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Supplementation of the protist Chilomonas
paramecium with a highly unsaturated fatty
acid enhances its nutritional quality for
the rotifer Keratella quadrata
IOLA G. BOËCHAT*, SEBASTIAN SCHURAN AND RITA ADRIAN
LEIBNIZ–INSTITUT FÜR GEWÄSSERÖKOLOGIE UND BINNENFISCHEREI, MÜGGELSEEDAMM
*CORRESPONDING AUTHOR:
301, D-12587 BERLIN, GERMANY
[email protected]
Received March 31, 2005; accepted in principle May 20, 2005; accepted for publication June 16, 2005; published online June 24, 2005
Communicating editor: K.J. Flynn
We tested whether a fatty acid supplementation technique using bovine serum albumin (BSA) as a
carrier, previously developed for autotrophic protists, is also appropriate for supplementation of
Chilomonas paramecium—a flagellated heterotrophic protist. Chilomonas paramecium was successfully enriched with eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), both known
to be essential for crustacean zooplankton. Preparing C. paramecium enriched with EPA and DHA
in concentrations similar to those found in Cryptomonas phaseolus, an alga known to support high
growth and reproduction of Keratella, allowed us to test the direct nutritional effects of EPA and
DHA on population growth and reproduction of the rotifer Keratella quadrata. Growth rates and
egg production were highest when K. quadrata was fed with C. phaseolus. Compared to nonsupplemented C. paramecium, egg production 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. We conclude that DHA is important for reproduction of K. quadrata but cannot
explain the majority of the difference in food quality between C. paramecium and C. phaseolus.
INTRODUCTION
Protists, such as ciliates and heterotrophic nanoflagellates,
occupy an intermediate position in aquatic food webs.
Heterotrophic flagellates and aloricate ciliates significantly
contribute to the biomass in the 5- to 20-mm size range in
marine (Sherr et al., 1986) and freshwater (Carrick et al.,
1991) pelagial environments. Since most metazoans are
not able to efficiently ingest food particles smaller than
10 mm, heterotrophic protists link bacterial production to
the energy flow through the classical food web (Pomeroy,
1974; Azam et al., 1983) as they are preyed upon by
mesozooplankton predators such as crustaceans (Klein
Breteler, 1980; Stoecker and Egloff, 1987; Klein Breteler
et al., 1999) and rotifers (Mohr and Adrian, 2001, 2002).
However, despite their fundamental importance within
the food web (Levinsen et al., 2000), research on the
nutritional quality of heterotrophic protists as prey for
mesozooplankton has been rather limited, especially in
freshwater systems. Most of the studies on the nutritional
quality of heterotrophic protists involve marine ciliate and
flagellate species (Tiselius, 1989; Gifford and Dagg, 1991;
Ederington et al., 1995; Harvey et al., 1997; Broglio et al.,
2003). It has been shown that the lipid composition (fatty
acids, neutral lipids, and sterols) of heterotrophic protists
resembles that of their bacterial or algal prey (Ederington
et al., 1995; Boëchat and Adrian, 2005). Thus, bacterivorous protists are expected to contain large amounts of
saturated (SAFA) and monounsaturated (MUFA) fatty
acids, which may together amount to >85% of the total
fatty acids (Ederington et al., 1995), and very low quantities of polyunsaturated fatty acids (PUFA), highly unsaturated fatty acids (HUFA), and sterols (Harvey and
McManus, 1991; Klein Breteler et al., 1999; Sul et al.,
2000). Phytoplanktivorous protists, on the contrary, generally contain large amounts of PUFAs and essential
amino acids (Boëchat and Adrian, 2005) as well as sterols
(I. G. Boëchat, unpublished results) when cultured on an
algal prey of high nutritional quality.
doi:10.1093/plankt/fbi040, available online at www.plankt.oupjournals.org
Ó The Author 2005. Published by Oxford University Press. All rights reserved. For Permissions, please email: [email protected].
