RESEARCH ARTICLE
The influence of bacteria-dominated diets on Daphnia magna
somatic growth, reproduction, and lipid composition
Sami J. Taipale1, Michael T. Brett2, Katja Pulkkinen1 & Martin J. Kainz3
1
Department of Biological and Environmental Sciences, University of Jyväskylä, Jyväskylä, Finland; 2Department of Civil and Environmental
Engineering, University of Washington, Seattle, WA, USA; and 3WasserCluster Lunz – Biological Station, Danube University Krems, Lunz am See,
Austria
Correspondence: Sami J. Taipale,
Department of Biological and Environmental
Sciences, University of Jyväskylä, Jyväskylä,
Finland. Tel.: +35 814 260 2344;
fax: +35 814 617 239; e-mail:
sami.taipale@jyu.fi
Received 26 August 2011; revised 14 March
2012; accepted 2 May 2012.
Final version published online 24 May 2012.
DOI: 10.1111/j.1574-6941.2012.01406.x
Editor: Riks Laanbroek
Keywords
bacteria; Daphnia; fatty acid; stable isotopes;
MICROBIOLOGY ECOLOGY
methane oxidizing bacteria; heterotrophic
bacteria.
Introduction
The importance of bacteria as conveyors of dietary energy
to upper trophic levels in lakes is the subject of much
conjecture (Wetzel, 1995; Jansson et al., 2007) because of
the observations that phytoplankton production is insuffi-
cient to solely support zooplankton production in some
systems (Hessen et al., 1990). Further, whether the bio-
chemical composition of bacteria is conducive to zoo-
plankton somatic growth and reproduction remains
poorly understood. Previous studies revealed that Daphnia
can consume bacteria and ingest them as well as phyto-
plankton; in fact, some studies have concluded that
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Abstract
We explored how dietary bacteria affect the life history traits and biochemical
composition of Daphnia magna, using three bacteria taxa with very different
lipid composition. Our objectives were to (1) examine whether and how bacte-
ria-dominated diets affect Daphnia survival, growth, and fecundity, (2) see
whether bacteria-specific fatty acid (FA) biomarkers accrued in Daphnia lipids,
and (3) explore the quantitative relationship between bacteria availability in
Daphnia diets and the amounts of bacterial FA in their lipids. Daphnia were
fed monospecific and mixed diets of heterotrophic (Micrococcus luteus) or met-
hanotrophic bacteria (Methylomonas methanica and Methylosinus trichosporium)
and two phytoplankton species (Cryptomonas ozolinii and Scenedesmus
obliquus). Daphnia neonates fed pure bacteria diets died after 6–12 days and
produced no viable offspring, whereas those fed pure phytoplankton diets had
high survival, growth, and reproduction success. Daphnia fed a mixed diet with
80% M. luteus and 20% of either phytoplankton had high somatic growth, but
low reproduction. Conversely, Daphnia fed mixed diets including 80% of either
methane-oxidizing bacteria and 20% Cryptomonas had high reproduction rates,
but low somatic growth. All Daphnia fed mixed bacteria and phytoplankton
diets had strong evidence of both bacteria- and phytoplankton-specific FA bio-
markers in their lipids. FA mixing model calculations indicated that Daphnia
that received 80% of their carbon from bacteria assimilated 46 ± 25% of their
FA from this source. A bacteria–phytoplankton gradient experiment showed a
strong positive correlation between the proportions of the bacterial FA in the
Daphnia and their diet, indicating that bacterial utilization can be traced in this
keystone consumer using FA biomarkers.
Daphnia have similar assimilation efficiencies for bacteria
and phytoplankton (Monakov & Sorokin, 1961; McMahon
& Rigler, 1965; Lambert, 1974; Peterson et al., 1978).
However, Daphnia feeding experiments with natural bac-
terial diets as well as bacteria monocultures (Pace et al.,
1983; Ojala et al., 1995; Kankaala et al., 2006a, b; Martin-
Creuzburg et al., 2011) indicate bacteria are poor-quality
resources compared with phytoplankton.
The different food quality of phytoplankton and bacte-
ria for zooplankton is because of their very different
amounts and composition of essential elements (e.g.
nitrogen and phosphorus) and biochemicals [e.g. fatty
acids (FA) and sterols]. While phytoplankton contain
FEMS Microbiol Ecol 82 (2012) 50–62
Bacterial diet influences on Daphnia
varying amounts of omega-3 (x-3) and x-6 polyunsatu-
rated fatty acids (PUFA) (Viso & Marty, 1993) that are
essential to support zooplankton somatic growth and
reproduction (Müller-Navarra et al., 2000), PUFA are
usually absent in bacteria (Harwood & Russell, 1984).
Bacterial FA are generally regarded as low in dietary qual-
ity for crustacean zooplankton (Goulden & Henry, 1984;
Napolitano, 1998). Additionally, phytoplankton synthesize
a wide variety of sterols, whereas bacteria do not contain
these essential components of eukaryotic membranes with
the exception of some methylotrophic and mycobacteria
(Volkman, 2003). Recent experiments with Daphnia using
five heterotrophic bacteria as food showed that a lack of
sterols might be a major food quality constraint for bac-
teria (Martin-Creuzburg et al., 2011).
Zooplankton production in freshwater systems depends
on the consumption of phytoplankton, bacteria, protozoa,
and detritus (Jones, 1992; Sherr & Sherr, 2002). While
monoculture experiments (e.g. Martin-Creuzburg et al.,
2011) suggest bacteria are insufficient diets for Daphnia
survival, experiments with natural bacteria suspensions
indicated bacteria to be at least an adequate food supple-
ment for Daphnia (Pace et al., 1983; Ojala et al., 1995;
