Y P E R R I E` R E * , †, CHRISTIAN DESVILETTES*,† AND GILLES BOURDIER*,† * Clermont universite´, Universite´ Blaise Pascal, LMGE, Clermont-Ferrand, France † CNRS, UMR 6023, LMGE, Clermont-Ferrand, France ‡ WasserCluster Lunz – Biologische Station, Dr. Carl Kupelwieser Promenade 5, Lunz am See, Austria
SUMMARY 1. Zooplankton are important in transferring dietary nutrients, including polyunsaturated fatty acids (PUFA), up through aquatic food webs. 2. We tested the hypothesis that the taxonomic composition of zooplankton affects the retention and subsequent transfer of PUFA from upwards through the food web. Using laboratory experiments, we investigated dietary PUFA accumulation and bioconversion capacities of six cladoceran species (Ceriodaphnia sp., Daphnia longispina, Daphnia magna, Daphnia pulex, Scapholeberis mucronata and Simocephalus vetulus) fed on two diets (Scenedesmus obliquus and Cryptomonas sp.) that differed in their PUFA profiles. We performed experiments at two different temperatures (14 and 20 C) to assess the role of temperature in the trophic transfer of PUFA. 3. There was little variation in the concentrations of PUFA in these cladocerans which were controlled by dietary PUFA supply. Moreover, as expected, the concentrations of PUFA in all cladoceran species were higher at low temperature. 4. However, even if the composition of PUFA in the cladoceran species generally corresponded to that in their diet, preferential accumulation of some PUFA was recorded in all these taxa. When fed on a highly unsaturated fatty acid-deficient diet, all the cladocerans showed some ability to convert C18-PUFA into arachidonic acid and eicosapentaenoic acid. Interspecific variation in the ability to accumulate and bioconvert PUFA in cladocerans was more pronounced at low temperature (14 C) for both diets. 5. Our results strongly suggest that in heterogeneous habitats with food partitioning between co-existing cladocerans, foraging behaviour may affect the transfer of PUFA more strongly than interspecific variation in accumulating and ⁄ or bioconverting dietary PUFA. Keywords: food webs, trophic transfer, trophic up-grading, zooplankton richness
Introduction Aquatic consumers require essential dietary compounds that are conducive to their somatic development and overall fitness. Among such compounds, polyunsaturated fatty acids (PUFA) play a key role for physiological and biochemical processes (Sargent et al., 1999) and must be supplied in the diet as animals cannot biosynthesize PUFA de novo (at least at rates sufficient to meet physiological requirements) (Sargent et al., 1999; Arts, Ackman
& Holub, 2001). There is experimental evidence that dietary PUFA are beneficial (i.e. they improve somatic growth, reproduction and survivial) for freshwater zooplankton (Bec et al., 2003b; Ravet, Brett & Mu¨ller-Navarra, 2003) and fish (Copeman et al., 2002; Tocher, 2003), which makes trophic transfer of PUFA crucial for aquatic food webs. In aquatic systems, microorganisms such as algae and heterotrophic protists are major producers of PUFA (Brett & Mu¨ller-Navarra, 1997; Desvilettes & Bec, 2009) that can
Correspondence: He´le`ne Masclaux, Universite´ Blaise Pascal, Baˆtiment de Biologie A, 24 avenue des Landais, BP 80026, 63170 Aubiere Cedex, France. E-mail:
[email protected]
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2012 Blackwell Publishing Ltd
