Nematology 15 (2013) 1-13
brill.com/nemy
Selective feeding in nematodes: a stable isotope analysis of
bacteria and algae as food sources for free-living nematodes
Tafesse Kefyalew E STIFANOS 1,2 , Walter T RAUNSPURGER 1 and Lars P ETERS 1,∗
1
University Bielefeld, Animal Ecology, Morgenbreede 45, D-33615 Bielefeld, Germany
2
Hawassa University, Department of Biology, P.O. Box 5, Hawassa, Ethiopia
Received: 7 January 2012; revised: 28 March 2012
Accepted for publication: 29 March 2012; available online: 22 August 2012
Summary – Laboratory experiments with stable isotopes (13 C and 15 N) were conducted to determine the importance of bacteria and
algae as food sources for free-living nematodes. All tested bacterivorous nematodes (Caenorhabditis elegans, Acrobeloides tricornis,
Poikilolaimus sp. and Panagrolaimus sp.) were found to be depleted in δ 13 C (on average by −1.71 ± 0.56h) and enriched in δ 15 N
(on average by 3.17 ± 1.27h) relative to their bacterial diets of Escherichia coli and Matsuebacter sp. The nematode species showed
considerable differences in their stable isotope composition with respect to food sources. Moreover, they differed significantly in δ 13 C
and δ 15 N values when placed on the same bacterial diet of E. coli, consistent with differences in their trophic shifts. Conversely, no
differences in δ 13 C values were observed among nematode species placed on the same Matsuebacter sp. diet. In mixed food sources
of E. coli and Matsuebacter sp., E. coli contributed 71% of the carbon to C. elegans and Matsuebacter sp. more than 90% of the
carbon to A. tricornis. An enrichment experiment based on 13 C-enriched NaH13 CO3 , 13 C6 -glucose and 15 N-enriched Na15 NO3 tracers
in a freshwater periphytic community showed the importance of micro-algae and diatoms over heterotrophic bacteria as the main
food sources of free-living periphytic nematodes. In this respect, direct grazing may predominate, possibly together with the use of
extracellular polymeric substances (EPS) from diatoms. In general, the use of stable isotopes to study nematode feeding ecology can
be useful to investigate directly the type of ingested food item(s), different bacteria and algae, and the contribution to nematode diet, in
addition to the conventional feeding type scheme.
Keywords – δ 13 C, δ 15 N, Caenorhabditis elegans, nematode diet, periphyton, tracers, trophic shift.
Nematodes are the dominating organism group of
meiofauna. Their ubiquity is evidenced by their numerical
predominance in estuarine, marine and freshwater soft
sediments (Heip et al., 1985; Traunspurger, 1996, 2002;
Moens et al., 2005) and the high densities (up to 1.6 ×
106 ind. m−2 ) of epilithic nematodes in freshwater habitats
(Peters & Traunspurger, 2005).
Consistent with their high species diversity (Heip et
al., 1985; Traunspurger, 1996; Traunspurger et al., 2006;
Hodda et al., 2009), nematodes occupy a variety of food
niches (Moens et al., 2006) and comprise diverse feeding
types (Traunspurger, 1997). The buccal morphology of the
various nematode species determines their food sources,
which include bacteria, unicellular eukaryotes, diatoms,
other microalgae, plants, fungi and other animals, including nematodes (Wieser, 1954; Yeates et al., 1993; Traunspurger, 1997; Moens et al., 2006). Although most aquatic
nematodes are known to graze on bacteria and micro∗ Corresponding
algae (Montagna, 1995; Moens & Vincx, 1997), they exhibit flexible feeding strategies in response to other potential food sources (Moens et al., 2004). However, whether
nematodes predominantly utilise bacterial or micro-algal
carbon cannot be answered by an examination of buccal
morphology and thus remains unclear (Montagna, 1984;
Montagna & Bauer, 1988). The limited methodological
options to study the feeding ecology of nematodes and address the many unanswered questions regarding nematode
food sources (Moens et al., 2004) has hampered research
to disentangle the complexity of nematode food selection
and consumption.
Carbon and nitrogen stable isotope ratios are standard
methods used in studies of food-web ecology (Peterson &
Fry, 1987; Couch, 1989). The isotope signatures of heterotrophs are a function of food composition, indicating
different food sources and their fractionation during food
processing (Peterson & Fry, 1987; Herman et al., 2000).
author, e-mail: [email protected]
© Koninklijke Brill NV, Leiden, 2013
DOI:10.1163/156854112X639900
T.K. Estifanos et al.