Kankaala et al., 2006b). For example, biomass accrual of
Daphnia longispina fed bacteria under experimental
conditions was similar compared with a green algae diet
(Ojala et al., 1995), which has been shown to be interme-
diate food quality for zooplankton (Brett et al., 2006).
Similarly, Kankaala et al. (2006a, b) concluded that
Daphnia can grow and reproduce on a mixed methane-
oxidizing bacteria (MOB) and phytoplankton diet. The
somatic growth of D. longispina fed on MOB was related
to increasing food quantity, whereas the dietary quality of
MOB for D. longispina seems to be low compared with
phytoplankton (Kankaala et al., 2006a, b). However,
experiments with natural bacterial suspensions may also
contain some flagellates that may be able to upgrade the
biochemical quality of the food they metabolize (e.g. Bec
et al., 2003a). Thus, in natural systems, the occurrence of
bacteria together with algal sources of essential FA and
sterols may allow bacteria to be used as an additional
resource for Daphnia more so than bacteria monoculture
experiments would indicate. It is thus ecologically impor-
tant to study how bacteria mixed with phytoplankton
affect Daphnia survival, somatic growth, and reproduc-
tion.
Stable isotopes are a common tool to investigate the
food sources of aquatic organisms (Grey et al., 2001).
However, bulk stable isotopes make it difficult to define
the contribution of phytoplankton, bacteria, protozoa,
and detritus in Daphnia, or among different types of phy-
toplankton or bacteria (but see Boschker & Middelburg,
2002). Conversely, the FA composition usually differs
FEMS Microbiol Ecol 82 (2012) 50–62
51
considerably among the major bacteria and phytoplank-
ton taxa, which provides the opportunity to use FA as
source-specific biomarkers (Desvillettes et al., 1997;
Dalsgaard et al., 2003). Although laboratory experiments
have shown that Daphnia FA profiles closely match those
of their algal diets (Brett et al., 2006), only a few studies
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have investigated the trophic transfer of bacterial FA
(Kates, 1964; Ratledge & Wilkinson, 1988). Ederington
et al. (1995) fed bacterivorous ciliates to herbivorous
copepods and found that iso- and anteiso-branched C15
and C17 FA were transferred from bacteria-fed ciliates to
copepods, showing that these FA were conveyed across
multiple trophic levels. Subsequently, Stevens et al. (2004)
carried out similar studies with the herbivorous copepod
Calanus glacialis, but did not find similar trophic transfer
for branched FA, and suggested that the monounsatu-
rated fatty acid (MUFA) 18:1x-7 might be a better bacte-
rial biomarker for marine copepods. However, Bec et al.
(2003b) showed that branched FA are accumulated in dif-
ferent lipid classes (phospholipid fatty acid and neutral
lipid fatty acid) of the benthic cladoceran Simocephalus
vetulus. There are fewer studies with gram-negative bacte-
ria that contain C16 or C18 MUFA. Deines et al. (2007)
fed Chironomus larvae 13C-labeled Methylosinus trichospo-
rium and demonstrated trophic transfer of C16 and C18
MUFA. Although there is some evidence indicating that
bacterial FA are transferred through aquatic food webs
and may be an important energy and lipid source for
upper trophic levels, quantitative relationships between
bacterial diets and lipids in zooplankton are needed to
show that this transfer is systematic.
In an effort to better understand the nature of bacte-
rial contributions to zooplankton production and bio-
chemical composition, we designed a series of laboratory
experiments to (1) examine how bacteria-dominated
diets affect Daphnia survival, growth, and fecundity; (2)
delineate how bacteria taxa-specific FA biomarkers
accrue in Daphnia (using bacteria taxa with very differ-
ent FA profiles); and (3) explore the quantitative rela-
tionship between bacteria availability in Daphnia diets
and the amounts of bacterial FA in Daphnia lipids. In a
life table experiment, we used three different types of
bacteria with distinct FA profiles combined with two
phytoplankton diets, which enabled us to study the
influence of bacterial diets on somatic growth and pro-
duction of Daphnia and the transfer of different types of
bacterial FA to Daphnia. Additionally, we fed Daphnia a
gradient of heterotrophic bacteria mixed with phyto-
plankton to obtain quantitative relationships between
bacterial diets and the concentration of bacterial FA in
Daphnia. This enabled us to assess the applicability of
bacterial FA as dietary biomarkers for this important
aquatic herbivore.
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52
Methods
Zooplankton and phytoplankton cultures
All experiments were conducted using a clone of Daphnia
magna (isolated at UC Davis) initially grown on Scenedes-
mus obliquus. Cryptomonas ozolinii LB 2782 (obtained
from the University of Texas Culture Collection of Algae)
and S. obliquus (from the Max Planck Institute of Lim-
nology) were cultivated using L16 growth medium (Lind-
ström, 1983), supplemented with B vitamins for
Cryptomonas. Both phytoplankton were grown at 18 °C
on a 14 h : 10 h light/dark cycle, and all experiments
were conducted at 19 ± 1 °C in a dark room.
Bacteria cultures
The heterotrophic gram-positive bacterium, Micrococcus
luteus (ATCC 4698), was cultivated using tryptic soy broth
media in serum vials (150 mL) at 30 °C for 48–60 h, with
new cultures started from plate colonies every second day.
MOB Type I Methylomonas methanica (LW13) and MOB