JOURNAL OF PLANKTON RESEARCH
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Information on the chemical compounds that confer
prey nutritional quality to mesozooplankton derives
mainly from correlative evidence. Several studies have
reported significant correlations between biochemical
compounds or the elemental stoichiometry of algae and
population growth rates of herbivorous zooplankton,
especially for daphniids (Kilham et al., 1997; Sundbom
and Vrede, 1997; Müller-Navarra et al., 2004). HUFA,
such as eicosapentaenoic acid (EPA) or docosahexaenoic
acid (DHA), were established to affect growth and reproduction of freshwater zooplankton (Müller-Navarra,
1995; Wacker and Von Elert, 2001; Müller-Navarra
et al., 2004). However, there is still a debate about which
HUFA of the class o3—such as EPA or DHA—primarily
limits zooplankton nutrition. Furthermore, sterols, especially cholesterol, have recently been reported to be
essential for zooplankton species such as Daphnia galeata
(Von Elert et al., 2003) and the marine copepods Acartia
tonsa, Acartia hudsonica, Calanus finmarchicus (Hasset, 2004),
Temora longicornis, and Pseudocalanus elongatus (Klein Breteler
et al., 2004).
An elegant way to verify correlative evidence is to
assess the role of individual HUFA directly by supplementing a diet artificially and then to evaluate the effects
of the supplementation on the performance of a predator. Current supplementation techniques involve the
addition of lipid microcapsules (Knauer and Southgate,
1997) 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,
previous research using these techniques frequently used
mixtures of HUFA, and therefore the effects of a single
fatty acid were not identified.
However, a new technique that allows algal fatty acid
profiles to be manipulated by using bovine serum albumin (BSA) as carrier of a single fatty acid that can then
be directly assimilated by algal cells has been recently
proposed (Von Elert and Wolffrom, 2001; Von Elert,
2002). The technique has two major advantages: (i) the
use of BSA prevents toxic effects of free fatty acids; and
(ii) 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 using the rotifer Keratella quadrata and different heterotrophic protists as prey,
we found correlative evidence that EPA and DHA promote egg production of this rotifer. Therefore, as a
prelude to defining the exact biochemical compound(s)
affecting the nutritional quality of heterotrophic protists,
we tested whether the supplementation technique developed for autotrophs (Von Elert and Wolffrom, 2001;
Von Elert, 2002) would also be suitable for heterotrophic
protists. To this end, we chose the heterotrophic
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flagellate Chilomonas paramecium as prey and EPA and
DHA as supplements. The rationale behind our choice
was that we expected to produce pronounced, and
thereby easily observable, effects, as C. paramecium comprised the lowest natural EPA concentrations out of four
examined heterotrophic protists (I. G. Boëchat, unpublished results) as well as lower DHA concentrations as
compared to the autotrophic flagellate Cryptomonas phaseolus (Boëchat and Adrian, 2005). Our intention was to
enhance EPA and DHA concentrations of C. paramecium
up to the natural concentrations observed in C. phaseolus,
which is considered a good nutritional quality alga (Boëchat and Adrian, 2005). In routine feeding experiments,
we then tested the effects of EPA and DHA supplementation on growth rates and egg production of K. quadrata.
Our study provides additional insights into the already
controversial issue of the possible roles of EPA and DHA
in regards to zooplankton reproduction and population
growth.
METHOD
Cultures
The heterotrophic flagellate C. paramecium (403 288 mm3,
mean biovolume SD; Strain number 977–2a,
Algal Culture Collection, University of Göttingen,
Germany) was cultured in Volvic water (a spring water,
poor in minerals, sold worldwide by Société des Eaux de
Volvic, Puy-de-Dôme, France) at 18 1 C under a 16:8 h
light : dark regime, and fed a mix of bacteria grown on
previously autoclaved polished rice corns (Mohr and
Adrian, 2002). The cryptomonad C. phaseolus (392 125
mm3; Strain number 2013, Algal Culture Collection,
University of Göttingen, Germany) was cultured in WC
(Woods Hole Chu-10) 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 <2%
of the total organic carbon contents in the cultures as
judged by our biomass to carbon content conversion calculations. Cryptomonas phaseolus and C. paramecium are both
cryptomonads with similar size and shape. The rotifer K.
quadrata was originally isolated from the lake Müggelsee,
Berlin, Germany. Rotifers were cultured in WC medium
in weekly diluted (60% dilution rate) batch cultures and
fed C. phaseolus (1.5 mg C L–1). Cultures were kept under
the same temperature and light : dark regimes as used
for C. phaseolus. Henceforth, species are referred to by
their genus names.
