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6  A Methodological Test For Supplementing Chilomonas paramecium With Essential Fatty Acids And Its Application For Evaluating Nutritional Quality for Keratella quadrata

Iola G. Boëchat, Sebastian Schuran, and Rita Adrian

(Submitted to Aquatic Microbial Ecology)

Keywords: Fatty acids; Supplementation; Heterotrophic protists; Nutritional quality; Rotifers

Abstract

A straightforward method to determine whether a single dietary biochemical compound is limiting for a predator is to use dietary supplements. We tested whether a fatty acid supplementation technique using bovine serum albumin as a carrier, previously developed for autotrophic protists, is also appropriate for fatty acid supplementation of Chilomonas paramecium – a flagellated heterotrophic protist. C. paramecium was successfully enriched with eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), both known to be essential fatty acids for crustacean zooplankton. Standardized feeding experiments were performed to test the nutritional value of EPA and DHA for the rotifer Keratella quadrata by following growth rates and egg numbers of the rotifer on a diet of supplemented and non-supplemented C. paramecium. The results were compared to the performance of the rotifer fed Cryptomonas phaseolus, an alga known to support growth and reproduction of Keratella. Preparing C. paramecium enriched with EPA and DHA in the same concentration range as found in C. phaseolus allowed us to test the direct nutritional effects of the two fatty acids for K. quadrata, qualitatively and quantitatively. Growth rates and egg production of K. quadrata were highest when fed the alga C. phaseolus. Among the different supplementation treatments with C. paramecium, egg development of K. quadrata was significantly enhanced on a diet of C. paramecium enriched with DHA, whereas no significant effects could be attributed to EPA enrichment. Thus, factors other than EPA or DHA limit food quality of C. paramecium for K. quadrata.


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6.1  Introduction

Heterotrophic protists such as ciliates and heterotrophic nanoflagellates occupy an intermediate position in aquatic food webs, linking bacterial production to the energy flow into the classical food web (Pomeroy 1974; Azam et al. 1983), as they are themselves preyed upon by the mesozooplankton such as crustaceans (Klein Breteler, 1980; Stoecker and Egloff, 1987; Klein Breteler et al., 1999) and rotifers (Mohr and Adrian, 2001, 2002a,b; chapter 5). Despite their important function as trophic linking, information on the quality of heterotrophic protists for mesozooplankton predators is rather limited to date (but see chapter 5).

Information on chemical compounds which confer prey nutritional quality mainly derived from correlative evidence relating biochemical composition such as fatty acids or the elemental stoichiometry of algae and corresponding growth rates of hervivorous zooplankton – mainly daphnids. Highly unsaturated fatty acids (HUFA) such as eicosapentaenoic acid (EPA) or docosahexaenoic acid (DHA) have been identified, among others, as being important in limiting growth and reproduction of freshwater zooplankton (Müller-Navarra, 1995; Wacker and Von Elert, 2001; Müller-Navarra et al., 2004). An elegant way to verify correlative evidence is to assess the role of individual HUFA directly by supplementing a diet artificially followed by the evaluation of the supplementation effects on the performance of a predator. Current supplementation techniques involve the addition of lipid microcapsules or liposomes (Ravet et al., 2003) and lipid emulsions (DeMott and Müller-Navarra, 1997; Boersma et al., 2001; Park et al., 2003) to an algal prey. Unfortunately, these techniques frequently used mixtures of HUFA, so that effects of a single fatty acid could not be identified. A new technique recently proposed by Von Elert (2002) allows the manipulation of algal fatty acid profiles by the addition of a single fatty acid, which is directly assimilated by algal cells using bovine serum albumin as a carrier. The use of bovine serum albumin prevents toxic effects of free fatty acids. Furthermore, the fatty acid is offered to the predator directly as part of its diet, by which problems related to inefficient ingestion or assimilation are avoided.

In previous feeding experiments with the rotifer Keratella quadrata offered a number of different protist prey species we found correlative evidence that EPA and DHA do also promote egg production of this rotifer (chapter 5). Given our interest in the biochemical nature of protist nutritional quality we tested whether the technique developed for autotrophs (Von Elert, 2002) is also applicable for heterotrophic protists. We chose the flagellate Chilomonas paramecium, which[page 85↓]was supplemented with EPA and DHA. By choosing this flagellate we expected pronounced supplemental effects because C. paramecium comprised thelowest natural EPA concentrations out of four examined heterotrophic protists (chapter 2) as well as lower DHA concentrations as compared to the autotrophic flagellate Cryptomonas phaseolus (chapter 2). Our intention was to enhance EPA and DHA concentrations of C. paramecium up to the natural concentrations observed in C. phaseolus, which is known as a good quality alga. In standardized feeding experiments we then tested the effects of EPA and DHA supplementation on growth rates and egg production of K. quadrata. Our study contributes to the overall controversial discussion about the role of EPA and DHA in zooplankton performance.

