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Evidence for biochemical limitation
of population growth and reproduction
of the rotifer Keratella quadrata fed with
freshwater protists
IOLA G. BOËCHAT*† AND RITA ADRIAN
LEIBNIZ-INSTITUT FÜR GEWÄSSERÖKOLOGIE UND BINNENFISCHEREI, MÜGGELSEEDAMM 301, D-12587 BERLIN, GERMANY
†
PRESENT ADDRESS: LABORATÓRIO DE FICOLOGIA, DEPTO. DE BOTÂNICA, ICB, UNIVERSIDADE FEDERAL DE MINAS GERAIS, AV. PRES. ANTÔNIO CARLOS 6627,
31270-090 BELO HORIZONTE, MINAS GERAIS, BRAZIL
*CORRESPONDING AUTHOR: [email protected]
Received March 13, 2006; accepted in principle July 10, 2006; accepted for publication August 17, 2006; published online August 21, 2006
Communicating editor: K.J. Flynn
The biochemical factors that determine the food quality of protists for rotifers are poorly understood.
We evaluated population growth rates and egg production of the rotifer Keratella quadrata fed with
four protist species growing on either an algal or a bacterial diet. The cryptomonad Cryptomonas
phaseolus, considered as a good quality prey, and assays without prey served as controls. Population
growth rates and egg numbers of K. quadrata were correlated with single biochemical compounds
(fatty acids, amino acids and sterols) of the protists. Feeding on the alga C. phaseolus or the
algivorous ciliates resulted in enhanced population growth rates and high egg production by
K. quadrata, whereas feeding on bacterivores supported only moderate egg production but no
population growth. The rotifers’ egg production was correlated with the protist biochemical
composition, including polyunsaturated fatty acids (PUFAs), the sterol desmosterol, ergosterol,
stigmastanol and the amino acid leucine. No significant relationships were observed between
population growth rates of the rotifers and the protists’ biochemistry, suggesting that population
growth and reproduction of K. quadrata may have different nutritional requirements. To our
knowledge, this is one of the first studies to analyze a large variety of biochemical compounds to
determine the food quality of protists for a zooplankton predator and the first study to analyze the
biochemical quality of protists to a rotifer species.
INTRODUCTION
Rotifers represent an important fraction of the zooplankton biomass in lakes (Arndt, 1993), and their prey size
spectrum (<1–200 mm) covers a wide range of microbes,
including bacteria and ciliated and flagellated protists
(Pourriot, 1977; Bogdan and Gilbert, 1987). In lakes,
during spring, rotifer density peaks shortly after protists’
density peak (Sommer et al., 1986); thus, rotifers and
ciliates compete for the same food resources, and rotifers
act as predators, potentially regulating protist population
in situ (Bogdan et al., 1980; Carrick et al., 1991; Gilbert
and Jack, 1993; Mohr and Adrian, 2002a). However,
despite the ecological significance of protist–rotifer interaction, it is far from understood how protists contribute
to growth and reproduction of rotifer populations.
The influence of protist prey on life history traits of
rotifers has been recently reported (Gilbert and Jack,
1993; Mohr and Adrian, 2001, 2002b; Weisse and
Frahm, 2001). Enhanced reproduction and growth
rates of Brachionus calyciflorus Pallas, 1766, were observed
when feeding the rotifers with a mixture of algae and
algivorous ciliates; however, no positive effect was
doi:10.1093/plankt/fbl036, available online at www.plankt.oxfordjournals.org
Ó The Author 2006. Published by Oxford University Press. All rights reserved. For Permissions, please email: [email protected]
JOURNAL OF PLANKTON RESEARCH
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reported when feeding the rotifers with a mixture of
algae and bacterivorous ciliates (Mohr and Adrian,
2002b). Moreover, enhanced growth of rotifer populations of the genus Keratella Müller, 1786, was observed
when the rotifers were fed a mixture of autotrophic
cryptophytes and algivorous ciliates (Weisse and Frahm,
2001). Taken together, these results suggest that the
trophic mode of ciliates—algivorous or bacterivorous—
shapes their food quality for rotifers. Indeed, previous
studies have shown that dietary biochemical composition
influences the fatty acid and amino acid to some extent
(Boëchat and Adrian, 2005), as well as the sterol composition (Boëchat et al., in press) of heterotrophic protists.
Nevertheless, essential compounds such as highly unsaturated fatty acids (HUFAs) and the sterol ergosterol
were detected in the bacterivore Chilomonas paramecium
Ehrenberg, 1832, although they were not present in
their bacterial prey (Boëchat and Adrian, this article).
Such disparities in the biochemical composition between
protists and their diet suggest species-specific differences
in protist metabolism, which could additionally affect
their food quality for mesozooplankton predators (Klein
Breteler et al., 1999; Boëchat and Adrian, 2005; Boëchat
et al., 2005, in press).
Studies on the food quality of planktonic prey organisms have emphasized the importance of essential biochemical compounds in promoting enhanced growth
and reproduction of zooplankton (Ahlgren et al., 1990;
Brett and Müller-Navarra, 1997; Weers and Gulati,
1997; Becker and Boersma, 2003). Among the substances
that have received considerable attention are the HUFAs
of the o3 and o6 families, such as eicosapentaenoic acid
(EPA) (20:5o3), docosahexaenoic acid (DHA) (22:6o3)
and arachidonic acid (20:4o6) as well as some polyunsaturated fatty acids (PUFAs) such as linoleic acid (18:2o6)
and linolenic acid (18:3o3). Enhanced growth and reproduction of cladocerans, especially Daphnia, have been
associated with higher contents of HUFAs and PUFAs
in cultured prey organisms (DeMott and MüllerNavarra, 1997; Park et al., 2002; Becker and Boersma,
2003) and in lake seston (Müller-Navarra et al., 2000,
2004; Park et al., 2003).