Accumulation of PUFA by cladocerans subsequently be retained in consumers and, in general, transferred conservatively along food chains (Kainz, Arts & Mazumder, 2004; Koussoroplis et al., 2011). As they constitute the major link between microorganisms and species higher in the food web, zooplankton play a key role in the transfer of PUFA to organisms such as fish. However, some recent studies have challenged the presumption that PUFA are transferred unmodified along food chains (Mu¨ller-Navarra, 2006; Persson & Vrede, 2006; Smyntek et al., 2008). These authors found taxon-specific differences in the PUFA composition of zooplankton, especially between cladocerans and copepods, which were not related to the PUFA composition of the edible seston. Ingested PUFA can be retained, metabolised or, to some extent, converted to other PUFA (a process called ‘bioconversion’). One might expect that zooplankton, even within the same taxon (such as the cladocerans), may differ in their PUFA accumulation, bioconversion and subsequent trophic transfer abilities. Therefore, in aquatic ecosystems with highly diverse zooplankton communities (Walseng et al., 2006), this diversity may affect the transfer of PUFA from microorganisms upwards through the food web. We investigated experimentally differences in the PUFA composition of six cladoceran species exposed to the same pool of dietary fatty acids (FA). They were fed on two algae differing in their FA composition: Cryptomonas sp., a diet rich in highly unsaturated fatty acids (HUFA; ‡20 carbon atoms and ‡3 double bonds), allowed us to test the capacity of the different cladoceran species to accumulate HUFA, whereas we assessed bioconversion capacity by feeding them on Scenedesmus, which is deficient in HUFA. It has also been shown that zooplankton retain more PUFA at lower temperatures (Schlechtriem, Arts & Zellmer, 2006) and that this maintains high membrane fluidity (Nishida & Murata, 1996). We thus performed feeding experiments at two temperatures, as we postulated that the different cladoceran species would have different biosynthetic abilities to adjust and maintain the PUFA necessary for survival at low temperature.
Methods Origin and maintenance of daphniids Six species of Daphniidae were used in the experiments: Ceriodaphnia sp., Daphnia longispina (O.F. Mu¨ller, 1776), Daphnia magna (Strauss, 1820), Daphnia pulex (Leydig, 1860), Scapholeberis mucronata (O.F. Mu¨ller, 1776), and Simocephalus vetulus (O.F. Mu¨ller, 1776). Daphnia magna, a species widely used in laboratory experiments 2012 Blackwell Publishing Ltd, Freshwater Biology, 57, 696–703
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(Dawidowicz & Loose, 1992; Mu¨ller-Navarra et al., 2000), were collected from a floodplain pond near the River Allier, France. The other species, among which S. mucronata and S. vetulus are common species in the littoral zone, were collected from a backwater of the River Allier, where they dominate the microcrustacean community. Females were maintained in glass containers (1 L) of ADaM medium (Klu¨ttgen et al., 1994) with a maximum density of 20 individuals L)1 and with a 13 : 11 h light : dark cycle, at 18 C for one month before the start of the experiment. They were fed daily ad libitum, using a mixture of Cryptomonas sp. strain SAG 26.80 and Scenedesmus obliquus strain SAG 276-3a (20%: 80% of respective biomass).
Cultures of autotrophic organisms Scenedesmus obliquus SAG 276-3a and Cryptomonas sp. SAG 26.80 were used as food for the six species of cladocerans. The two algae were grown in modified WC (Woods Hole modified CHU-10) medium with vitamins (Von Elert & Wolffrom, 2001) at 18 C and cultured semi-continuously at a dilution rate of 0.25 per day in aerated vessels (3 L). Stock solutions of the autotrophic organisms for the growth experiments were prepared by centrifuging and resuspending the cultured cells in WC medium lacking vitamins. The carbon concentrations in the food suspensions were estimated by photometric light extinction (800 nm) and previously determined carbon-extinction regressions (D. Martin-Creuzburg, pers. comm.).