6.2 Methodology

6.2.1 Cultures

The flagellate Chilomonas paramecium (403 ± 288 µm3, mean biovolume ± SD) was cultured in Volvic water at 18 ± 1°C under a 16:8 h light : dark regime, and fed bacteria grown on previously autoclaved polished rice corns. The cryptomonad Cryptomonas phaseolus (392 ± 125 µm3) was obtained from the Algal Culture Collection of the University of Göttingen, Germany, and cultured in WC medium (Guillard and Lorenzen, 1972) at 17 ± 1°C under a 16:8 h light : dark regime. Algal cultures were non axenic, but bacteria accounted for less than 2 % of the total organic carbon contents in the cultures. The rotifer Keratella quadrata was originally isolated from Müggelsee, an eutrophic lake located in Berlin, Germany. Rotifers were cultured in WC medium in weekly diluted batch cultures and fed C. phaseolus (1.5 mgC L-1). Cultures were kept under the same temperature and light : dark regimes applied for C. phaseolus. Henceforth, species are referred to by their genus names.

6.2.2 Supplementation with Essential Fatty Acids

Chilomonas was enriched with EPA and DHA following the protocol given in Von Elert (2002). Chilomonas cultures were incubated with EPA (Sigma E2011, purity ≥99%) and DHA (Sigma D2534, purity ≥98%) along a concentration range between 70 and 360 µg mgC-1 of EPA or DHA in the incubation medium. An ethanolic solution containing each single fatty acid was added to a bovine serum [page 86↓]albumin solution (BSA, Sigma A7906, 4 mg mL-1). Subsequently, 10 mL WC medium and a minimum Chilomonas’ biomass of 4 mgC were added to the solution. Chilomonas were previously separated from their culture medium by filtration through a 10 μm silk net and resuspended in Volvic water. Resuspended cells were concentrated to 4 mgC by repeated centrifugation (3000 rpm for 5 min). The resulting suspensions (fatty acids + BSA + WC medium + 4 mgC Chilomonas biomass) were incubated under rotation (100 rpm) at 18°C for 4 h in the light (100 μmol m-2 s-1). After incubation, cells were repeatedly rinsed with WC medium in order to remove excess BSA and free fatty acids, and then collected on pre-incinerated GF/C glass fibre filters (Whatman) and stored at – 20°C until analysis. All treatments were run in five replicates from at least three different Chilomonas batches (n=15 – 20). The integrity of Chilomonas cells was checked by standard microscopy. Control treatments were incubated without fatty acids (non-supplemented Chilomonas). An additional treatment consisting of Chilomonas incubated with BSA only- was carried out in order to eliminate the influence of essential amino acids on Keratella’s performance.

6.2.3 Fatty Acid Analysis

Filters containing material for analysis were extracted in a 2:1 v/v chloroform-methanol solution at 20°C for 4 h (Folch et al., 1957). An internal standard was added to the samples (tricosanoic acid, 0.2 mg mL-1).Fatty acid methyl-esters (FAME) were formed by addition of a methanolic sulphuric acid solution (5% v/v) and heating the samples for 4 h at 80°C (Weiler, 2001). An aliquot of 0.2 μL of the samples was injected into a Varian Star 3600 CX series gas chromatograph, equipped with a fused silica capillary column (Omegawax 320, SUPELCO, 30 m x 0.32 mm). An initial temperature of 180°C (2 min) was applied, subsequently heated at 2°C min-1 to 200°C, which was held isothermally for 33 min. Injector and FID detector temperatures were 250°C and 260°C, respectively. Helium was used as a carrier gas. FAMEs identification were made by comparing the retention times with retention times of a standard solution (Supelco FAME Mix 47885–4, PUFA Nr. 3–47085–4 and PUFA Nr. 1–47033) and values were expressed as concentrations per carbon biomass of Chilomonas. The concentrations of EPA and DHA are given for samples incubated with EPA, DHA, BSA only, and in non-supplemented samples as well.