Along with essential o3 and o6 fatty acids, amino
acids (Kleppel et al., 1998; Klein Breteler et al., 1999;
Guisande et al., 2000; Boëchat et al., 2005), and more
recently the sterol composition of prey organisms (Von
Elert et al., 2003; Hasset, 2004), have been reported to be
important in limiting zooplankton nutrition. Other studies have suggested that prey mineral composition and
stoichiometry, especially the P : C and N : C ratios, also
play an important role in influencing zooplankton life
history (Sterner et al., 1992; DeMott et al., 1998; Plath
and Boersma, 2001).
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Here, we investigated how algivorous and bacterivorous protists contribute to the nutrition of rotifers.
Moreover, we specifically identified the biochemical
components of the protists that may determine their
quality as prey for rotifers. For these purposes, we performed population growth and reproduction experiments using the common rotifer Keratella quadrata
Müller, 1786, as a predator and four protist prey species
growing on either an algal or a bacterial diet. Cryptomonas
phaseolus Ehrenberg, 1832, considered a good food quality alga, and treatments without prey served as experimental controls. The previously estimated biochemical
composition of the protist prey was correlated with the
results on population growth rates and egg numbers of
K. quadrata.
To our knowledge, this is the first study to consider a
wide range of biochemical parameters to explore protist
food quality for rotifers. Our findings highlight the
importance of protists as prey for rotifers and suggest
many limiting biochemical factors driving protist food
quality for K. quadrata’s nutrition.
METHODS
Protist and rotifer cultures
The algivorous ciliates Balanion planctonicum Wullf, 1922
(3256 ± 1331 mm3, average cell volume ± SD), and
Urotricha farcta Claparède and Lachmann, 1858 (2778 ±
1707 mm3), both isolated from Lake Constance, were
cultured in Woods Hole Chu-10 (WC) medium
(Guillard and Lorenzen, 1972) in weekly 1:5 (culture :
medium) diluted batch cultures incubated at 178C under
a 12:12-h light/dark regime. The algivorous ciliates were
fed the cryptophyceae C. phaseolus Ehrenberg, 1832 (392 ±
125 mm3; strain number 2013, Algal Culture Collection,
University of Göttingen, Germany), cultured in WC medium at 17 ± 18C under a 16:8-h light/dark regime.
The bacterivorous ciliate Cyclidium sp. Müller, 1786
(1315 ± 617 mm3; obtained from the culture collection of
the University of Constance, Germany), and the bacterivorous flagellate C. paramecium Ehrenberg, 1832 (403 ± 288
mm3; strain number 977-2a, Algal Culture Collection,
University of Göttingen, Germany), were cultivated in
Volvic mineral water (a spring water, poor in minerals,
sold worldwide by Société des Eaux de Volvic, Puy-deDôme, France) and fed a mix of bacteria grown on previously autoclaved polished rice corns (Oryza sativa). Bacteria
generally comprised 20–30% of the total carbon content
in the cultures of the bacterivores. Carbon (C) concentrations of protists and their diet were derived from cell
volume estimates using carbon : cell volume conversion
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factors of 0.10 pg C mm3 for Cryptomonas (Montagnes et al.,
1994), 0.19 pg C mm3 for ciliates (Putt and Stoecker, 1989)
and 0.12 pg C mm3 for bacteria (Pelegrı́ et al., 1999). By
knowing the total C content of the protistan cultures, which
was measured using a thermal conductivity elemental analyzer (Vario EL, Elementar, Hanau, Germany), and the
bacterial C concentration derived from bacterial biovolume
in the cultures, the proportion of bacterial C was estimated.
The cultures were kept at 18 ± 18C under a 16:8-h light/
dark regime.
The rotifer K. quadrata was originally isolated from
Müggelsee, a lake located in Berlin, Germany (588130
N, 348080 E). Rotifers were cultured in WC medium in
weekly diluted (60% dilution rate) batch cultures and fed
C. phaseolus (1.5 mg C L1). Cultures were kept under the
same temperature and light/dark regimes as used for
C. phaseolus. Henceforth, our prey and predator species
are referred to by their genus names only.
Feeding experiments with K. quadrata
We performed a series of feeding experiments in which
reproduction and population growth of Keratella fed with
algivorous and bacterivorous protists were investigated.
By choosing protist prey species of similar size, shape and
mobility, we have minimized the influence of morphological and behavioral features of the protists on Keratella
population growth and reproduction. Functional
response experiments for Keratella fed with each protist
prey type (data not shown) were conducted before the
feeding experiments to determine the prey-specific incipient limiting level (ILL) and to assure that the rotifers
indeed ingest the protists. Four prey treatments were
conducted, each one with five replicates—treatment 1:
Balanion + Cryptomonas (2 103 Balanion cells mL1 + 5 103 Cryptomonas cells mL1); treatment 2: Urotricha +
Cryptomonas (2 103 Urotricha cells mL1 + 5 103
Cryptomonas cells mL1); treatment 3: Cyclidium (3.5 103 Cyclidium cells mL1) and treatment 4: Chilomonas (5
103 Chilomonas cells mL1). Control assays were run in
parallel with Cryptomonas as sole prey (5 103 cells mL1)
as well as controls without prey.