Fractionation in the heavier isotope between consumer
and diet is small for carbon (0.4-1h) as an indicator of
carbon flow but high for nitrogen (3-4h) as an indicator of trophic position (Minagawa & Wada, 1984; Post,
2002).
In the study of food-web interactions, stable isotope
studies have mainly been conducted in macrofaunal organisms, including birds (Hobson et al., 1994; Pearson et
al., 2003), estuarine mussels (Peterson et al., 1985), juvenile crabs (Fantle et al., 1999), intertidal mollusc species
(Riera et al., 2002) and macrobenthos deposit feeders
(Herman et al., 2000). However, the use of stable isotope
measurements has recently found application in meiofauna ecological studies as well, although there are only
a few reports, mostly describing in situ enrichment experiments in marine and estuarine habitats, on the food
sources of nematode communities (e.g., Riera & Hubas,
2003; Moens et al., 2005; Evrard et al., 2010). The authors
specifically showed that for estuarine nematodes the microphytobenthos is the dominant carbon source (Riera et
al., 1996; Middelburg et al., 2000; Moens et al., 2002), together with spartinal detritus (Couch, 1989). By contrast,
information on freshwater nematodes is scarce and, except for the studies on Caenorhabditis elegans by DeNiro
and Epstein (1978, 1981), there are no stable isotope data
from laboratory studies of the food sources of free-living
nematodes.
Thus, in the present work we investigated the contribution of different food sources (bacteria and algae) to the
diet of free-living nematodes, using a stable-isotope approach to examine the food source consumption of nematodes in laboratory experiments. Enrichment in the stable
isotopes 13 C and 15 N in free-living nematodes grown on
two bacterial species, Escherichia coli and Matsuebacter
sp., was investigated in controlled laboratory experiments
as single and mixed diet. Under the mixed diet, the relative
contribution of the different bacterial food sources was
examined. A 13 C and 15 N enrichment experiment with a
natural periphyton nematode community was carried out
to determine the preference of free-living nematodes for
bacterial vs algal food sources.
Materials and methods
N EMATODE CULTURES
The bacterivorous nematode species C. elegans (wildtype, N2 Bristol strain), Acrobeloides tricornis, Panagrolaimus sp. and Poikilolaimus sp. were obtained from lab2
oratory cultures at the Department of Animal Ecology,
Bielefeld University, Germany. These nematodes were
grown on a thin lawn of E. coli strain OP50 as food source.
Acrobeloides tricornis and C. elegans were grown on
standard Nematode Growth Medium (NGM), and Panagrolaimus sp. and Poikilolaimus sp., isolated from the
chemoautotrophic Movile Cave in Romania, on Nematode Growth Gelrite (NGG) medium. A detailed description of NGG and the culture condition can be found in
Muschiol and Traunspurger (2007). The fresh cultures of
each nematode species used in this study were obtained
from stock cultures.
F OOD SOURCES FOR NEMATODES
The stock bacteria E. coli (OP50) and Matsuebacter sp.
(frozen at −20°C) were obtained from the Department
of Animal Ecology, Bielefeld University, and tested as
food sources for the nematodes; E. coli OP50 is a
uracil auxotroph that is typically used as a food source
for culturing bacterivorous nematodes. Gram-negative
Matsuebacter sp. was isolated from soil (Park et al.,
1999).
Escherichia coli strain OP50 was cultured in E. coli
standard liquid Lysogeny Broth (LB): peptone, 10 g;
yeast extract, 5 g; and NaCl, 10 g l−1 . The cultures were
shake-incubated overnight at 200 rpm and a temperature
of 37°C. Matsuebacter sp. was grown on modified Bmedium (Widdel et al., 1983) for 96 h at room temperature (24°C) at 200 rpm. Sterile K-medium (3.1 g NaCl and
2.4 g KCl in 1 l deionised water) was used for washing the
bacterial food sources and culturing the nematodes.
Bacterial cultures were prepared for use as the nematode diet and for stable isotope analysis under similar growth conditions. Cultured bacterial food sources
were washed (2×) with sterile K-medium by centrifugation (Sigma laboratory centrifuge 3-15) in order to remove bacterial metabolites and media nutrients. Escherichia coli was centrifuged at 3333 g and Matsuebacter
sp. at 10 000 g, in each case for 20 min. The bacterial
food source was supplied based on the nematodes’ optimal food density requirements for their life cycle and
for growth (Jager et al., 2005; Muschiol & Traunspurger,
2007; Muschiol et al., 2009), as verified in preliminary
experiments. In addition, parts of washed bacterial pellets
of E. coli and Matsuebacter sp. were stored at 4°C for 3,
5, 7 and 9 days and were later on used as a food source
for the nematode species during their growth period. Bacterial densities were adjusted to 2 × 109 cells ml−1 (using
Cary 50 Bio UV visible spectrophotometer) at OD600 (opNematology
Stable isotope analysis of food sources of free-living nematodes
tical density at 600 nm wavelength). This density determination was based on a previously determined curve of absorption (at OD600 ) vs cell density. The curve was derived
from DAPI (4 , 6-diamidino-2-phenylindole)-stained bacterial cell densities visualised using epifluorescence microscopy as described by Schallenberg et al. (1989).