Type II M. trichosporium (OB3b) were cultivated using
nitrate mineral salts medium (NMS; 30 mL) (Whittenbury
et al., 1970) under a methane and air gas phase (50 : 50 v/
v) and incubated at 30 °C for 2 days. The purity of these
cultures was checked using solid NMS medium supple-
mented with 10% LB Broth Miller medium (Luria–Bertan-
i; Difco). New inoculates were started every third day. For
the experiments, bacteria suspensions were centrifuged
and the pellets diluted into L16 medium. For calculating
the mass of food suspensions, it was assumed that their
carbon content was 50% of dry weight.
Experimental design of life table experiment
with bacterial diets
For all experiments, D. magna neonates (5 ± 3 h old)
were divided randomly among treatments to minimize
maternal effects. Neonates were placed singly into glass
vials (40 mL) with each treatment using 10 or 15 repli-
cates. The medium was changed and the animals fed
every 2 days (see below).
Table 1. Length, carbon, and nitrogen content, and major FAs of used bacteria and phytoplankton species
Bacteria or phytoplankton Length (µm)
Methylomonas methanica 0.5–3.0
Methylosinus trichosporium 2.1–3.0
Micrococcus luteus 0.5–3.5
Scenedesmus obliquus 6–18
Cryptomonas ozolinii 14–80
DHA, docosahexaenoic acid.
ª 2012 Federation of European Microbiological Societies
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S.J. Taipale et al.
Daphnia were fed pure suspensions (5 mg C L 1) of
M. luteus (ATCC 4698), M. methanica (LW13), and
M. trichosporium (OB3b, see Table 1), intermediate-
quality phytoplankton diet (100% Scenedesmus), or a
high-quality diet (100% Cryptomonas) at a food concen-
tration of 5 mg C L 1 (‘high dietary phytoplankton
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contribution’) and 1 mg C L 1 (‘low dietary phytoplank-
ton contribution’). In freshwater and marine ecosystems,
consumers are often exposed to different dietary supplies
of bacteria and phytoplankton. Therefore, to test for the
influence of bacteria-dominated diets, D. magna were fed
with 80% (4 mg C L 1) bacteria and 20% (1 mg C L 1)
phytoplankton for each of the three species of bacteria
and two phytoplankton species, resulting in a total of six
different diet mixtures of bacteria and phytoplankton.
Thus, in addition to the pure diet supply of either bacte-
ria or phytoplankton (see above), we used the following
diet mixes:
• Scenedesmus (20%) and M. luteus (80%),
• Scenedesmus (20%) and M. methanica (80%),
• Scenedesmus (20%) and M. trichosporium (80%),
• Cryptomonas (20%) and M. luteus (80%),
• Cryptomonas (20%) and M. methanica (80%), and
• Cryptomonas (20%) and M. trichosporium (80%).
The body size (from the top of the head to the base of
the tail spine) of D. magna was measured 14 days after
the start of the experiments and the cumulative number
of offspring (including eggs and neonates) recorded. All
Daphnia were then placed into Eppendorf® vials
(1.5 mL), freeze-dried, and stored at 80 °C until FA
analysis. Dry weights of phytoplankton and bacteria were
estimated using a Hach® turbidity meter that was cali-
brated against gravimetric dry weight measurements.
Thus, 50 mL of bacteria or phytoplankton was centri-
fuged after which the pellet was freeze-dried and weighed.
A calibration curve was generated using four different
phytoplankton concentrations. A single calibration curve
was generated for all bacteria cultures.
One-way ANOVA was used to compare treatment means
of Daphnia size. For offspring results, we used the
Brown–Forsythe test (Brown & Forsythe, 1974) because
of unequal variance between treatments, and the Tukey’s
% of C % of N C:N FAs
36.5 8.6 4.2 16:1x7, 16:1x8, 16:1x6 and 14:0
38.4 7.9 4.9 18:1x8 and 18:1x7
43.8 11.6 3.6 iso -15:0 and anteiso -15:0
46.2 8.7 5.3 LIN, ALA, SDA, 16:4x3 and 16:3x3
44.5 9.6 4.6 LIN, ALA, SDA, EPA and DHA
FEMS Microbiol Ecol 82 (2012) 50–62
Bacterial diet influences on Daphnia
range test as our post hoc test to determine which treat-
ment means differed. We used correlation analysis to
determine how size and reproduction correlated with x-3
FA in Daphnia. We calculated the proportion of Daphnia
FA in the mixed diet treatments originating from the bac-
teria diet by comparing the actual Daphnia FA profiles in
the mixed treatments to hypothetical Daphnia FA profiles
obtained by ‘mixing’ actual bacteria and phytoplankton
FA profiles. We then used solver to find the proportion
of bacteria that maximized the fit between the observed
and predicted profiles according to Brett et al. (2009a).
Specifically, we used the algorithm: Predicted mixed
diet FA profile = X 9 bacteria FA profile + (1 X) 9
phytoplankton FA profile, where X equals the hypotheti-
cal proportion of bacterial FA incorporated by D. magna.
We then used the Solver® function in Excel® to find the
value of X that maximized r2 and/or minimized the error
sum of squares (Error SS) between the observed and pre-
dicted FA profiles for these diets.
Bacterial gradient experiment
To study whether bacterial biomarker FA were quantita-
tively transferred to D. magna, a M. luteus diet gradient
experiment was carried out. For this experiment, 15
D. magna juveniles (5 ± 3 h old) were preconditioned for
7 days in glass jars (altogether 10 jars) with 300 mL media
and fed ad libitum with S. obliquus (which has no eicosa-
pentaenoic acid; EPA, 20:5x3). After 7 days, D. magna
were randomly divided into groups of nine individuals into
glass jars with 300 mL media. There were five experimental
treatments: 95% bacteria (M. luteus)–5% phytoplankton