Bacterivorous protists were separated from their bacterial prey by filtering the cultures through a 10-mm mesh
net. Owing to their similar size dimensions, we could not
separate algivorous protists from their algal food, which
explains why treatments 1 and 2 contained mixed diets of
protists and Cryptomonas. To a lesser extent, this was also
the case for the protist cultures growing on bacteria and
rice corns, although previously filtration through a 10mm net decreased bacterial biomass to <2% of the total
carbon content in the cultures. To test whether differences in rotifer growth rates and egg production in these
treatments were because of the additional presence of the
algivorous protists, we kept algal density in the treatments as in the control treatment with Cryptomonas as
sole prey (5 103 cells mL1, treatment 3). The protists
were offered in concentrations above the ILL (1.0 mg C
L1; I. G. Boëchat, unpublished results) to rule out
limitation by food quantity instead of food quality. The
ILL is defined as the threshold prey concentration below
which the predator’s consumption rate increases with
increasing prey concentration. It is important to note
that algal densities present in the algivorous suspensions
during feeding experiments were in the same range as
those present in the algivorous cultures during the functional response experiments. Under these mixed prey
conditions, Keratella efficiently ingested the algivorous
protists at a rate of eight protist cells per rotifer per hour.
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 15mL chambers in macrotiter plates containing the different diets at concentrations above the ILL (1.0 mg C L1).
Microtiter plates were incubated at 18 ± 18C under a
16:8-h light/dark regime. The addition of cetyl-alcohol
pellets decreased the superficial tension in the experimental chambers, thus preventing rotifer mortality
through adherence to the surface film. The rotifers
received fresh prey suspensions daily over a 5-day experimental period. As common practice in such feeding
experiments, the predators are transferred 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 biovolume of 4 mL and adding prey suspension 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. Rotifers
and eggs were enumerated daily at 20 magnification
(stereoscope). Intrinsic population growth rates of the
rotifers were calculated for daily intervals, assuming
exponential growth according to
r ¼ lnðNt Þ lnðNt1 Þ;
where r is the intrinsic growth rate (day1) and Nt, t–1 is
the number of rotifers on consecutive days (Rothhaupt,
1995). Keratella numbers were log-transformed to assure
normality of data distribution. The overall growth rate
for the experimental period was calculated as the mean
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of the daily intrinsic growth rates. Egg numbers are
represented as cumulative curves, which indicate daily
increases in egg numbers, fitted by non-linear regression
models using the program Table Curve 2D, version 5.0
(Systat Software Inc., Richmond, California, USA).
Biochemical data
Sterol analyses were performed using gas chromatography coupled to mass spectrometry (GC–MS). Fatty acids
were analyzed using GC; however, the identities of fatty
acids were subsequently confirmed using GC–MS. A
detailed description of the biochemical analyses as well
as the biochemical composition of algivorous ciliates and
the alga Cryptomonas used in the present study is provided
elsewhere (Boëchat and Adrian, 2005; Boëchat et al., in
press). Here, we present data on the fatty acid, sterol and
amino acid composition of the bacterivorous ciliate
Cyclidium sp. and the flagellate C. paramecium. Absolute
concentration of biochemical compounds is expressed
as micrograms per carbon biomass of the protists
(protist C).
Statistical analyses
Differences in total population growth rates of Keratella
were tested for significance using one-way analysis of
variance (ANOVA) followed by Dunnett’s t-tests,
which pairwise compare experimental treatments
against control treatments (Statistica for Windows,
5.01). Increase in rotifer egg numbers in experimental
treatments was tested against control treatments using
the non-parametric Mann–Whitney U-test (Statistica
for Windows, 5.01). Rotifer growth rates and cumulative egg numbers were correlated with prey absolute
and relative amounts of fatty acids, amino acids and
sterols using the non-parametric Spearman’s rank correlation method in cases where positive growth and
enhanced egg production of the rotifers were observed.
Because of their relevance for food quality determinations,
we additionally calculated some biochemical variables
from the original data, such as the o6:o3 fatty acid ratio
and the total amounts of saturated, monounsaturated,
polyunsaturated, o6 and o3 fatty acids.
RESULTS
Feeding experiments with K. quadrata
Population growth rates of Keratella fed with algivorous
protists (treatments 1 and 2) and the alga Cryptomonas as a
sole prey (treatment 3) were significantly higher than
population growth rates of Keratella fed with bacterivorous
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protists (treatments 4 and 5; one-way ANOVA,
F = 24.01, Dunnetts t-test P < 0.001; Fig. 1). No significant differences were observed when comparing population growth rates of Keratella fed with bacterivorous
protists versus growth rates in the control treatment without prey (one-way ANOVA, F = 24.01, Dunnetts t-test,
all P > 0.05; Fig. 1).
Curves of cumulative egg numbers of Keratella were
similar for rotifer fed with Balanion and Cryptomonas (treatments 1 and 3; Mann–Whitney U-test, P > 0.05; Fig. 2).
However, the shape of the curves differed slightly, which
suggests different daily accumulation rates (Table I). A diet
of Urotricha (treatment 2) resulted in lower cumulative egg
numbers than those observed on a diet of Cryptomonas and
Balanion (Mann–Whitney U-test, P < 0.05) but higher than
those observed in the control treatment without prey
(Mann–Whitney U-test, P < 0.05). Cumulative egg numbers of Keratella in the Cyclidium- and Chilomonas-feeding
treatments (treatments 4 and 5) were lower than those
observed on a diet of Balanion or Cryptomonas as sole prey
(treatments 1 and 3; Mann–Whitney U-test, P < 0.05), but
they were higher than the cumulative egg numbers
observed in the control treatment without prey (Mann–
Whitney U-test, P < 0.05).