For the bacterial food selectivity experiment, in which
the contributions of the various food sources to the
nematode species were determined, an overnight culture
of E. coli and a 96 h culture of Matsuebacter sp. were
mixed in an equivalent ratio (1 : 1) to a final density of
2 × 109 cells ml−1 . This density was used in the singlefood source tests.
For the preparation of labelled bacterial carbon source,
E. coli was cultured in modified LB medium containing
13
C6 -enriched D-glucose (5 mg l−1 ) (99.4 atom% 13 C;
Campro Scientific). The enriched bacterial density was
adjusted to 1010 cells ml−1 after the cells were washed
by centrifugation (3333 g for 20 min) to remove the
unused enriched compound. One portion of the bacterial
pellet was stored at −20°C for stable isotope analysis and
the other used as a readily available food source, at a
density of 108 cells ml−1 , for field nematodes during the
enrichment test.
E XPERIMENTAL DESIGN
Culturing nematodes with a single food source
The nematode test species were cultured in sterile
multi-well suspension culture plates (Cellstar® ) containing 4 ml of sterile liquid K-medium and the bacterial
species food suspension per well. In single food sources,
nematode species were cultured in monoxenic medium
of E. coli or Matsuebacter sp. Four replicates were prepared for each condition. Twenty (six adults and 14 juveniles) clean and active individuals from each species of
Acrobeloides sp., Panagrolaimus sp. and Poikilolaimus sp.
were hand-picked, rinsed in sterile K-medium and transferred to the food source. The exception was C. elegans,
for which ten individual nematodes (two adults and eight
juveniles) were used. Nematode cultures were incubated
at 20°C for 7, 28, 36 and 45 days (C. elegans, A. tricornis, Poikilolaimus sp. and Panagrolaimus sp., respectively) until enough biomass (1500-2000 individual nematodes per sample and a dry biomass of 330-2600 μg) was
achieved for stable isotope analysis. To avoid shortage of
food sources during nematode population growth, nematodes were fed with bacterial food sources (based on their
life cycle: C. elegans after 3 and 5 days, and A. tricornis,
Poikilolaimus sp. and Panagrolaimus sp. after 3, 7 and
Vol. 15(1), 2013
9 days from the time they were transferred to the multiwells). A final density of 5 × 108 bacterial cells ml−1 for
Poikilolaimus sp., and 2 × 109 bacterial cells ml−1 were
used for the other three nematode species.
Nematodes in mixed food sources
Caenorhabditis elegans and A. tricornis were cultured
in the mixed food sources of E. coli and Matsuebacter
sp., with a final bacterial density of 2 × 109 cells ml−1 .
Equivalent numbers and the same proportion of nematode
life stages were used as in the single food source test. The
culture conditions were also the same.
Natural abundance of (C and N) stable isotopes samples
Bacterial food source-stable isotope samples were taken
when nematodes were introduced into the food sources
during culturing. Cultured nematodes were sieved through
a 5-μm sieve and non-utilised food sources were rinsed
with sterile K-medium. Cleaned nematodes were kept
in K-medium for 3 h to purge their guts and then
rinsed with Milli-Q water. Finally, the nematodes were
inspected under a stereomicroscope (Leica S6E; maximum magnification 40×) for adhering debris and then
transferred to tin capsules (4.75 × 11 mm dimensions;
IVA-Analysetechnik) for analysis; C. elegans with a noncleaned gut was preliminarily examined and compared
with cleaned samples. No significant differences in their
stable isotopes were noted.
E NRICHMENT EXPERIMENT
Study site, sampling and experimental procedures
The enrichment experiment was conducted at Erken
Laboratory, Uppsala University, Sweden, in April 2011.
Lake Erken is situated in south-east Sweden (59°50 N,
18°35 E; surface area 24 km2 ; mean depth 9 m), where
most of the shoreline is stony (Kahlert et al., 2002). The
lake is considered mesotrophic to eutrophic, with mean
phosphorus and nitrogen concentrations of 27 and 657 μg
l−1 , respectively (Peters et al., 2007). The sampling site
was located in the south-eastern part of the lake with
high exposure due to the predominant westerly winds
(59°50.218 N, 18°38.094 E). The study site was chosen
following a preliminary assessment of periphytic nematode communities (e.g., Peters & Traunspurger, 2005).