Experimental setup Thirty individuals of each zooplankton species were randomly isolated from the population and transferred to glass containers (1 L) in ADaM medium with a 13 : 11 light : dark cycle for each treatment. Experiments were performed at 14 and 20 C with two food sources, resulting in a 2 · 2 factorial design for each species, with three replicates per treatment. The food sources were Cryptomonas sp. SAG 26.80, a HUFA-rich diet, and S. obliquus 276-3, a HUFA-deficient diet (Ahlgren et al., 1990; Brett & Mu¨ller-Navarra, 1997). During the experiments, individuals were transferred every second day to fresh medium and fed every day under non-limiting food conditions (2 mg C L)1, i.e. well above the incipient limiting level that is reported to be c. 0.5 mg C L)1; Lampert, 1978). The experiments were stopped after 10 days, allowing the turnover of cladoceran fatty acids (Fraser et al., 1989; Bychek et al., 2005). All individuals were collected, freeze-dried, weighed on a microbalance
698 H. Masclaux et al. (Mettler Toledo UMX2 balance ± 1 lg), and subsequently frozen at )80 C until further analysis. With Cryptomonas, there were between 30 and 1176 individuals in each container at the end of the experiment, and with Scenedesmus between 30 and 746, depending on the temperature and the cladoceran species considered.
Fatty acid analysis Fatty acid analyses of phytoplankton were performed on 2 mg triplicate samples of particulate organic carbon of Scenedesmus and Cryptomonas sp. filtered on pre-combusted GF ⁄ F filters (WhatmanTM, Maidstone, England). At the end of the feeding experiments, FA analyses were performed on the zooplankton in each container, i.e. in triplicates for each treatment. Lipids were extracted using chloroform ⁄ methanol, following the method of Folch, Less & Stanley (1957). Fatty acids of total lipid extracts were converted into fatty acids methyl-esters (FAME) after the addition of non-methylated 13 : 0 and 23 : 0 as internal standards. FAME were generated by acid catalysed trans-esterification according to a modified protocol of Christie (1982) (4% H2SO4 in methanol at 75 C for 2 h) and subsequently analysed on a gas chromatograph (Agilent technologiesTM 6850, Santa Clara, CA, USA) equipped with a DB-Wax column (J&W Scientific, Folsom, CA, USA), and a flame ionisation detector (250 C; split injection; carrier gas: helium; oven temperature ramp 150–240 C at 3 C min)1). FAME were identified by comparing retention times with those obtained from Supelco standards (Bellefonte, PA, USA) (37-Component
FAME mix, Bacterial FAME Mix) and laboratory standards (Cod liver oil FAMEs) and quantified using internal standards (13 : 0 and 23 : 0).
Data analysis Differences in FA concentrations among species, as well as FA concentration ratios of cladocerans reared at 14 and 20 C, were analysed by one-way analysis of variance (A N O V A ). Pair-wise comparisons were performed using a post hoc test [Tukey’s Honestly Significant Difference (HSD)] with the Bonferroni adjustment (a = 0.003). All data were log-transformed prior to analysis to meet the assumptions of normal data distribution.
Results PUFA of phytoplankton Scenedesmus was rich in C18-PUFA, with a-linolenic acid (ALA, 18:3n-3) and linoleic acid (LIN, 18:2n-6) concentrations being the most abundant PUFA (Fig. 1; see Table 1 for fatty acid abbreviations), but did not contain any HUFA; i.e. arachidonic acid (ARA; 20:4n-6), eicosapentaenoic acid (EPA, 20:5n-3), or docosahexaenoic acid (DHA, 22:6n-3). As was the case for Scenedesmus, Cryptomonas sp. was also rich in C18-PUFA concentrations, especially in ALA and stearidonic acid (SDA, 18:4n-3). However, PUFA concentrations of Cryptomonas sp. clearly differed from those of Scenedesmus by the presence of SDA, EPA and DHA (Fig. 1).
FA concentration (µg mg C–1)
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14
:0
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: 0 n-7 n-7 n-4 n-3 n-1 : 0 n-9 n-7 n-6 n-6 n-3 n-3 n-6 n-3 n-3 n-3 : 1 : 1 : 3 : 3 : 4 18 : 1 : 1 : 2 : 3 : 3 : 4 : 4 : 3 : 5 : 6 16 17 16 16 16 18 18 18 18 18 18 20 20 20 22
HUFA PUFA
Fig. 1 Fatty acid concentrations (+SD) of Scenedesmus and Cryptomonas. PUFA, polyunsaturated fatty acids; HUFA, highly unsaturated fatty acids.