Biochemical composition of bacterivores
Data on the biochemical composition of algivorous protists and the algae Cryptomonas are documented in the
Fig. 1. Population growth curves of Keratella quadrata on a diet of the
alga Cryptomonas phaseolus, the protists Balanion planctonicum, Urotricha
farcta, Cyclidium sp., Chilomonas paramecium and in the control treatments
without prey. Logarithmic numbers of K. quadrata individuals
(average ± upper SD) represent at least five replicates per treatment.
Same letters indicate similar population growth rates (r) of the rotifers
when compared with the growth rates obtained in the control treatments with C. phaseolus and without prey [one-way analysis of variance
(ANOVA) followed by the Dunnetts t-test]. Population growth rates of
K. quadrata fed with C. paramecium and the alga C. phaseolus are from
Boëchat et al. (Boëchat et al., 2005).
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diploptene) were identified by their mass spectrometric
patterns. Squalene, the sterol precursor in all biosynthetic pathways, was detected in similar concentrations
in all heterotrophic protists and in their diets.
Cycloartenol, an intermediate precursor in the biosynthesis of phytosterols (Moreau et al., 2002), was identified in
Chilomonas and Cyclidium. The alcohol tertepenoid tetrahymanol, the hopanoids hopan-22-ol (diplopterol) and
hop-22(29)-ene (diploptene) were detected in Cyclidium
(11.5, 30.2 and 2.5% of the total neutral lipids fraction,
respectively).
Protist biochemical composition and
Keratella population growth rates
Fig. 2. Cumulative curves of Keratella quadrata egg numbers on a diet
consisting of the alga Cryptomonas phaseolus, the protists Balanion planctonicum, Urotricha farcta, Cyclidium sp., C. paramecium and in the control treatments without prey. Curves were fitted according to non-linear
regression models (see Table I for equations). Keratella quadrata cumulative
egg numbers (average ± upper SD) represent at least five replicates per
treatment. Cumulative egg curves of K. quadrata fed with C. paramecium
and the alga C. phaseolus are from Boëchat et al. (Boëchat et al., 2005).
studies of Boëchat and Adrian (Boëchat and Adrian, 2005)
and Boëchat et al. (Boëchat et al., in press). The concentrations of biochemical components in bacterivores (PUFAs,
amino acids and sterols) used for the correlation analyses
with Keratella egg production are summarized in Table II.
Moreover, Cyclidium presented high concentrations of saturated (116.7 mg SFA mg1 protist C) and monounsaturated
fatty acids (MUFAs) (125.8 mg MUFA mg1 protist C),
which together amounted to 70% of the total fatty acid
content of this ciliate. Chilomonas also showed high concentrations of saturated (98.9 mg SFA mg1 protist C) and
monounsaturated fatty acids (93.3 mg MUFA mg1
protist C), which together represented 57% of the total
fatty acid content of this flagellate. Besides sterols, two
sterol precursors, the alcohol tertepenoid tetrahymanol
and two tertepenoids of the hopane class (diplopterol and
Because algivorous protists and the alga Cryptomonas were
the only prey capable of sustaining population growth of
the rotifers throughout the experiments, we performed
non-parametric Spearman’s rank correlation between
population growth rates and biochemical composition
considering only feeding experiments on algivorous protists and Cryptomonas. Nevertheless, we did not detect any
significant correlation between Keratella population
growth rates and the fatty acid, amino acid nor sterol
composition of the algivorous protists and the alga
Cryptomonas (Spearman’s rank correlation, P > 0.05).
Protist biochemical composition and
Keratella cumulative egg numbers
Significantly positive correlations were found between
cumulative egg numbers and both the absolute concentration and the relative amounts of total PUFAs, glinolenic acid (18:3o6), 20:3o6, arachidonic acid
(20:4o6) and docosapentaenoic acid (DPA) (22:5o3).
Significantly positive relationships were additionally
found for the absolute EPA (20:5o3) concentration
and the relative amounts of DHA (22:6o3) and 22:o6
PUFAs (22:2o6, 22:5o6). Significantly negative
Table I: Non-linear regression parameters calculated for the cumulative egg production (y) versus
experimental time (x) (see Fig. 2 for fitting curves)
Prey organism
Equation
Fstat
r2
Dunnett’s t-test
Cryptomonas phaseolus
y = –0.28 + 6.33x ln(x)
187.91
0.82
A
Balanion planctonicum (fed the algae C. phaseolus)
ln(y) = 3.96 – 11.59ex
238.81
0.91
A
Urotricha farcta (fed the algae C. phaseolus)
y = –16.10 + 15.79x0.583
18.84
0.63
B
Cyclidium sp. (fed bacteria)
y = –14.30 + 14.03x0.533
22.02
0.48
B
Chilomonas paramecium (fed bacteria)
y = –4.11 + 4.11x0.923
15.79
0.40
B
Control without prey
y = –2.46 + 2.35x0.472
9.54
0.46
C
Same letters indicate similar cumulative egg numbers when protist prey treatments are compared to the treatments with C. phaseolus and the control
without prey (Mann–Whitney U-test). Fstat value represents the goodness-of-fit statistic. F-statistic goes toward infinity as a fit becomes better.
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Table II: Absolute concentrations of polyunsaturated fatty acids (PUFAs),
amino acids and sterols in the carbon biomass of bacterivorous protists
Cyclidium sp. and Chilomonas paramecium
Cyclidium sp.