To obtain baseline information on the natural nematode
densities and community assemblages, we used a brush
sampler to collect samples of periphyton from hard substrates (Peters et al., 2005). The samples were sieved
3
T.K. Estifanos et al.
Table 1. Experimental setup and description of the treatments in labelling experiment, the type of label(s) used, and the test type
conducted in the study. Four treatments with single labelled diet were used to trace label uptake, two treatments with selectivity testing
on the preference of nematodes food sources, and the control without the labelling (to trace the natural background level stable isotopes).
Treatment name
Label(s), 13 C and/or 15 N source
Test types
13 C-SoBiCarb
NaH13 CO3
Na15 NO3
13 C -glucose
6
13 C -enriched Escherichia coli
6
13 C -enriched E. coli and NaH13 CO
6
3
Na15 NO3 and 13 C6 -enriched E. coli
Ambient abundance
Single labelling (13 C)
Single labelling (15 N)
Single labelling (13 C)
Single labelling (13 C)
Mixed labelling (2 × 13 C)
Mixed labelling (13 C + 15 N)
Ambient background
15 N-SoNitr
13 C6-Gluc
13 C-Ecoli
13 C-Ecoli
13 C-Ecoli
Control
+ 13 C-SoBiCarb*
+ 15 N-SoNitr
* In
this treatment, the 13 C enrichment level (label uptake) of nematodes was determined relative to the respective 13 C label uptake in
the single label enrichments; 13 C-SoBiCarb and 13 C6 -Ecoli.
through a 10-μm sieve and the nematode community assemblage (abundance and species composition) was determined. From this sample, 119 individual nematodes were
randomly handpicked followed by species identification
and for feeding type classification based on buccal morphology (Traunspurger, 1997). The main food sources of
the periphytic nematodes from the littoral zone of the lake
were studied using stable isotope (13 C and 15 N) tracers.
For this purpose, small periphyton-covered stones/hard
substrates (ranging in area cover from 45 to 250 cm2 )
were carefully sampled from a 50 cm depth of the lake,
with maximum precautions taken to avoid loss or contamination of the material. Finally, the samples were carefully
transported to the aquarium room of our laboratory.
Twenty-one small plastic aquaria (each 11.5 × 11 ×
17 cm) were filled with 1 l of filtered lake water (GF 6
glass fibre filter, diam. 47 mm; Whatman) and arranged
in the aquarium room (20 ± 0.5°C). Stable isotopes
substrates of 13 C-enriched NaH13 CO3 (99 atom% 13 C),
[13 C6 ]D-glucose (99.4 atom% 13 C) and 15 N-enriched
Na15 NO3 (98 atom% 15 N) (all Campro Scientific) were
prepared to final concentrations of 100, 200 and 2 mg l−1 ,
respectively.
13
C6 -labeled E. coli was also used as a readily available
bacterial food source for the nematodes. 13 C6 -labelled
E. coli mixed with [13 C]NaHCO3 (to test the relative
contribution of 13 C-labelled carbon) and 13 C6 -labelled E.
coli mixed with 15 N-enriched Na15 NO3 (as dual tracer)
were used to determine nematode food source selectivity
based on algae and bacteria. A triplicate control without
enrichment was used to trace the natural abundance of
the stable isotopes. The experiment was conducted using
4
six enrichment treatments, each with three replicates
(Table 1).
One to two small periphyton-covered stones were
transferred into each treatment and control aquarium. In
the treatments containing 13 C6 -labelled E. coli, the labelled bacteria were evenly distributed (using a 5-ml micropipette) among the periphytic community to a density
of 5 × 108 cells cm−2 , corresponding to a final bacterial
density, on average, of 3.5 × 107 cells−1 . All treatments
and the control were equally illuminated with a fluorescent lamp for 96 h.
13
C and 15 N incorporation into nematodes after 96 h
was determined by carefully scraping off the periphytic
community from the surface of the stones. The samples were sieved through a 20-μm sieve and all material that had passed through the sieve (periphyton fraction
<20 μm) was collected for stable isotope analysis. Nematode samples (>20 μm) were rinsed with filtered lake water to remove the unincorporated enrichment compounds
and immediately preserved with formalin (6% final concentration) after which 1000-1400 individual nematodes
(dry biomass 114-194 μg) were handpicked, cleaned with
ultrapure water, and further checked for adhering debris
under a stereomicroscope at 40× magnification. Contaminated nematodes were excluded from the analyses.