2012 Blackwell Publishing Ltd, Freshwater Biology, 57, 696–703
Accumulation of PUFA by cladocerans Table 1 Nomenclature and abbreviations of the fatty acids used in the text Abbreviation
Full name
Structure
FA SAFA MUFA PUFA HUFA
Fatty acid Saturated fatty acid Monounsaturated fatty acid Polyunsaturated fatty acid Highly unsaturated fatty acid
LIN ALA SDA ARA EPA DHA
Linoleic acid a-linolenic acid Stearidonic acid Arachidonic acid Eicosapentaenoic acid Docosahexaenoic acid
– No double bond 1 double bond ‡2 double bonds ‡20 carbon atoms and ‡3 double bonds 18:2n-6 18:3n-3 18:4n-3 20:4n-6 20:5n-3 22:6n-3
Fatty acid concentrations of cladocerans fed on Scenedesmus At both temperatures, concentrations of LIN, SDA, ALA, and total PUFA were not significantly different among species, but slight differences were recorded in monoun-
saturated fatty acid (MUFA) concentrations (Fig. 2). Total saturated fatty acid (SAFA) concentrations were not different at 20 C, but some differences were recorded among species reared at 14 C (Fig. 2). Although EPA and ARA were not detected in Scenedesmus, these two HUFA were present in the six cladoceran species fed on Scenedesmus. However, ARA and EPA concentrations were significantly different among species at both temperatures. Scapholeberis had the highest concentrations of ARA and EPA at both temperatures (Fig. 2).
Fatty acid concentrations of cladocerans fed on Cryptomonas No significant difference in the concentrations of LIN, ALA, SDA, EPA and total PUFA were determined among the six cladoceran species fed on Cryptomonas at either temperature (Fig. 3). However, significant differences in SAFA and MUFA concentrations were found among the species. For ARA, concentrations were not significantly different when species were reared at 20 C, but were different when reared at 14 C. At this lower temperature,
FA concentration (µg mg DW–1)
Accumulated FA at 14 °C (a) a 80
Scapholeberis Ceriodaphnia Simocephalus D. longispina D. pulex D. magna
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Fig. 2 Fatty acid concentrations at (a) 14 C and (b) 20 C by cladoceran species fed on Cryptomonas. Data are mean concentrations (+SD; three replicates per treatment). Distinct letters for the comparisons of each fatty acid indicate a significant difference among cladoceran species (oneway A N O V A with a Tukey’s HSD tests at a = 0.003). SAFA, saturated fatty acids; MUFA, monounsaturated fatty acids; PUFA, polyunsaturated fatty acids; LIN, linoleic acid (18:2n-6); ALA, a-linolenic acid (18:3n-3); SDA, stearidonic acid (18:4n-3); ARA, arachidonic acid (20:4n-6); EPA, eicosapentaenoic acid (20:5n-3). 2012 Blackwell Publishing Ltd, Freshwater Biology, 57, 696–703
700 H. Masclaux et al. Scapholeberis Ceriodaphnia
FA concentration (µg mg DW–1)
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Accumulated FA at 14 °C aa b
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0 SAFA
MUFA
PUFA
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0.4
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a
a a b bb b
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aa a bab a b bb
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EPA
Fig. 3 Fatty acid concentrations at (a) 14 C and (c) 20 C and fatty acids (FA) conversion at (b) 14 C and (d) 20 C by cladoceran species fed on Scenedesmus. Data are mean concentrations (+SD; three replicates per treatment). Distinct letters for the comparisons of each FA indicate a significant difference among cladoceran species (one-way A N O V A with a Tukey’s HSD tests at a = 0.003). Fatty acid abbreviations are as in Fig. 2.
Scapholeberis had the highest ARA concentrations (Fig. 3). Even though SDA concentration in Cryptomonas was up to 19 times that in Scenedesmus (Fig. 1), the concentrations in cladocerans feeding on Cryptomonas were only 4–12 times higher at 14 C, and 5–11 times higher at 20 C, than in cladocerans feeding on Scenedesmus (Figs 2 & 3). Similarly, ALA concentrations were almost three times higher in Cryptomonas than in Scenedesmus, but quite similar in cladocerans feeding on Cryptomonas or Scenedesmus.