C. paramecium
16:2o4
36.3 (2.1)
31.4 (2.8)
16:3o4
4.0 (0.2)
1.7 (0.3)
18:2o6
8.0 (1.0)
27.3 (17.6)
18:3o6
1.7 (0.2)
1.2 (0.7)
18:3o4
0.6 (0.1)
3.9 (1.7)
18:3o3
2.7 (0.3)
3.4 (0.9)
18:4o3
8.5 (0.3)
17.4 (14.0)
20:2o6
3.8 (0.3)
18.9 (17.3)
20:3o6
0.7 (0.1)
0.9 (0.5)
20:4o6
1.5 (0.3)
1.2 (0.2)
20:3o3
2.0 (0.2)
1.6 (0.2)
20:4o3
2.8 (0.2)
14.1 (12.2)
PUFAs (mg PUFA mg1 protist C)
20:5o3 [eicosapentaenoic acid (EPA)]
8.2 (0.2)
4.6 (0.5)
22:5o3 [docosapentaenoic acid (DPA)]
0.4 (0.0)
0.6 (0.0)
0.9 (0.1)
0.7 (0.4)
22:6o3 [docosahexaenoic acid (DHA)]
–
o6/o3
1.1 (0.69)
Sterols (mg sterol mg1 protist C)
Cholesterol
31.3 (14.8)
1.8 (0.9)
Dihydrocholesterol
2.1 (1.5)
Desmosterol
0.4 (0.2)
Campesterol
1.0 (0.2)
6.2 (4.5)
Stigmasterol
2.3 (0.7)
17.4 (7.3)
Sitosterol
4.1 (2.3)
15.9 (10.4)
Lathosterol
1.7 (0.4)
Ergosterol
–
Stigmastanol
–
1.5 (0.3)
1.2 (0.1)
2.1 (0.7)
1.2 (0.7)
0.7 (0.3)
Amino acids (pmol AA fg1 protist C)
His
0.4 (0.2)
1.9 (0.7)
Thr
6.7 (4.4)
11.0 (3.9)
Arg
3.0 (1.3)
6.9 (1.6)
Try
15.5 (11.4)
36.0 (6.6)
Met
2.0 (1.2)
3.8 (1.5)
Val
0.7 (0.1)
2.1 (0.2)
Phen
3.8 (2.7)
7.6 (1.8)
Isol
2.5 (1.5)
6.2 (0.4)
Leu
7.4 (5.1)
3.6 (1.4)
Lys
1.8 (1.2)
4.5 (0.6)
Values are means (±SD) of at least six analyses (n = 6); –, not detected.
correlations were observed between cumulative egg
numbers and the absolute concentration and relative
amount of linoleic acid (18:2o6) as well as the absolute
stearidonic acid (18:4o3) concentration and the 22:3o6
relative amount (Table III).
Desmosterol and ergosterol absolute concentrations
and relative amounts as well as stigmastanol relative
amounts were positively correlated with the cumulative
egg numbers of Keratella (Table III). A significantly positive relationship was observed between the relative
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Table III: Spearman’s rank correlation coefficients (R) for cumulative egg numbers of Keratella quadrata
versus prey absolute biochemical concentration (abs. conc.) and relative amounts (%)
Biochemical variables (abs. conc.)
SPolyunsaturated fatty acids (PUFAs)
RSpearman
Biochemical variables (%)
0.45*
SPUFAs
RSpearman
0.78*
0.49*
18:2o6
0.49*
18:3o6
0.56*
18:3o6
0.39*
18:4o3
0.58*
20:3o6
0.71*
20:3o6
0.54*
20:4o6
0.50*
20:4o6
0.69*
22:2o6
0.84*
20:5o3 [eicosapentaenoic acid (EPA)]
0.69*
22:3o6
0.56*
22:5o3 [docosapentaenoic acid (DPA)]
0.52*
22:5o3 (DPA)
Desmosterol
0.67*
22:5o6
0.65*
Ergosterol
0.79*
22:6o3 [docosahexaenoic acid (DHA)]
0.84*
Desmosterol
0.67*
Ergosterol
0.79*
18:2o6
0.52*
Stigmastanol
0.55*
Leucine
0.77*
No significant relationship was detected between Keratella population growth rates and prey biochemical composition.
*Significant at 95% confidence.
amount of the amino acid leucine and the cumulative
egg numbers of Keratella (Table III).
DISCUSSION
Biochemical composition of bacterivores
In general, the biochemical composition of both the
bacterivores under study reflected that of their diet,
exemplified by the high concentrations of SFAs and
MUFAs present in both the bacterivores Cyclidium and
Chilomonas and the bacterial diet. Moreover, all amino
acids analyzed were present in the bacterial diet.
However, the lipid composition found for bacterivores
diverged to some extent from most biochemical data
published for other protist species fed with bacteria. We
found that Cyclidium and especially Chilomonas contained
lipids that were not present in their bacterial diet at all or
present only in very small amounts. For example, the
presence of EPA in Cyclidium and Chilomonas as well as of
DHA and the sterols ergosterol and brassicasterol in
Chilomonas was surprising because those lipids were not
present in the bacterial diet. However, it should be kept
in mind that the bacterial assemblages used here were
grown on polished rice corns (O. sativa). Except for linoleic and linolenic acid, polished rice corns do not contain
measurable amounts of long-chain fatty acids (Souci
et al., 1994); fatty acid analyses of the rice corns used
in our study confirmed this result (I. G. Boëchat,
unpublished data). Moreover, polished rice corns contained small amounts of saturated fatty acids and
MUFAs and of the sterols sitosterol, stigmasterol, stigmastanol, campesterol and cholesterol. Neither rice nor
bacterial diet contained EPA, DHA and the sterols ergosterol and brassicasterol. Thus, polished rice may have
provided the bacterivores with some PUFAs and sterols
but not with EPA, DHA, ergosterol or brassicasterol.