Supplementation with essential fatty acids
Chilomonas was enriched with EPA and DHA following
standard protocols (Von Elert and Wolffrom, 2001;
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Von Elert, 2002). Chilomonas cultures were incubated with
EPA (Sigma E2011; purity, 99%) and DHA (Sigma
D2534; purity, 98%) at selected concentrations between
70 and 360 mg EPA or DHA g–1Chilomonas C added to the
incubation medium. An ethanolic solution containing
each single fatty acid was added to a 4 mg mL–1 BSA
solution (Sigma A7906). Subsequently, 10 mL WC medium and a minimum protistan biomass of 4 mg C were
added to the solution. To obtain a minimum of 4 mg C
protistan biomass, Chilomonas cultures were filtered
through a 10-mm silk net and then resuspended in 0.5mL Volvic water. Resuspended cells were concentrated to
4 mg C by repeated centrifugation (604 g for 5 min). The
resulting suspensions (fatty acids + BSA + WC medium +
4 mg C Chilomonas biomass) were incubated under rotation
in a plankton wheel (100 r.p.m.) at 18 C for 4 h in the light
(100 mmol m–2 s–1). After incubation, cells were repeatedly
rinsed with WC medium in order to remove excess BSA
and free fatty acids. Cells for fatty acid analysis were then
collected on ashed 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). Chilomonas cells used in the feeding
experiments with Keratella were resuspended in WC Medium and immediately used for feeding the rotifer (see
‘Feeding experiments with K. quadrata’). The integrity of
Chilomonas cells was verified by light microscopy. Control
treatments were incubated without fatty acids (non-supplemented Chilomonas). An additional treatment consisting of Chilomonas incubated with BSA without the fatty
acid supplement was carried out to examine any influence the essential amino acids may have on Keratella’s
performance.
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 of 0.2 mg mL–1
tricosanoic acid was added to the samples. Fatty acid
methyl esters (FAME) were formed by addition of a 5%
v/v methanolic sulphuric acid solution followed by heating the samples for 4 h at 80 C (Weiler, 2001). An
aliquot of 0.2 mL 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 0.32 mm). An initial
temperature of 180 C (2 min) was applied, and then
raised by 2 C min–1 to 200 C, which was held isothermally for 33 min. Injector and Flame Ionization Detector
(FID) temperatures were 250 C and 260 C, respectively.
Helium was used as a carrier gas. FAMEs were identified
by comparing the obtained retention times with those of a
calibration standard solution (SUPELCO FAME Mix
47885–4, PUFA Nr. 3-47085-4 and PUFA Nr. 1–
47033). Quantification was performed by referring to
the internal standard and to response factors determined for each FAME from the SUPELCO mixtures
of known composition cited above. Values were
expressed as concentrations per carbon biomass of the
protist. For this purpose, carbon concentrations were
derived
from cell
volume estimates using
carbon : biovolume conversion factors of 0.10 pg C
mm–3 for Cryptomonas (Montagnes, 1994) and 0.19 pg C
mm–3 for Chilomonas (Putt and Stoecker, 1989). The
concentrations of EPA and DHA are given for samples
incubated with EPA, DHA, and BSA as well as for nonsupplemented samples.
Feeding experiments with K. quadrata
We assessed the importance of EPA and DHA by following growth rates and egg production of Keratella in
prey treatments with supplemented and non-supplemented Chilomonas. The rotifers were separated from their
algal food over filtration through a 70-mm silk net and
resuspended in Volvic water 12 h before the onset of the
feeding experiments. Twenty Keratella without eggs were
placed into 15-mL chambers in macrotiter plates containing the different diets at concentrations above the
incipient limiting level (ILL: 1 mg C L–1), previously
determined in functional experiments. The rotifers
received fresh prey suspensions daily for 5 days, and
the experiments were repeated three times. A common
practice in such feeding experiments is to transfer the
predators daily into a new chamber with fresh prey
suspension. However, owing to the high fragility of Keratella to manual handling, we did not 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 adding the prey
densities daily to a final volume of 12 mL at day 5.