(Cryptomonas), 85–15%, 70–30%, 50–50%, and 0–100%,
with three replicates each. This created a bacterial concen-
tration gradient of 4.75, 4.25, 3.5, 2.5, and 0 mg C L 1,
with a total food concentration of 5 mg C L 1. The Scene-
desmus diet was switched to Cryptomonas to quantify the
effect of EPA and to have a more distinctive carbon stable
isotope values for diet calculations. The number of eggs
and neonates was counted to monitor reproduction suc-
cess. After 7 days of being fed these diet mixtures, the size
of four Daphnia from each jar was measured. Thereafter,
all Daphnia were kept for 2 h in algae-free media to evacu-
ate their guts and subsequently freeze-dried and kept at
80 °C until FA and stable isotope analyses.
One-way ANOVA was used to compare the treatment
means for Daphnia size, and a Dunnetts one-way t-test
was used to determine which treatments reduced Daphnia
size compared with pure Cryptomonas. We also used the
Brown–Forsythe test (Brown & Forsythe, 1974) for num-
ber of juveniles because the treatment variances were not
equal. We used Pearson correlation and linear regression
analysis to characterize relationships between iso- and
FEMS Microbiol Ecol 82 (2012) 50–62
53
anteiso-branched FA concentrations of Daphnia and their
bacterial diet. We also used Pearson correlation analysis
to determine how the size and number of offspring corre-
lated with the x-3 concentrations of Daphnia.
We calculated the proportion of M. luteus FA in Daphnia
in the M. luteus–C. ozolinii gradient experiment by
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comparing the actual Daphnia FA profiles for the mixed
diet treatments to hypothetical Daphnia FA profiles
obtained by ‘mixing’ the FA profiles obtained for the
M. luteus and Cryptomonas diets to find the proportion
of M. luteus FA that maximized the fit between the
observed and predicted Daphnia FA profiles. Specifically,
we used the algorithm: Predicted mixed diet FA pro-
file = X 9 (M. luteus FA profile) + (1 X) 9 (Crypto
FA profile), where X equals the hypothetical proportion
of M. luteus FA retained by Daphnia. We then used the
Solver® function in Excel® to find the value of X that
maximized the correlation coefficient (r) between the
observed and predicted FA profiles. These mixing models
generally resulted in good fits (i.e. r2 = 0.78–0.86)
between the observed and hypothetical FA profiles.
FA analyses
Lipids from freeze-dried, homogenized phytoplankton
(1–4 mg), bacteria (0.4–1.5 mg), and zooplankton (0.3–4 mg)
samples were extracted using a 4 : 2 : 1 chloroform/meth-
anol/water mixture as reported elsewhere (Heissenberger
et al., 2010). In brief, samples were sonicated and vortexed
(3X), and the organic phases were removed and pooled.
For the formation of FA methyl esters (FAME), toluene
and a sulfuric acid–methanol solution was used. FAME
were analyzed with a gas chromatograph (HP 6890)
equipped with a flame ionization detector and separated
using an Agilent® DB-23 column (30 m 9 0.25 mm 9
0.15 lm). FA concentrations were calculated using calibra-
tion curves based on known standard solutions (i.e. 2.75,
55, 125, 250, 500, 1000, and 2000 ng lL 1) of a FAME
standard mixture. The Pearson correlation coefficient was
> 0.99 for each individual FA calibration curve.
Stable isotope analyses
Carbon and nitrogen isotope analyses were used to
calculate the assimilated carbon and nitrogen from the
different diets. Freeze-dried, homogenized phytoplankton,
bacteria, and zooplankton were weighed (0.6–1.0 mg) in
tin cups for d13C and d15N analyses, which were carried
out on a Carlo-Erba Flash 1112 series Element Analyzer
connected to a Thermo Finnigan Delta Plus Advantage
Isotope Ratio Mass Spectrometry (IRMS) at the Univer-
sity of Jyväskylä, Finland. These samples were com-
pared with the NBS-22 standard using fish muscle as a
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Published by Blackwell Publishing Ltd. All rights reserved
54
laboratory-working standard. The precision of the d13C
and the d15N analyses was 0.2& and 0.3&, respectively,
for all samples.
The d13C and d15N values of Daphnia and diet sources
were measured to calculate ingested carbon and nitrogen
content of the diet sources. Actual calculation was per-
formed using a two-source mixing model (Phillips &
Gregg, 2003; Fry, 2006). In these mixing model calcula-
tions, the d13C and d15N of the phytoplankton diet com-
ponents were measured directly from Daphnia fed on
either phytoplankton diet, thus including isotopic frac-
tionation. However, the fractionation of bacterial carbon
and nitrogen to Daphnia was calculated to be 0.5& and
2.0&, respectively, when Daphnia were fed M. luteus for
6 days. Nitrogen fractionation from bacteria to Daphnia
was also estimated using the trophic enrichment equation
by Adams & Sterner (2000): y = 0.15x 0.10, where y is
the trophic enrichment of d15N from bacteria to Daphnia
and x the carbon-to-nitrogen ratio of bacteria. The d13C
values of Daphnia were used directly as well as lipid cor-
rected using the modified equation of Smyntek et al.
(2008): d13C of sample + 6.4 [(initial C : N ratio of sam-
ple 4)/initial C : N ratio of sample].
(a)
(b)
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S.J. Taipale et al.
Results
Life table experiment with bacterial diets
Somatic growth and offspring
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All Daphnia fed the three different pure bacteria diets
died between 6 and 12 days and produced no viable off-