However, they might also have been the source of
PUFAs and sterol precursors, which may have been
used by the protists to synthesize other fatty acids and
sterols.
Synthesis of biochemical compounds such as fatty
acids and sterols by protists has already been described
(Koroly and Connor, 1976; Klein Breteler et al., 1999).
First evidence for the synthesis of HUFAs, such as EPA
and DHA, and also sterols by the heterotrophic dinoflagellate Oxyrrhis marina (Klein Breteler et al., 1999) suggests
reconsidering the assumption that heterotrophic protists
are unable to synthesize complex lipid molecules. For
instance, because DHA was not observed in the bacterial
diet of Chilomonas, a possible explanation for the presence
of DHA in this flagellate may be DHA synthesis through
elongation of linolenic acid obtained from the rice corns.
However, synthesis of unsaturated fatty acids may be less
efficient in phagotrophic protists than in autotrophic
protists. Cryptomonas and Chilomonas are both cryptomonads, and they both contained DHA (Boëchat and
Adrian, 2005; this study, Table II). Interestingly, DHA
concentrations were higher in Cryptomonas than in
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Chilomonas. Possibly, DHA synthesis in Chilomonas is not as
efficient as in Cryptomonas because of the trophic mode of
the organisms, because Cryptomonas was cultured under
autotrophic and Chilomonas under heterotrophic conditions, but possibly also because of species-specific differences in fatty acid metabolism.
Surrogate lipids such as tetrahymanol and hopanoids
are usually found in bacterivorous ciliates, which may
be able to synthesize those neutral lipids (Harvey and
McManus, 1991; Ederington et al., 1995; Klein Breteler
et al., 1999). Our results corroborate this hypothesis,
because the triterpenoids tetrahymanol, diplopterol
and diploptene represented an important fraction of
the neutral lipid fraction found in Cyclidium but were
not found in the bacterial diet or in the polished rice
corns. Interestingly, we detected ergosterol and brassicasterol in Chilomonas although these sterols were present neither in the bacterial diet nor in the polished rice
corns used to raise bacteria. De novo synthesis of sterols
has been described for some amoebae species belonging
to the genera Acanthamoeba and Naegleria (Raederstorff
and Rohmer, 1987a, 1987b). In those species, ergosterol was the predominant sterol, with cycloartenol serving as a precursor. Cycloartenol was also found in
Chilomonas, which may have used it to synthesize
ergosterol.
Our results on the lipid composition of bacterivores
may stimulate further studies involving single-labeled
lipids in heterotrophic protists and their diet. Such studies are necessary to elucidate the mechanisms of lipid
synthesis and metabolism in freshwater heterotrophic
protists.
daphnids (Sanders and Porter, 1990; Lair and Picard,
2000). On the contrary, the inadequacy of bacterivorous
protists as food has already been demonstrated for the
rotifer B. calyciflorus fed with the flagellates C. paramecium
or ciliate Tetrahymena pyriformis Ehrenberg, 1830, grown
on a bacterial diet (Mohr and Adrian, 2002b) and for
cladocerans fed with the ciliate Cyclidium glaucoma Müller,
1773 (Bec et al., 2003), and Cyclidium sp. (De Biase et al.,
1990). When feeding on bacterivorous protists, the
biochemical variables we investigated had obviously no
primary influence on rotifer population growth rates,
because growth rates were as negative as in the control
treatments without prey. Even in cases of positive growth
rates of Keratella populations (feeding on Balanion,
Urotricha and Cryptomonas), we did not observe significant
relationships with prey biochemical composition, which
led us to conclude that Keratella’s population growth may
not be limited by the protists’ biochemical composition in
our study.
The slightly higher population growth rates of Keratella
fed with Balanion compared with Keratella fed with
Cryptomonas and Urotricha, along with significantly higher
cumulative egg numbers supported by the diet of Balanion
than those supported by the diet Urotricha, suggest a
supplementary effect of Balanion to a diet consisting
only of Cryptomonas. Because the supplementation effect
of Balanion was not because of its biochemical features,
other factors such as the protists’ elemental composition,
feeding behavior of the rotifers or interactions among
different food quality aspects might be important.
Protist biochemical composition and
Keratella population growth rates
A different picture emerged with respect to the egg
production (Table III). The significantly positive relationships between Keratella egg numbers and protists’
biochemical composition imply a primary influence of
prey biochemistry on Keratella’s nutrition. For instance,
Keratella egg numbers were positively related to the
absolute concentration and relative amounts of total
PUFAs. Indeed, feeding experiments of Keratella fed
with the bacterivorous flagellate Chilomonas supplemented
with single fatty acids support this finding (Boëchat et al.,
2005). Particularly, essential o3 PUFAs, such as EPA
(20:5o3), DPA (22:5o3) and DHA (22:6o3), have been
often found to limit growth and reproduction of many
planktonic predators. For instance, limitation of growth
and reproduction of daphnids (DeMott and MüllerNavarra, 1997; Von Elert, 2002; Bec et al., 2003) and
copepods (Jónasdóttir, 1994) was associated with low
dietary o3 fatty acid contents, for instance EPA (20:5o3)
or a-linolenic acid (18:3o3).