Adjustments of prey concentration were performed by
previously counting prey densities in the experimental
chambers and subsequent addition of new prey suspensions in a concentration sufficient to reach the ILL in the
given volume. Prey treatments were as follows: Chilomonas previously supplemented with EPA (Chilomonas +
EPA) or DHA (Chilomonas + DHA) with BSA carrier,
BSA only (Chilomonas + BSA), non-supplemented Chilomonas (Chilomonas), and a starved control treatment (no
food). A diet of non-supplemented Cryptomonas was used
as a reference because Cryptomonas is known to support
growth and reproduction of Keratella (I. G. Boëchat,
unpublished results). Chilomonas were separated from
the culture medium and incubated with fatty acids as
described above (see ‘Supplementation with essential
fatty acids’). Incubation concentrations of EPA and
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DHA were 90 mg mg–1 protist C, which resulted in
increased concentrations of EPA (13.3 mg EPA mg–1 C)
and DHA (33.6 mg DHA mg–1 C) in Chilomonas equivalent to the concentrations found in Cryptomonas (11.2 mg
EPA mg–1 C and 40.2 mg DHA mg–1 C) (Boëchat and
Adrian, 2005). This allowed us to directly test the nutritional effects of EPA and DHA on the performance of
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
daily at 20 magnification. Daily population growth rates
(r) of the rotifers were calculated assuming exponential
growth according to:
r¼
lnðNt2 Þ lnðNt1 Þ
Dt
µg mg– 1 C
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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. While feeding
on Chilomonas supplemented with DHA improved the
rotifer population growth rates and egg production
(Fig. 2) when compared to non-supplemented Chilomonas
(Dunnett t-test, P < 0.05) or controls without food (Dunnett
t-test, P < 0.01), growth rates were still negative. Cumulative egg numbers of rotifers fed DHA-supplemented
Chilomonas were significantly higher than egg numbers of
*
100
75
EPA
DHA
Chilomonas + DHA
*
*
*
50
*
*
*
25
10
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Feeding experiments with K. quadrata
Chilomonas + E PA
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The natural concentration of EPA in Chilomonas was 5.9
1.2 mg mg–1 C (Fig. 1). EPA concentrations were
significantly elevated in EPA supplemented Chilomonas
(23.3 7.7 mg mg–1 C) 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 mg EPA mg–1 C, EPA uptake by Chilomonas reached
saturation (Fig. 1).
The natural DHA concentration in Chilomonas was
0.94 0.53 mg mg–1 C (Fig. 1). DHA concentrations
in supplemented Chilomonas increased with increased
DHA concentrations in the incubation medium, without
saturation in the concentration range tested, and were
significantly elevated to up to 63.5 17.5 mg mg–1 C, as
compared to non-supplemented Chilomonas (ANOVA
F = 10.73, P < 0.01, Dunnett t-test, P < 0.01; Fig. 1).
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). Microscopic examination revealed
no cell damage to supplemented Chilomonas.
To detect any significant differences in the concentrations of either EPA or DHA in the total fatty acid profile
of supplemented and non-supplemented Chilomonas, we
employed one-way ANOVA followed by the Dunnett
t-test. This allowed supplemented and non-supplemented treatments to be compared in a pairwise manner
(Statistica, version 5.01, Stat Soft). The same statistical
procedure was applied to test differences in total population growth rates and cumulative egg production of
Keratella among feeding treatments.
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Data analysis
EPA
DHA
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RESULTS
where Nt1 and Nt2 are rotifer numbers at the start (t1)
and end (t2) of each experimental day, respectively.
Keratella’s numbers were log transformed to assure normality of data distribution. The overall growth rate for
the entire experimental period (5 days) was calculated
as the mean of the daily growth rates. Egg numbers are
represented as cumulative curves, which indicate daily
increases in egg numbers, fitted by nonlinear regression
models using the program Table Curve 2D, version 5.0
(Systat Software).