spring. No biochemical analyses were performed on these
dead individuals as rapid organic matter degradation
postmortem may have altered their biochemical composi-
tion. Daphnia fed the mixed diets had similar survival (80
–100%) compared with Daphnia fed only pure phyto-
plankton diets, except for the mixed diet of Methylosinus
and Scenedesmus that only had 30% survival. Daphnia
size differed significantly among the treatments (ANOVA;
F9,78 = 39.11, P = 0.001) and formed five homogenous
groups (Tukey’s range test, Fig. 1a). Daphnia fed a high
concentration (5 mg C L 1) of 100% Cryptomonas were
the largest after 14 days (Fig. 1a). The size of Daphnia
fed with a high concentration of 100% Scenedesmus (dif-
ference = 0.2 mm) or a mixed diet of Micrococcus and
Scenedesmus (difference = 0.3 mm) was statistically
Fig. 1. Results of the life table experiment
with bacterial diet. (a) Size of adult Daphnia
(mm) and (b) number of offspring (eggs or
neonates present in the brood pouch at the
end of the experiment summed together per
individual) after 14 days of the different diets:
low concentration of phytoplankton (low),
mixed bacteria (80% of total C) and
phytoplankton (20% of total C, phyto), and
high concentration of phytoplankton. Bacteria:
Micrococcus luteus (ML), Methylomonas
methanica (MOB1), and Methylosinus
trichosporium (MOB2). Phytoplankton:
Cryptomonas ozolinii and Scenedesmus
obliquus. Homogenous subsets (Tukey’s range
test) are marked as small letters (a–e in size
and a–c in offspring).
FEMS Microbiol Ecol 82 (2012) 50–62
Bacterial diet influences on Daphnia
similar to the Cryptomonas diet (100%, high concentra-
tion) and belonged to the same homogenous subset
(Fig. 1a), whereas the other Daphnia treatments were sig-
nificantly smaller (Tukey’s range test, P < 0.05). Daphnia
fed a mixed diet of Scenedesmus and either MOB type
formed a homogenous subset, and they were significantly
smaller than Daphnia fed on any other diet.
The number of offspring differed significantly among
treatments (ANOVA; F9,86 = 10.67, P = 0.001; the Brown–
Forsythe test F9,18.57 = 9.26, P = 0.001), forming three
homogenous subsets (Tukey’s range test, Fig. 1b). The
highest reproduction occurred when Daphnia were fed
either high Cryptomonas (23.7 ± 18.8 neonates) or Methy-
lomonas mixed with Cryptomonas (25.1 ± 9.9 neonates;
Fig. 1b). Methylosinus mixed with Cryptomonas and high
Scenedesmus had an intermediate reproduction grouping
together with the highest reproduction group, but also
with Micrococcus mixed with either phytoplankton
(Tukey’s range test, P < 0.05, Fig. 1b). The lowest fecundity
was measured when Daphnia were fed a low concentra-
tion of either type of phytoplankton (4.4 ± 3.6 neonates)
or Scenedesmus mixed with any type of bacteria
(3.6 ± 8.2 neonates). FA groups did not correlate signifi-
cantly with either size of Daphnia or number of offspring
(Pearson correlation, P > 0.05). However, the contribu-
tion of x-3 in Daphnia correlated marginally (r = 0.68,
P = 0.063) with the body size of Daphnia.
Stable isotopes
The differences in d13C values were larger among the bac-
teria diets than between the two phytoplankton (Fig. 2).
Even after accounting for within culture variation, all of
the bacteria and phytoplankton used for these experiments
Fig. 2. The d13C and d15N values of bacterial
diet (white circle): Micrococcus luteus (ML),
Methylomonas methanica (MOB1), and
Methylosinus trichosporium (MOB2);
phytoplankton diet (white square):
Cryptomonas ozolinii (Crypto) and
Scenedesmus obliquus (Scene); and Daphnia
[Dph, black circle (bacterial dominated diet) or
black square (phytoplankton diet)].
FEMS Microbiol Ecol 82 (2012) 50–62
55
could be clearly distinguished from each other according
to their d13C values, except for Micrococcus and Scenedes-
mus. As expected, the d13C values were most depleted in
MOB. The two MOB species additionally varied in their
d15N values, making it possible to separate these diets.
The d13C and d15N values of Daphnia showed diet
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influenced isotopic values (Fig. 2). Based on these results,
we calculated that Daphnia trophic enrichment was
1.7& for d13C and 1.2& for d15N when they consumed
Scenedesmus and 0.8& and 1.0&, respectively, when they
consumed Cryptomonas. According to the d13C results,
the contribution of bacteria to Daphnia in the mixed
diets was 40–64% and 45–59% of carbon source without
and with lipid correction, respectively (Table 2). Accord-
ing to the d15N calculations, Daphnia obtained 37–80%
of their nitrogen from bacteria.
FA composition of bacteria and phytoplankton
The three bacteria and two phytoplankton used for these
diet mixing experiments all had very distinct FA profiles
(see Fig. 3): iso- and anteiso-branched 15:0 the dominant
FA (90%) of M. luteus, whereas C16 and C18 MUFA,
respectively, dominated (> 60%) the FA of Methylomonas
and Methylosinus; M. methanica also had considerable pro-
portions of the saturated fatty acid (SAFA) 14:0 (18.6%),
16:0 (8.2%), and 18:0 (8.5%), whereas M. trichosporium
contained 18% of 18:0. The MUFA 16:1x-8 was diagnostic
for Type I MOB as was 18:1x-8 for Type II MOB. The
three bacteria species used for these experiments contained
only traces or no x-6 and x-3 PUFA. Because linoleic acid
(LIN; 18:2x-6) contributed < 1% of all bacterial FA, it was
assumed that fungal contamination of the bacterial cultures
was minimal (Ratledge & Wilkinson, 1988).
ª 2012 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
56 S.J. Taipale et al.