6.2.4 Feeding Experiments with Keratella quadrata

We tested the importance of EPA and DHA by following growth rates and egg production of Keratella in prey treatments with supplemented and [page 87↓]non-supplemented Chilomonas. The rotifers were separated from their algal food and resuspended in Volvic water 12 hours prior to the start of the experiments. Twenty Keratella without eggs were placed into 15 mL chambers in macrotiter plates containing the different diets, offered in concentrations above the incipient limiting level, previously determined in functional experiments (1 mgC L-1, Boëchat unpublished data). The rotifers received fresh prey suspensions daily over a 5 day experimental period. As a common practice in such feeding experiments, the predators are daily transferred into a new chamber with fresh prey suspension. However, due to the high fragility of Keratella to manual handling, we decided not to remove them from the experimental chambers. Instead, we added fresh prey suspensions to the chambers, starting the experiments with an initial volume of 4 mL and adjusting the prey densities daily to a final volume of 12 mL at day 5. Prey treatments were as follows: Chilomonas previously supplemented with EPA (Chilomonas + EPA) and DHA (Chilomonas + DHA), BSA only (Chilomonas + BSA), non-supplemented Chilomonas (Chilomonas), and a control treatment without food (no food). A diet of non-supplemented Cryptomonas was used as a reference, since Cryptomonas is known to support growth and reproduction of Keratella (chapter 5). Incubation concentrations of EPA and DHA were 90 μg mgC-1, which resulted in an increase in EPA (13.3 μg EPA mgC-1) and DHA (33.6 μg DHA mgC-1) concentrations in Chilomonas equivalent to the range found in Cryptomonas (11.2 μg EPA mgC-1 and 40.2 μg DHA mgC-1; see chapter 2). This allowed us to directly test the nutritional effects of EPA and DHA for Keratella. Macrotiter plates containing each treatment in five replicates were incubated at 18 ± 1°C under a 16:8 h light : dark regime. Rotifers and eggs were enumerated at x20 magnification. Population growth rates (r) of the rotifers were calculated assuming exponential growth according to:

Where Nt is the number of rotifers at the end of the experimental time interval (Δt) and Nt-1is the number of rotifers at the beginning of the experimental time interval (Δt). Keratella’s numbers were log-transformed to assure normality of data distribution. The overall growth rate for the entire experimental period (five days) was calculated as a mean of daily growth rates. Egg numbers are presented as cumulative curves, which represent the daily increase in egg numbers, fitted by non-linear regression models using the program Table Curve 2D, version 5.0.


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6.2.5  Statistical Analysis

Differences in the percentages of each fatty acid (EPA or DHA) in the total fatty acid profile of supplemented versus non-supplemented Chilomonas were tested for significances using one-way ANOVA followed by the Dunnett t-test, which pairwise compared supplementation treatments against non-supplemented treatments (Statistica for Windows, version 5.01, Stat Soft). The same statistical procedure was applied for testing differences in total population growth rates and cumulative egg production of Keratella among feeding treatments.

6.3 Results

6.3.1 EPA and DHA Supplementation of Chilomonas paramecium

The natural concentration of EPA in Chilomonas was 5.9 ± 1.2 μg mgC-1 (Fig. 1). EPA concentrations were significantly elevated in EPA supplemented Chilomonas (23.3 ± 7.7 μg mgC-1) as compared to non supplemented Chilomonas (ANOVA F=10.73, P<0.01, Dunnett t-Test, P<0.05; Fig. 1). Beyond an incubation concentration of 140 µg EPA mgC-1, EPA uptake by Chilomonas reached saturation (Fig. 1).

Fig. 1 – Eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) concentrations in Chilomonas paramecium supplemented with different incubation concentrations of either EPA or DHA versus concentrations in non-supplemented Chilomonas (control). Significant differences between supplemented and non-supplemented (control) treatments are indicated by asterisks (ANOVA followed by Dunnett t-Test).


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The natural DHA concentration in Chilomonas was 0.94 ± 0.53 μg mgC-1 (Fig. 1). DHA concentrations in supplemented Chilomonas increased with increased DHA concentrations in the incubation medium and were significantly elevated to up to 63.5 ± 17.5 μg mgC-1 (ANOVA F=10.73, P<0.01, Dunnett t-Test, P<0.01; Fig. 1) as compared to non-supplemented Chilomonas. No significant changes were observed in DHA concentrations when supplementing Chilomonas with EPA (ANOVA F=0.85, P=0.51) and vice-versa (ANOVA F=3.09, P=0.08). Microscopy revealed no cell damage of supplemented Chilomonas.

6.3.2 Feeding Experiments with Keratella

Keratella fed Cryptomonas exhibited higher population growth rates (ANOVA F=14.74, P<0.01) and cumulative egg numbers (ANOVA F=3.25, P<0.01; Fig. 2) than rotifers fed supplemented Chilomonas.

Fig. 2 – Keratella quadrata lognumbers and population growth rates (upper panel) and cumulative egg numbers (lower panel). Treatments were: rotifers fed non-supplemented Cryptomonas phaseolus (Cryptomonas), Chilomonas paramecium supplemented with docosahexaenoic acid (Chilomonas + DHA), eicosapentaenoic acid (Chilomonas + EPA), with bovine serum albumin without fatty acids (Chilomonas + BSA) as well as non-supplemented Chilomonas paramecium (Chilomonas), and a control treatment without food (no food).