Interestingly, protist species growing on different diets
had similar concentration ranges of various biochemical
compounds (Boëchat and Adrian, 2005; Boëchat et al., in
press; Table II). Nevertheless, offered as prey to Keratella,
they led to significantly different population growth rates
and egg production of the rotifer. For instance, relative
amounts of PUFAs and absolute sterol concentrations in
the bacterivores Cyclidium and Chilomonas (Table II) were
partially within the same range or even higher (e.g.
cholesterol, sitosterol and campesterol) than found in at
least one algivorous species Balanion and Urotricha
(Boëchat and Adrian, 2005; Boëchat et al., in press).
However, both the bacterivores led to negative population growth rates, which did not significantly differ from
the rotifers’ growth rates in control treatments without
prey (Fig. 1). This finding contrasts previous reports that
bacterivorous protists are prey of good quality for
Protist biochemical composition and
Keratella cumulative egg numbers
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Interestingly, several positive relationships were
detected with o6 PUFAs, suggesting the importance of
those fatty acids for Keratella’s reproduction. A limited
ability for elongation and desaturation of 18-o6 precursors such as g-linolenic acid (18:3o6) into arachidonic
acid (20:4o6) has been suggested for the rotifer Brachionus
plicatilis (Lubtzens et al., 1985). It would be interesting to
follow the fate of dietary o6 PUFAs in Keratella metabolism to test whether rotifers can synthesize PUFAs.
However, the negative relationship between the Keratella
cumulative egg numbers and the absolute concentrations
and relative amounts of linoleic acid (18:2o6) as well as
with the relative amounts of 22:3o6 may indirectly suggest a limiting effect of o3 fatty acids. Owing to their
molecular resemblance, some o3 and o6 fatty acids may
compete for the same bonding sites in the membranes
(Singer, 1994). Hence, an increase of certain o6 fatty
acids and the resultant decrease in the relative amount of
their concurrent o3 fatty acids may lead to a shortfall of
active bonded o3 fatty acids, which become limiting
even though they are available in high absolute concentrations. Ratios of o6/o3 did not differ considerably
among the protists studied (Cryptomonas, Balanion,
Cyclidium and Chilomonas had a o6/o3 ratio 1, whereas
Urotricha presented a ratio of 2.1; Boëchat and Adrian,
2005; Table II, this study); however, it is possible that
concurrence mechanisms are caused by more specific
interactions between single fatty acids rather than by
the total contents of o6 and o3 fatty acids. Because we
used living prey organisms instead of artificial diets,
complex co-limitation mechanisms involving synergistic
and antagonistic interactions among biochemical substances may have affected the correlations with single
fatty acids in our study.
Sterol limitation of growth and reproduction of zooplankton predators has been found to be mainly caused
by dietary cholesterol shortage (Von Elert et al., 2003;
Hasset, 2004). Cholesterol plays an important role in
membrane stabilization and acts as a precursor in hormone synthesis (Moreau et al., 2002). Decreased growth
of copepods (Hasset, 2004) and Daphnia (Von Elert et al.,
2003) and retarded development of crustacean larvae
(Teshima, 1991) have been associated with cholesterol
deficiency. Using our experimental set-up, we detected
no direct influence of cholesterol on population growth
and reproduction of Keratella. However, we did observe a
strong positive relationship between Keratella cumulative
egg numbers and the C-27 sterol desmosterol. Assuming
that cholesterol concentrations were limiting rotifers reproduction, we hypothesize that Keratella may have converted
dietary desmosterol to obtain cholesterol. Conversion of
desmosterol into cholesterol has been described for a
wide range of organisms, including crustaceans (Teshima
et al., 1982), mollusks (Knauer et al., 1998) and insects
(Ikekawa, 1985).
The positive relationship with ergosterol, a C-29 sterol
only detected in Cryptomonas and Urotricha (Boëchat et al.,
in press) as well as Chilomonas (Table II) in our studies,
suggests an important role of this sterol for Keratella’s
reproduction. Ergosterol is widespread in fungi, and its
physiological functions have been mainly elucidated in
studies on yeast. Enhanced growth and reproduction of
crustacean larvae have been attributed to high dietary
ergosterol levels (Kanazawa et al., 1971; Teshima and
Kanazawa, 1986), suggesting a possible C-24 dealkylation of ergosterol to cholesterol in crustaceans. Teshima
(1982) proposed a pathway of dealkylation of C-28 to C29 sterols via desmosterol to cholesterol in crustaceans.
Whether ergosterol is converted into cholesterol in
Keratella, thus diminishing dietary cholesterol deficiency
is still an open question.
The positive relationship observed between the
Keratella egg numbers and the relative amounts of stigmastanol, the only saturated sterol we found in the
studied prey organisms, is rather difficult to explain.
It could indicate a limitation originating from
increasing sterol : stanol ratios. The effect of another
phytostanol—sitostanol—on the solubilization of cholesterol by model dietary-mixed micelles was examined
under in vitro conditions (Mel’nikov et al., 2004). Free
sitostanol was shown to decrease the concentration of
cholesterol in the dietary-mixed micelles via a dynamic
competition mechanism (Mel’nikov et al., 2004).
However, because stigmastanol was the only saturated
sterol observed in our samples, such a limitation
mechanism is unlikely in our prey organisms.
Studies on the effect of dietary amino acids on life
history traits of zooplanktonic predators are rare. Top
predators, such as the rotifer B. plicatilis Müller, 1786,
showed rather constant amino acid composition when
fed different algal species or the yeast Saccharomyces cerevisae Meyen and Hansen, 1883 (Frolov et al., 1991).