Table 2. Experimental setup of life table experiment with bacterial diet and the contribution of added bacteria (bact) and calculated assimilated
(assim) contribution by d13C without and with lipid correction (lipid cor.), by d15N with measured (from Daphnia fed Micrococcus luteus for
6 days) and estimated nitrogen fractionation (frac, based on carbon and nitrogen ratio) between bacteria and Daphnia and finally by FA profiles

% Assim
% Assim % Assim % Assim bact (d 15N,
% Added bact bact (d 13C, bact estimated % Assim

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Bacteria bact (d 13C) lipid cor.) (d 15N frac) frac) bact (FA) r2 Phytoplankton

Methylomonas methanica 80 55 52 nd 77 32 0.86 Scenedesmus obliquus

Methylosinus trichosporium 80 46 45 42 38 57 0.24 Scenedesmus obliquus
Micrococcus luteus 80 nd nd 37 47 20 0.98 Scenedesmus obliquus
Methylomonas methanica 80 64 59 80 nd 77 0.66 Cryptomonas ozolinii
Methylosinus trichosporium 80 62 59 52 46 68 0.48 Cryptomonas ozolinii
Micrococcus luteus 80 40 50 55 47 20 0.84 Cryptomonas ozolinii

The two phytoplankters contained moderate amounts
of the SAFA 18:0 (14–17%) and the x-6 PUFA LIN
(10–13%). These cultures also contained very high
proportions of x-3 PUFA. However, the FA profiles of
the two phytoplankters could be readily distinguished by
a high proportion (12%) of the C16 PUFA 16:3x-3 and
16:4x-3 in Scenedesmus, and a high proportion of EPA
(13%) in Cryptomonas. The FA profiles of these phyto-
plankters also differed because Scenedesmus had considerably
more LIN and a-linolenic acid (ALA; 18:3x-3), while
Cryptomonas had considerably more stearidonic acid
(SDA; 18:4x-3). The total FA concentration was dramati-
cally lower for M. luteus (0.8 ± 0.8 lg FA mg 1 dry wt)
than MOB or phytoplankton, which all had FA concen-
trations between 45 and 70 lg FA mg 1 dry wt.
FA composition of Daphnia
We used the FA composition of Daphnia fed on the
mixed diets to infer the proportion of their FA that were
obtained from the two sources. In all cases, Daphnia fed
on mixed diets showed obvious markers of bacteria con-
sumption within their FA profiles. Daphnia fed on Micro-
coccus mixed with either Cryptomonas or Scenedesmus
were clearly enriched with iso- and anteiso-15:0
(12–16%). Daphnia that consumed Methylomonas mixed
with phytoplankton were enriched with C16 MUFA
(18–34%). Similarly, Daphnia fed on the Type II MOB
Methylosinus were enriched with C18 MUFA (15–27%)
and especially 18:1x-8. All Daphnia fed on mixed diets
also contained considerable amounts of phytoplankton
FA, including x-6 and x-3 PUFA.
We calculated the proportion of Daphnia lipids in the
mixed diet treatments originating from the bacteria by
comparing the actual Daphnia FA profiles in the mixed
treatments to hypothetical Daphnia FA profiles obtained
by ‘mixing’ the actual bacteria and phytoplankton FA
profiles. These calculations produced strong fits
(i.e. r2  0.90) and small bacteria contributions (i.e. 20%)
for the Daphnia fed Micrococcus as part of their mixed
diets. The fit between the observed and predicted FA pro-
files declined (i.e. r2  0.75) when the Daphnia were fed
Type I MOB in their mixed diets, and the estimated bac-
terial contribution increased to  55%. Finally, the fit
between the predicted and observed FA profiles was weak
(i.e. r2  0.35) when they were fed Type II MOB, and
the estimated bacterial contribution was high (i.e. 63% of
total FAs).
Overall, the FA examined in these experiments can be
divided into four groups: (1) ubiquitous FA that were
found in the heterotrophic bacteria, phytoplankton, and
Daphnia. This group consisted of the SAFA 16:0; (2) FA
that were dominant in the heterotrophic bacteria as well
as in Daphnia consuming the mixed diets: i-15:0 and
ai-15:0 branched FA for Micrococcus, C16 MUFA for
Methylomonas, and C18 MUFA for Methylosinus; (3) FA
dominant in phytoplankton and Daphnia that consumed
phytoplankton: x-3 and x-6 PUFA, including LIN, ALA,
SDA, EPA in Cryptomonas, and LIN, 16:4x-3, ALA, and
SDA in Scenedesmus; and (4) FA that were generally more
prevalent in Daphnia than their diet that included the
SAFA 18:0 and the MUFA 18:1x-9.
Bacterial gradient experiment
Somatic growth and offspring
Daphnia were first fed pure Scenedesmus for 7 days and
then an additional 7 days with a gradient of M. luteus
and C. ozolinii. After 7 days with the diet mixture (when
Daphnia where 14 days old), the body size of adult Daph-
nia and the number of eggs and neonates per individual
were lowest for the diet containing 95% bacteria and
highest for the pure Cryptomonas diet (Table 3). Daphnia
body size differed significantly among treatments (ANOVA;
F1,78 = 2.71, P = 0.04) such that groups fed with 70%
and 95% of bacteria differed significantly (Dunnett’s test
P < 0.05), the group fed with 15% differed marginally

ª 2012 Federation of European Microbiological Societies FEMS Microbiol Ecol 82 (2012) 50–62
Published by Blackwell Publishing Ltd. All rights reserved
Bacterial diet influences on Daphnia 57

(P < 0.1) and the group fed with 50% did not differ from
the group fed pure Cryptomonas. The number of eggs
and neonates did not differ among treatments (Brown–
Forsythe F4,27.042 = 2.294, P = 0.085), because of high
within-treatment variation. Nevertheless, there was signifi-
cant correlation (r = 0.96, P < 0.01) between the number

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of offspring and the concentration of x-3 FA.

Stable isotopes
The d13C and d15N values of Micrococcus (d13C =
22.2 ± 0.2 and d15N = 6.0 ± 0.3) and Scenedesmus
(d13C = 22.8 ± 0.1 and d15N = 4.0 ± 0.1) were too sim-
ilar for mixing model analysis, and thus after 7 days of
preconditioning with Scenedesmus, the Daphnia were
switched to Cryptomonas (d13C = 30.5 ± 0.3 and
d15N = 2.1 ± 0.1). The d13C and d15N values of Daphnia
fed with pure Cryptomonas were 29.2 ± 0.7 and
0.7 ± 0.2, respectively. The d13C and d15N values of
Daphnia fed mixed bacteria–phytoplankton diets were
25.2 ± 1.0 and 0.7 ± 1.0, respectively. The contribution
of assimilated carbon using mixing model calculations is
presented in Table 3. The contribution of assimilated
nitrogen could not be calculated because the d15N values
were too similar among the treatments.

Fig. 3. FA profiles of Daphnia (Dph) for the different diets. Bacterial
diet: Micrococcus luteus (ML), Methylomonas methanica (MOB1), and
Methylosinus trichosporium (MOB2); phytoplankton diet: Cryptomonas
ozolinii (Crypto) and Scenedesmus obliquus (Scene).
FA composition of Daphnia
Total FA concentrations were low in M. luteus in relation
to C. ozolinii, which did not affect the total FA concen-
trations of Daphnia (184 ± 24 lg FA mg DW) among
treatments (Supporting Information, Table S1). The FA
profiles of Daphnia among treatments reflected their diet
sources qualitatively and quantitatively. The abundance of
C16 PUFA in Daphnia was a residual FA ‘signature’ of the
preconditioning diet (Scedenesmus).
The relative composition of FA in bacteria and Cryptomonas
diets used for the M. luteus–C. ozolinii gradient experiment
was entirely different (r2 = 0.02 for a comparison of
M. luteus and Cryptomonas profiles). In contrast, Daphnia
that consumed Cryptomonas had a FA composition that very

Table 3. Experimental setup of the bacterial gradient experiment and the size of adult Daphnia (mm) and number of eggs and neonates per
individual (mean ± SD)

Added % Added % Assim % Assim Size of adult Eggs and neonates

Bacteria bact (mg C L 1) bact bact (d 13C) bact (FA) r2 Daphnia (mm) per individual

Micrococcus luteus 4.75 95 87 ± 2 54 0.78 3.7 ± 0.1 8.5 ± 1.5

Micrococcus luteus 4.25 85 79 ± 2 38 0.81 3.8 ± 0.3 11.3 ± 3.2
Micrococcus luteus 3.5 70 64 ± 0 30 0.85 3.8 ± 0.2 11.9 ± 3.8
Micrococcus luteus 2.5 50 52 ± 9 18 0.86 4.0 ± 0.2 13.1 ± 4.7
Micrococcus luteus 0 0 0 0 0.89 4.1 ± 0.1 17.2 ± 4.3

The calculated assimilated carbon is presented using carbon stable isotopes in mixing model analysis and the calculated assimilations by FA.