Effect of temperature on fatty acid concentrations of cladocerans Except for LIN, ARA and EPA concentrations in D. longispina fed on Scenedesmus, and ARA concentrations in Ceriodaphnia fed on Cryptomonas, all FA concentration ratios based on the two temperatures (FA14 C ⁄ FA20 C) were >1, indicating that cladocerans retained more FA at the lower temperature (Fig. 4). However, differences in FA concentrations between 14 and 20 C varied according to FA, cladoceran species and food source. When fed on Cryptomonas, differences in LIN, ALA, EPA and PUFA accumulation between 14 and 20 C were not significantly different among these cladoceran species. Some slight
differences were detected among SAFA and MUFA, as well as in SDA and ARA concentrations between the two temperatures. When cladocerans were fed on Scenedesmus, significant differences in FA concentrations between 14 and 20 C were recorded for all the FA and FA groups except ALA (Fig. 4).
Discussion It is still uncertain how zooplankton species vary in their ability to modify their PUFA profiles relative to their dietary PUFA. Whatever the cladoceran species and the temperature, our results showed that cladoceran PUFA composition varied with diet. The high concentrations of ALA, SDA, and EPA in cladocerans fed on Cryptomonas clearly reflected the PUFA profile of their food source. The same was true for cladocerans fed on Scenedesmus that contained high ALA concentrations. There is laboratory evidence that PUFA concentrations in Daphnia generally match those in their diet (Brett et al., 2006; Burns, Brett & Schallenberg, 2011), although it evidently has some intrinsic ability to modify dietary PUFA (Weers, Siewertsen & Gulati, 1997; Von Elert, 2002; Bec et al., 2003a). Our study corroborates these results by showing that the six 2012 Blackwell Publishing Ltd, Freshwater Biology, 57, 696–703
Accumulation of PUFA by cladocerans Scapholeberis
Simocephalus
D. pulex
Ceriodaphnia
D. longispina
D. magna
8 (a) Scenedesmus
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FA14 °C/FA20 °C
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a
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MUFA
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Fig. 4 Fatty acid concentrations ratios of cladocerans reared at 14 C (FA14) and 20 C (FA20) on, (a) Scenedesmus, and, (b) Cryptomonas. The error bars represent ±SD. Bars labelled with the same letters are not significantly different (Tukey’s HSD, a = 0.003). Values >1 represent increased accumulation of the respective fatty acids in cladocerans fed at 14 C compared with cladocerans fed at 20 C. Fatty acid abbreviations as in Fig. 2.
cladocerans accumulated dietary PUFA and, in addition, contain non-dietary HUFA such as ARA and EPA. The retention of non-dietary HUFA in all of these cladocerans suggested that they are able partly to regulate their PUFA through bioconversion. Our results showed that even if low concentrations of DHA were detected in dietary Cryptomonas, DHA was never detected in these cladocerans. Similar results on the inability of daphniids to accumulate DHA have been found in field studies (Persson & Vrede, 2006; Smyntek et al., 2008; Kainz et al., 2009) and through laboratory supplementation experiments (Weers et al., 1997; Von Elert, 2002). Moreover, even if the green alga Scenedesmus lacks HUFA, these six cladocerans fed on Scenedesmus contained ARA and EPA, which indicates that these daphniids might are able to bioconvert dietary precursors, such as LIN and ALA, into ARA and EPA, respectively, probably through the successive use of D6 and D5 desaturases and elongases (Bec et al., 2003a). In contrast to previous single-species laboratory experiments, our results provide experimental 2012 Blackwell Publishing Ltd, Freshwater Biology, 57, 696–703
evidence that Ceriodaphnia sp., D. longispina, D. magna, D. pulex, S. mucronata and S. vetulus, and perhaps cladocerans in general, may all be able to convert some dietary FA into EPA. This suggests that cladocerans may be able to use a poor quality food source to produce EPA, an important PUFA necessary for cladocerans (Mu¨ller-Navarra et al., 2000; Von Elert, 2002). Finally, the lower variability in PUFA concentrations (e.g. ALA and SDA) in daphniids compared with dietary phytoplankton suggests that for cladocerans fed on Scenedesmus, the lack of HUFA could be compensated for by active accumulation of C18PUFA. In field studies, some authors have failed to find correlations between the PUFA composition of zooplankton and their food sources, and suggested that the PUFA composition could be taxon-dependent (Persson & Vrede, 2006; Smyntek et al., 2008). However, field studies make it difficult to specify the diet assimilated by zooplankton. Here, we assessed interspecific variability of cladocerans in the accumulation and bioconversion of PUFA under