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Feeding on Chilomonas supplemented with DHA improved rotifers’ population growth rates and reproduction based on egg numbers (Fig. 2), when compared to non-supplemented Chilomonas (Dunnett t-Test, P<0.05) or controls without food (Dunnett t-Test, P<0.01). Although we found a slight tendency of higher population growth rates and cumulative egg numbers on a daily time scale in EPA supplemented treatments, EPA enriched Chilomonas did not significantly improve population growth rates of the rotifer, when compared to non-supplemented Chilomonas (Dunnett t-Test, P=0.97) or to the control treatments without food (Dunnett t-Test, P=0.09). Egg numbers were unaffected by EPA supplementation (Dunnett t-Test, P=0.69). In the Chilomonas + BSA treatments population growth rates of Keratella were higher than those found in non-supplemented Chilomonas or the control treatments without food (Dunnett t-Test, P<0.05). Egg numbers remained unaffected in the Chilomonas + BSA treatments (Dunnett t-Test, P=0.35).

6.4 Discussion

The fatty acid supplementation technique proposed by Von Elert (2002) for autotrophic protists proved to be equally appropriate for supplementing Chilomonas with EPA and DHA. The concentration dependent relationship between fatty acid concentrations in the incubation medium and fatty acid uptake by Chilomonas enabled us to prepare prey organisms of defined fatty acid contents. This generally allows the evaluation of direct effect of dietary specific fatty acids on zooplankton performance, qualitatively and quantitatively. As no significant changes were observed in the EPA concentrations in DHA supplemented Chilomonas and vice-versa, interconversion of those molecules, which may have complicated the interpretation of single EPA and DHA effects on Keratella’s performance, could be excluded. Although cellular EPA and DHA concentrations of Chilomonas had been elevated to concentrations found in Cryptomonas, Chilomonas did not support rotifers’ growth and high egg numbers as Cryptomonas did. This indicates that EPA and DHA were not the major factors limiting the performance of Keratella fed Chilomonas. Since both Cryptomonas and Chilomonas are morphologically very similar (similar size and shape, both flagellated cryptomonads) differences in the trophic mode may relate to the differences in their nutritional quality.


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Nevertheless, DHA supplementation of Chilomonas significantly improved its nutritional quality as prey for Keratella, when compared to non-supplemented Chilomonas. This suggests that DHA was present in limiting concentrations in non-supplemented Chilomonas. This is supported by the positive correlation between Keratella’s egg numbers and DHA dietary concentrations derived from laboratory experiments (chapter 5). Especially reproduction of Keratella seems to be positively affected by DHA concentrations. Our results observed for Keratella are consistent with findings found for other zooplankton organisms. Dietary DHA, together with EPA and other long chained fatty acids, has been found in high amounts in copepod’s eggs (Sargent and Falk-Petersen, 1988). Moreover, in some crustaceans EPA and DHA have been found in 2 – 5 fold higher amounts in eggs and ovaries Than in other female tissues (Hayashi, 1976), suggesting the importance of these fatty acids for embryonic survival and growth. DHA is also believed to play a significant role during larval development and metamorphosis of marine molluscs (Delaunay et al., 1993) and to promote enhanced larval growth of the zebra mussel Dreissena polymorpha (Wacker et al., 2002). The fact that DHA supplementation lead to increased egg numbers but only slightly enhanced Keratella population growth rates suggests that reproduction and population growth have different metabolic demands, which is in accordance with the stoichiometric theory (Sterner and Hessen, 1994). Supplementation of Chilomonas with only BSA improved population growth of the rotifers when compared to non-supplemented cells or to the control treatments without food, suggesting that Chilomonas may be short in amino acid concentrations. Amino acid limitation has indeed been shown to constrain egg production and hatching success of copepods (Kleppel et al., 1998; Guisande et al., 2000).

There is a debate about which HUFA of the ω3 class – such as EPA or DHA – primary limits zooplankton nutrition. Although field studies have shown a significant correlation between sestonic EPA concentrations and growth rates of daphnids (Müller-Navarra, 1995), supplementation of an algal diet with EPA did not improve growth and reproduction of Daphnia magna (Von Elert and Wolffrom, 2001). In a study combining EPA supplementation and mineral limitation, Becker and Boersma (2003) showed that EPA limiting effects on growth rates of Daphnia magna were only detectable below a certain nutrient ratio (C:P = 350) in the algal diet. Other studies, in contrast, showed evidence for the positive effects of DHA supplementation for the zebra mussel Dreissena polymorpha (Wacker et al., 2002) and for the calanoid Acartia tonsa (Kleppel et al., 1998). Our study adds to the controversial discussion about the role of HUFA in zooplankton nutrition and suggests DHA limitation at the interface [page 92↓]heterotrophic protists – zooplankton. However, DΗA supplementation could not completely compensate the low nutritional quality of Chilomonas for Keratella, suggesting the role of other limitation sources.


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