FEMS Microbiol Ecol 82 (2012) 50–62 ª 2012 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
58
strongly reflected its diet (r2 = 0.89). We used these dra-
matic differences in dietary FA composition and Daph-
nia’s strong dependence on its dietary lipids to back-
calculate the relative contribution of M. luteus and Cryp-
tomonas FA to Daphnia when these zooplankters were
fed mixed diets. These calculations showed a very strong
relative contribution of Cryptomonas FA to Daphnia
when they consumed a mixed diet. When Daphnia
obtained 95% of their dietary carbon from M. luteus,
they acquired 46% of their FA from Cryptomonas
(Fig. 5). Similarly, when Daphnia obtained 70% of their
diet from M. luteus, they acquired 70% of their FA from
Cryptomonas. The bacterial FA group was by itself a
strong indicator of the overall contribution of M. luteus
to Daphnia lipid composition (Fig. 4). Similarly, ALA,
SDA, and EPA strongly tracked Cryptomonas contribu-
tions to Daphnia, and when Daphnia only consumed
Cryptomonas, these FA accounted for 62% of the total
FA in Daphnia.
The contribution and concentrations of bacterial
iso- and anteiso-branched FA in Daphnia correlated sig-
nificantly (P < 0.01) with the bacterial gradient, r = 0.96
and r = 0.97, respectively (Fig. 4). Only x-3 FA, but not
SAFA or x-6 FA, in Daphnia correlated significantly
(a)
(b)
Fig. 4. The contribution (a) and concentration (b) of bacterial FA (iso-
and anteiso -branched FA) and phytoplankton (sum of x-3 FA) FA in
Daphnia after bacterial gradient experiments. There was a significant
(P < 0.01) correlation between the contribution and the concentration
of bacterial (r = 0.96 and r = 0.97) and phytoplankton (r = 0.96 and
r = 0.97) FA and the bacteria–phytoplankton diet gradient.
ª 2012 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
S.J. Taipale et al.
(r = 0.96 and r = 0.97; P < 0.01) with the increase of
phytoplankton in the diet (Fig. 4). The contribution and
concentration of C16 MUFA in Daphnia correlated signifi-
cantly (r = 0.95 and r = 0.92; P < 0.05) with the bacterial
gradient, whereas C16 MUFA correlated positively
(r = 0.90 and r = 0.90; P < 0.05) with increased phyto-
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plankton in the diet. The relationship between bacterial
FA concentrations in Daphnia and their diet could be
characterized by linear regression analysis, y = 11.3x +
4.83, R2 = 0.92, where y is bacterial FA concentration in
Daphnia and x is the FA concentration of bacterial diet.
The polynomial regression between assimilated carbon by
Daphnia using stable isotopes and FA methods is repre-
sented in Fig. 5 (y = 0.0069x2 0.0076x + 0.0739, R2 =
0.98), where y is assimilated carbon by FA analysis and x
is assimilated carbon by d13C analysis.
Discussion
Somatic growth and reproduction of Daphnia
on bacterial dominated diet
Bacteria as sole diet supply were nutritionally incomplete
for Daphnia survival and resulted in premature death.
This is most likely due to the absence of essential dietary
nutrients such as sterols (Martin-Creuzburg et al., 2011)
and x-3 or x-6 PUFA which bacteria lack and Daphnia,
or animals in general, are not able to biosynthesize de
novo (Cook & McMaster, 2004). However, Daphnia sur-
vival differed markedly when exposed to different bacte-
ria diets. Daphnia survived 12 days on the MOB Type I
diet, but only 6 days on the MOB Type II diet and
8 days on Micrococcus. High mortality on the MOB 2
diet was similar to experiments when Daphnia were fed
Polynucleobacter sp. (Hahn et al., 2009; S.J. Taipale,
unpublished) or Pseudomonas sp. and Hydrogenophaga
sp. (Martin-Creuzburg et al., 2011). All of these four
bacteria genera are gram negative, contain C18 MUFA
(Oyaizu & Komagata, 1983; Willems et al., 1989), and
yet still appear to be biochemically inadequate to sustain
Daphnia survival.
The three types of bacteria mixed with phytoplankton
affected the somatic growth and reproduction of Daphnia
differently, indicating that dietary quality varies among
the different types of bacteria. In contrast, Daphnia
reached similar body sizes and reproduction success when
phytoplankton was mixed with bacteria as when fed on
pure phytoplankton diets suggesting that Daphnia were
able to use bacteria to support their reproduction when
also supplied with phytoplankton. We also demonstrate
that Daphnia fed 20% Cryptomonas and 80% MOB fared
better than Daphnia fed 20% Scenedesmus and 80%
MOB, which confirms that taxa-specific differences of
FEMS Microbiol Ecol 82 (2012) 50–62
Bacterial diet influences on Daphnia
60
Assimilated C (based on FA analysis)
50
40
30
20
10
Fig. 5. Polynomial regresions
(y = 0.0069x2 0.0076x + 0.0739,
0
R2 = 0.98) explained connection between the
0 20
assimilated (%) carbon using carbon stable
isotopes and FAs analysis.
phytoplankton affect Daphnia reproduction (Brett et al.,
2006; Vargas et al., 2006; Striebel et al., 2008). The high
variability between individuals in the Cryptomonas treat-
ment might be due to maternal effects on the age at first
reproduction accentuated under high-quality conditions.
Possibly, this variability would have decreased if the
experiment had been continued beyond the production of
the first clutch, which generally has higher variability than
subsequent clutches.
According to stable isotope and FA analyses, the per-
centage of C, N, and FA of bacterial origin assimilated
by Daphnia varied among the bacterial treatments.
Altogether, 53 ± 10% of C and 51 ± 15% of N origi-
nated from bacteria, whereas 46 ± 25% of FA were of
bacterial origin when 80% of the diet was comprised
of bacteria. When three types of bacteria were mixed
with Scenedesmus, the contribution of bacterial FA, C,
and N in Daphnia was lower than in diets mixed with
Cryptomonas. This indicates that after Daphnia have
obtained physiologically important EPA from their diet
they are more able to utilize bacterial FA, C, and N
than would be the case for an EPA-deficient diet. Bac-
terial FA retention in Daphnia differed greatly between
bacterial diets being lowest (20%) on the Micrococcus
diet and highest (77%) on the Methylomonas diet. The
low contribution of bacterial FA may be due to the
fact that the total FA concentration of Micrococcus was
only one-tenth that of the other bacteria and phyto-
plankton used for these experiments. However, when
Daphnia were fed Micrococcus and Cryptomonas, 41–