40
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Control 70
140
210
0
360
Control 70
140
210
360
–1
Fatty acid concentration range in incubation medium ( µg mg C)
Fig. 1. Eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) concentrations in Chilomonas paramecium supplemented with different
incubation concentrations of 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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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).
Log Keratella numbers
1.8
1.5
1.2
0.6
0.3
0.0
Cumulative egg numbers
DISCUSSION
0.9
Cryptomonas (r = 0 .13)
Chilomonas + DHA (r = –0.08)
Chilomonas + BSA (r = – 0.15)
Chilomonas + EPA (r = –0.22)
Chilomonas (r = – 0.25)
Control, no food (r =–0.40)
1
2
3
4
5
4
5
Cryptomonas
Chilomonas + DHA
Chilomonas + EPA
Chilomonas
Chilomonas + BSA
Control (no food)
60
50
40
30
20
10
0
1
2
3
Time (days)
Fig. 2. Keratella quadrata log numbers and population growth rates
(upper panel) and cumulative egg numbers (lower panel). Curves are
adjusted according to nonlinear regression models. Treatments were
rotifers fed supplemented and non-supplemented Chilomonas paramecium
(Chilomonas), non-supplemented Cryptomonas phaseolus (Cryptomonas) and a
control treatment without food (no food). Chilomonas were supplemented with the polyunsaturated fatty acids eicosapentaenoic acid (EPA)
(Chilomonas + EPA) and docosahexaenoic acid (DHA) (Chilomonas +
DHA) as well as with bovine serum albumin without fatty acids
(BSA) (Chilomonas + BSA).
rotifers fed Chilomonas supplemented with only BSA
(Dunnett t-test, P < 0.05), whereas no difference was
found for growth rates. Although we observed a slight
tendency of higher population growth rates and cumulative egg numbers 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),
the control treatments without food (Dunnett t-test,
P = 0.09) or Chilomonas supplemented with only BSA
(Dunnett t-test, P = 0.42). 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
The fatty acid supplementation technique proposed by
Von Elert and Wolffrom (Von Elert and Wolffrom,
2001) and Von Elert (Von Elert, 2002) for autotrophic
protists proved to be equally suitable and efficient for the
supplementation of 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 with known fatty acid contents. This allows the
evaluation both qualitatively and quantitatively of the
direct effect of dietary specific fatty acids on zooplankton
performance. As no significant changes were observed in
the EPA concentrations in DHA-supplemented Chilomonas and vice versa, interconversion of these molecules in
Chilomonas during the experimental period, which may
have complicated the interpretation of single EPA and
DHA effects on Keratella’s performance, can be excluded.
Although cellular EPA and DHA concentrations of Chilomonas were increased up to those found in Cryptomonas,
Chilomonas did not support rotifer growth and high egg
numbers to the extent that Cryptomonas did. This indicates that EPA and DHA were not the major factors
limiting the performance of Keratella fed with Chilomonas.
Because both Cryptomonas and Chilomonas are morphologically very similar (similar size and shape, both
flagellated cryptomonads) differences in their nutritional
quality may relate to the differences in other biochemical
or mineral compounds as well as in the trophic mode—
autotrophy versus heterotrophy. Indeed, we have found
that the fatty acid composition of the flagellate Ochromonas sp. is strongly affected by the trophic mode of the
flagellate, cultured autotrophically, heterotrophically or
mixotrophically. However, we cannot rule out the possibility that the biochemical form of the fatty acids (as
BSA complexes) used for the supplementation of Chilomonas may have not been sufficient to ensure a full
response of Keratella when comparing with the performance of rotifers fed Cryptomonas. Although the use of
BSA as carrier enhances the solubility of the fatty acids,
thereby avoiding the formation of micelles and toxic
effects (Von Elert, 2002), no data are available on the
bioavailability of BSA-fatty acid complexes for zooplankton species.
Nevertheless, DHA supplementation of Chilomonas significantly improved its nutritional quality as prey for Keratella, when compared to non-supplemented Chilomonas.