Guisande et al. (1999) concluded that the selective retention of amino acids in Euterpina acutifrons Dana, 1848, was
based on a chemical homeostasis of essential amino acids
in this copepod. In our study, we found a positive correlation between the egg production of Keratella and the
relative amounts of leucine. Leucine has been found to
be an essential amino acid for the egg production and
larval development of many species including fish (Fyhn,
1989; Dayal et al., 2003), crustaceans (Reddy, 2000),
copepods (Laabir et al., 1999) and insects (Chang,
2004). Furthermore, the biochemical composition of
copepod eggs is correlated with relative amounts of
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leucine in the copepod’s diet (Laabir et al., 1999). To our
knowledge, this study is the first to report the effects of
leucine on reproduction of a rotifer species.
Keratella population growth rates versus
egg production
The fact that biochemical compounds were not correlated with population growth rates but with cumulative
egg numbers suggests that biochemical requirements for
population growth differ from those for egg production,
in the case of Keratella fed with protists. This is in accordance with the stoichiometric theory that states that
growth, reproduction and survivorship have different
metabolic demands (Sterner and Hessen, 1994). In our
study, the observed effects of biochemical compounds
were rather related to Keratella reproduction, measured
as the cumulative numbers of produced eggs. Fatty acid
and sterol effects may be related to egg production and
maintenance until hatching. Increased egg production of
the copepod Acartia tonsa Dana, 1848, was found to be
positively influenced by the o3 fatty acid composition of
its algal diet (Jónasdóttir, 1994). Teshima et al. (Teshima
et al., 1982) showed that cholesterol is indispensable for
the normal metamorphosis from nauplii to post-larvae
and the survival of the larval prawn, Panulirus japonica
Von Siebold, 1824.
In a recent study, limitation effects of an essential fatty
acid on Daphnia life history could only be detected below
a specific prey nutrient threshold (Becker and Boersma,
2003). Above this threshold, the daphnids were limited
by elemental stoichiometric ratios. It seems that, depending on the developmental stage of daphnids, mineral
limitation may prevail over biochemical limitation.
However, such limitation effects are more apparent in
studies in which limitation of either mineral or biochemical compounds is artificially induced, generally by supplementing prey organisms with one compound in
detriment to another. Here, we found support for nutritional limitation in a trophic relationship between a
rotifer species and five different prey organisms, whose
original biochemical or mineral composition was not
artificially modified. Our data suggest a primary role of
protist biochemistry for Keratella reproduction but only a
secondary role for Keratella population growth, although
we could not identify the primary limitation factors in the
latter case. Müller-Navarra et al. (Müller-Navarra et al.,
2004) elegantly showed how limitation of o3 fatty acids
for Daphnia may be predicted in situ when coupled to the
trophic state and the phytoplankton composition of lakes,
thus demonstrating how coupled factors and mechanisms
drive nutritional limitation in nature. Furthermore,
under laboratorial conditions, phosphorus limitation
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was observed to cause significantly decreased growth
rates of the rotifer Anuraeopsis Lauterborn, 1900 (CondePorcuna, 2000). Also, higher growth rates and enhanced
survival of the rotifer Keratella cochlearis Gosse, 1851, were
found by feeding the rotifers with nitrogen- and phosphorus-limited algae (Ramos-Rodrı́guez and CondePorcuna, 2003). Finally, differences in the elemental
stoichiometry of N and P between bacterivorous protists
and Keratella as well as the presence of toxic secondary
metabolites in the protists (Mitra and Flynn, 2005) may
have negatively affected assimilation rates and therefore
growth and reproduction of Keratella fed with bacterivorous protists. Whether mineral constraints are of primary
importance for rotifers’ population growth is still a question to be answered.
By selecting protist species of similar size, morphology
and mobility, we intended to limit the variability of food
quality to aspects related to the protist biochemical composition. Nevertheless, the influence of non-quantified
factors, such as vitamins and elemental composition,
and especially of synergetic and/or antagonistic interactions among biochemical compounds and the effects of
secondary metabolites cannot be excluded. Our data
indeed suggest nutritional limitation of Keratella fed with
protists, although biochemical limitation effects were
only evident for Keratella’s reproduction but not for population growth. Synergistic and/or antagonistic aspects of
simultaneous mineral and biochemical limitation may
have been responsible for the absence of significant correlations between Keratella population growth and prey
biochemistry in our study.
In a broader ecological sense, our study emphasizes
protists as an alternative source of essential substances for
mesozooplankton predators and as an important link
between microbial and classic food webs. However,
more research is necessary to understand and to couple
biochemical composition and limitation mechanisms
with food quality of protists for zooplankton predators.
Although correlation analysis between the chemical composition of prey organisms and the life history parameters
of predators has proved extremely valuable to understand the nutritional quality of prey organisms in aquatic
food webs, it became clear that the specific nutritional
quality of distinct biochemical compounds cannot be
detected only by correlation analyses, given the complex
nature of metabolic pathways. Labeling techniques are
favorable to overcome these shortcomings.
ACKNOWLEDGEMENTS
We thank C. Enderes, D. Pflanz and M. Harz for helping
with sample processing and maintenance of the cultures.
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We thank A. Krüger, J. Ogunji and M. Wirth for their
assistance with the biochemical analyses. This article
benefited from helpful comments of B. Gücker and R.
Willmott. T. Weisse is acknowledged for providing the
cultures of B. planctonicum and U. farcta and K. O.
Rothhaupt for the cultures of Cyclidium sp. This research
was supported by a PhD grant to I.G.B from the
Deutscher Akademischer Austauschdienst (DAAD).
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