51% of C and 76–77% of N were retained from Micro-
coccus despite of the low retention of their FA. This
difference between bacteria-specific FA retention and
bacterial C and N contributions to Daphnia suggests
that FA provide bacteria source information, but bulk
C analysis, including stable isotopes, are needed to fully
assess C contributions.
FEMS Microbiol Ecol 82 (2012) 50–62
59
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40 60 80 100
Assimilated C (based on 13C analysis)
Phytoplankton and bacterial FA as biomarkers
Our different feeding experiments clearly indicate that
dietary FA can be tracked in Daphnia. When Daphnia
were fed pure Cryptomonas or bacteria mixed with Cryp-
tomonas, high accumulation of EPA was observed,
whereas EPA was only a minor component of the FA for
those Daphnia fed pure or mixed Scenedesmus diets. Simi-
larly, the C16 PUFA that were prevalent in Scenedesmus
but absent in Cryptomonas were found only in Daphnia
fed Scenedesmus. Furthermore, the total FA profiles of the
Daphnia strongly reflected their pure phytoplankton diets
(r2 > 0.9), which suggests that the FA profiles of Daphnia
indicate the phytoplankton contribution in their diets
(Taipale et al., 2011).
In addition to phytoplankton FA, iso- and anteiso-
branched FA, and C16 or C18 MUFA were the predom-
inant FA in our three different types of bacteria and
clearly were transferred to Daphnia. Dietary supply of
iso- and anteiso-branched FA (biomarkers for Actinobacteria)
predicted the presence of these bacterial FA in Daphnia
also quantitatively (r = 0.97, P < 0.01). The iso- and
anteiso-branched FA are only synthesized by bacteria;
thus, their presence in zooplankton is unequivocal
evidence of Actinobacteria FA retention. However,
our experiments showed that these FA were less
retained compared with other dietary bacterial and phy-
toplankton FA. Moreover, Type I MOB-specific C16
MUFA was transferred to Daphnia especially when
mixed with EPA-rich phytoplankton. Although the
physiological implication of bacterial FA retention in
aquatic consumers still remains unclear, low contribu-
tions of iso- and anteiso-branched FA were previously
reported for Daphnia in a dystrophic lake (Taipale
et al., 2009a), as well as for upper trophic levels such
as fish (Rasoarahona et al., 2004) and seals (Strandberg
et al., 2008).
ª 2012 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
60
Ecological significance
In freshwater systems, the abundance and available bio-
mass of bacteria and phytoplankton can vary over very
short time scales (Taipale et al., 2011), which can affect
zooplankton somatic growth and reproduction. Our
results with bacterial dominated diets indicate that the
structure of microbial communities clearly affect zoo-
plankton somatic growth and reproduction. Microbial
communities with gram-positive Actinobacteria (common
heterotrophic bacteria) and gram-negative bacteria con-
taining C16 MUFA, together with high-quality phyto-
plankton (e.g. Cryptophyceae), support zooplankton
production, whereas lack of high-quality phytoplankton
and a high biomass of bacteria containing C18 MUFA
results in a biochemically poor diet for Daphnia and may
even inhibit growth and reproduction. Dystrophic lakes
are an example of systems that sometimes have low phy-
toplankton and high bacteria biomass (Taipale et al.,
2009a), especially MOB. Results from the mixed MOB
Type I and Cryptomonas diets clearly show a strong
reproduction response to even low amounts of EPA in
the bacteria-dominated diets, which lends support to the
observed high biomass of Daphnia during fall mixing in
the lakes with high methane concentrations (Kankaala
et al., 2010). Cryptophyceae occur globally and are espe-
cially common in the boreal humic lakes (Kankaala et al.,
1996, 2010; Lepistö & Rosenström, 1998). Our result that
even low amounts of dietary Cryptomonas strongly
enhance somatic growth of Daphnia provides experimen-
tal evidence that this primary producer may be crucial for
herbivorous zooplankton in boreal humic lakes.
This study shows that FA of zooplankton provide
more detailed information regarding their ingested diet
sources than do stable isotope signatures of bulk C or
N alone. Stable isotope and FA results show that the
low contribution of iso- and anteiso-branched FA does
not necessarily mean that these bacteria are minor
sources of carbon and nitrogen for zooplankton. How-
ever, although the assimilation of MOB-derived carbon
by Daphnia was similar from both types of bacteria, it
is still unclear why reproduction was higher with the
Methylomonas (Type I MOB)-dominated diet than with
the Methylosinus (Type II MOB)- or heterotrophic bac-
teria-dominated diets.
In conclusion, these bacterial diet experiments show
that Daphnia cannot survive solely on a bacterial diet,
demonstrating that pure bacterial diets are of low dietary
quality for Daphnia. However, when bacteria are mixed
with phytoplankton that contain EPA, bacteria may be an
adequate supplement to Daphnia diets to support somatic
growth and reproduction. Our experiments showed that
bacteria provide carbon and nitrogen to Daphnia and
ª 2012 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
S.J. Taipale et al.
their biomarker FA were retained. Thus, FA analyses
provide detailed information for studying bacterial diet
retention in zooplankton. Further research should be
aimed at clarifying the physiological importance of bacte-
rial FA in herbivorous consumers.
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Acknowledgements
We thank Marina Kalyuzhnaya from the Department of
Microbiology, University of Washington, for inocula of
M. methanica (LW13) and M. trichosporium (OB3b). This
project was supported by a National Science Foundation
(0075591) grant to M.T.B.
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Supporting Information
Additional Supporting Information may be found in the
online version of this article:
Table S1. Fatty acid profiles (%) and total FA concentra-
tions of diets (ML = Micrococcus luteus and Crypto =
Cryptomonas) and adult Daphnia among treatments.
Please note: Wiley-Blackwell is not responsible for the
content or functionality of any supporting materials sup-
plied by the authors. Any queries (other than missing
material) should be directed to the corresponding author
for the article.
FEMS Microbiol Ecol 82 (2012) 50–62