702 H. Masclaux et al. controlled experimental conditions. In general, our results showed low variability of PUFA concentrations among these six zooplankton species, all non-selective feeders, when exposed to the same dietary PUFA pool. However, it seems that dietary Scenedesmus exposed some differences in PUFA bioconversion ability among these cladocerans, with ARA and EPA concentrations varying among species (Fig. 3). Among all species tested, Scapholeberis accumulated ARA and EPA most efficiently, which also may indicate that Scapholeberis can bioconvert precursors to ARA and EPA most efficiently at both temperatures. These results suggest that Scapholeberis is able to regulate its PUFA composition better than the other cladoceran species. It is important to note that interspecific variability in PUFA accumulation and bioconversion was more pronounced at the lower temperature for both food sources. One of the important roles of PUFA is to help maintain membrane fluidity at low temperatures (Nishida & Murata, 1996). Temperature is thus an important factor affecting PUFA concentrations (Farkas & Herodek, 1964; Schlechtriem et al., 2006), which tend to increase with decreasing temperatures in all cladocerans tested (see also Farkas & Herodek, 1964; Jiang & Gao, 2004; Schlechtriem et al., 2006). Furthermore, the accumulation and bioconversion of some PUFA, such as LIN, ALA, SDA, ARA and EPA, were on average higher at the lower temperature on both food sources. However, interspecific differences in the ability to regulate metabolically PUFA concentrations at the lower temperature were found (Fig. 4), which suggests interspecific differences in the ability to adapt to temperature that may consequently improve survival success. When individuals were fed on Scenedesmus, the difference in PUFA concentrations between the two temperatures was largest for Simocephalus and Scapholeberis, particularly with respect to their SDA and EPA concentrations. Simocephalus and Scapholeberis are two littoral cladocerans and, as such, can be exposed to substantial temperature variations. These results suggest that littoral cladocerans may be able to regulate their PUFA content to overcome environment constraints, as was proposed by Lemke & Benke (2003), suggesting that these two species could be more adapted to cold conditions. In conclusion, our study indicates that all six cladocerans showed some ability to regulate their PUFA concentrations through bioconversion, and might therefore be able to compensate for poor quality food. Using controlled experimental conditions, we showed that the variability in PUFA accumulation and bioconversion among species increases with decreasing temperature. Such differences
in PUFA regulation are likely to depend on nutritional requirements and biochemical limitation of each species in response to constraints of their diet and habitat. However, under warmer conditions, the cladocerans showed only minor differences in their PUFA accumulation and bioconversion. In littoral habitats with a high diversity of zooplankton, there may be some dietary specialisation and food partitioning with the cladocerans exploiting diets that may differ in their biochemical ⁄ lipid quality. Hence, the foraging behaviour of cladoceran species (exploiting benthic, epiphytic, planktonic and neustonic food sources) may affect PUFA transfer upthrough food webs more strongly than metabolic effects.
Acknowledgments This research was supported by the French Ministry of Education and Research and the Rivie`re Allier PPF research programme. Thanks to Emilie Duffaud for her technical support during lipid analysis. We are also very grateful to Alan Hildrew for his editing work that greatly improved an earlier version of this manuscript.
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(Manuscript accepted 22 December 2011)