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This suggests that DHA was present in limiting concentrations in non-supplemented Chilomonas and is supported
by the positive correlation between Keratella’s egg numbers
and DHA dietary concentrations derived from our previous laboratory experiments. DHA concentrations had a
positive effect especially on the reproductive capacity of
Keratella. Our results for DHA observed for Keratella (Fig. 2)
are consistent with findings for marine zooplankton species, such as the copepods A. tonsa and A. hudsonica
( Jónasdóttir, 1994; Jónasdóttir et al., 1995) and Calanus
helgolandicus (Anderson and Pond, 2000). Dietary DHA,
together with EPA and other long chained fatty acids, has
been found in high concentrations in copepod’s eggs
(Sargent and Falk-Petersen, 1988; Jónasdóttir et al.,
1995). Moreover, in some crustaceans, EPA and DHA
were estimated to be present in 2- to 5-fold higher concentrations in eggs and ovaries compared to 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) and
fish larvae (Copeman et al., 2002). Moreover, DHA is vital
to the metabolism and vision efficiency of fish larvae
(Koven, 2003).
As DHA supplementation led to increased egg numbers, but only slightly enhanced Keratella population growth
rates, this suggests that reproduction and population
growth have different metabolic demands. Egg development may be favoured through dietary DHA supplementation, but survival of young individuals is probably
unaffected. Thus, the negative growth rates of Keratella
fed with supplemented and non-supplemented Chilomonas
may be a result of high mortality rates, which were only
slightly decreased by supplementing the flagellate with
fatty acids. Supplementation of Chilomonas with BSA only
improved population growth of the rotifers when compared to non-supplemented cells or the control treatments
without food, suggesting that untreated Chilomonas may
have low concentrations of certain amino acids. 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 still a debate about which HUFA of the o3
class—such as EPA or DHA—primarily limits zooplankton nutrition under which conditions. Although field
studies have shown a significant correlation between
sestonic EPA concentrations and growth rates of daphnids (Müller-Navarra, 1995), supplementation of the
cyanobacteria Synechococcus with EPA did not improve
growth and reproduction of Daphnia magna (Von Elert
and Wolffrom, 2001). Supplementation of the green
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algae Scenedesmus with EPA, however, improved the
nutritional quality of this alga as prey for D. galeata
(Von Elert, 2002). In a study combining EPA supplementation and mineral limitation, Becker and Boersma
(Becker and Boersma, 2003) demonstrated that EPA
limitation effects on D. magna growth were only detectable below a certain nutrient ratio (C:P = 350) in the
algal diet Scenedesmus. In contrast, other studies reported
positive effects resulting from DHA supplementation for
the zebra mussel D. polymorpha (Wacker et al., 2002) and
the calanoid A. tonsa (Kleppel et al., 1998), and from EPA
supplementation for Daphnia fed a mixture of well edible
cyanobacteria (Ravet et al., 2003). Our study suggests
DHA limitation at the interface of heterotrophic
protists–zooplankton in the food chain, thereby adding
to the already controversial issue of the role of HUFA in
zooplankton nutrition. However, DHA supplementation
could not completely compensate for the low nutritional
quality of Chilomonas for Keratella. Thus, factors other
than DHA are probably driving nutritional quality of
this heterotrophic protist as prey for the rotifer. Possible
candidates are sterols, especially cholesterol (Von Elert
et al., 2003; Hasset, 2004; Klein Breteler et al., 2004), and
amino acids (Kleppel et al., 1998; Guisande et al., 2000).
Last but not least, differences in the elemental stoichiometry of N and P between Chilomonas and Keratella as well
as the presence of toxic secondary metabolites in Chilomonas (Mitra and Flynn, 2005) may have negatively
affected assimilation rates and therefore growth and
reproduction of Keratella fed supplemented and non-supplemented Chilomonas.
ACKNOWLEDGEMENTS
We thank C. Enderes for helping with sample processing
and maintenance of the cultures. We owe thanks to
M. Wirth and W. Weiler for helpful methodological
advice. This article also benefited from comments of
B. Gücker and three anonymous reviewers as well as from
English-language editing by R. Willmott. This research
was supported by a PhD grant to I.G.B. from the Deutscher
Akademischer Austauschdienst (DAAD).
REFERENCES
Anderson, T. R. and Pond, D. W. (2000) Stoichiometric theory
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