The periphytic material (<20 μm) considered to contain potential food resources for nematodes was stored
in the climate room (4°C) of the laboratory until further
treatment. For each treatment, 10 ml of well mixed periphytic material was filtered through a pre-combusted
(550°C for 6 h) and pre-weighed GF 6 glass-fibre filter (diam. 25 mm, Whatman) for stable isotope analysis.
The filtered samples were freeze-dried, weighed, carefully
Nematology
Stable isotope analysis of food sources of free-living nematodes
scraped off the glass fibre filter, and then transferred to
pre-weighed tin capsules until further analysis.
comparison with Tukey’s HSD test (α = 0.05). Statistical
software for Social Sciences (SPSS Version 7, SPSS) and
Statistica software (version 9; StaSoft) were used.
S TABLE ISOTOPE ANALYSIS
Nematodes, media chemicals and bacterial food sources
were transferred into pre-weighed tin capsules, oven dried
at 55°C for 24-48 h, weighed, and pinched closed. All
dried samples in the capsules were stored in a desiccator until stable isotope analysis. Carbon and nitrogen stable isotopes were expressed in delta notation as parts per
thousand (h) according to the standard formula: δX =
((Rsample /Rstandard ) − 1) × 1000, where X is 13 C or 15 N
and R denotes the corresponding 13 C/12 C or 15 N/14 N ratio. The Rstandard for 13 C is Vienna PeeDee Belemnite and
that for 15 N atmospheric N2 (air).
Fractionation of stable isotopes between bacterial food
source (A) and nematode consumer (B) were described
in terms of the difference in delta (δ 13 C) values using
the “delta” notation, where δ 13 C = δ 13 CB − δ 13 CA .
A similar procedure was also applied to 15 N. A positive
δ-value indicates a relatively greater concentration of
the heavier isotope in B. The relative contribution of
the two bacterial food sources to the nematodes’ diet
was determined based on the two-source mixing model
reported in McCutchan et al. (2003):
Kcarbon = 1 − ((δ 13 CA − δ 13 Cconsumer + δ 13 C)
/(δ 13 CA − δ 13 CB )),
where δ 13 CA and δ 13 CB are the isotope ratios of the
potential food sources, δ 13 Cconsumer the isotope ratio of
the consumer, δ 13 C the trophic shift for C, and K the
proportionate contribution of food source A to consumer
growth.
The enrichment experiment examined the label uptake
by natural peripthyton organisms. Thus, the enrichment
level of samples was compared with that of the natural
background value in the control and presented as δ 13 C
(h) (δ 13 Csample − δ 13 Cbackground ). An increase in δ 13 C
values compared with the background means the introduced labelled stable isotopes were taken up by nematodes and periphytic organisms. A similar analysis was
done for δ 15 N values.
S TATISTICAL ANALYSIS
Differences in isotopic signatures were compared based
on one-way analysis of variance (ANOVA) and Student’s
t-test. After the samples were tested for homogeneity
of variances, the data were analysed using a post-hoc
Vol. 15(1), 2013
Results
S TABLE ISOTOPE VALUES OF BACTERIAL FOOD
SOURCES
The two food sources, E. coli and Matsuebacter sp.,
with mean ± SD δ 13 C values of −20.25 ± 0.36 and
−18.47 ± 0.51h, respectively, and mean δ 15 N values
of 3.76 ± 0.39 and −7.79 ± 0.43h, respectively, were
significantly different with respect to stable isotope composition (t-test, t(4) = −6.41, P < 0.01 for δ 13 C; and
t(4) = 42.13, P < 0.001 for δ 15 N).
S TABLE ISOTOPE VALUES OF NEMATODES
The stable isotope values of the nematode species
depended on the type of bacterial food source, i.e., E.
coli vs Matsuebacter sp. (Table 2). On the E. coli diet
the four nematode species varied significantly in their
δ 13 C values, which ranged from −23.33 ± 0.13h in A.
tricornis to −21.19 ± 0.12h in C. elegans (one-way
ANOVA, F3,12 = 344.3, P < 0.001; Tukey’s HSD, P <
0.05). Interestingly, Poikilolaimus sp. and Panagrolaimus
sp. showed small but significant differences in their δ 13 C
values (Tukey’s HSD, P < 0.05). Likewise, when fed
on E. coli, the nematode species differed significantly in
their δ 15 N values, with the highest value determined in
C. elegans (8.15 ± 0.08h) and the lowest in A. tricornis
(4.85 ± 0.56h; one-way ANOVA, F3,12 = 40.45, P <
0.001; Tukey’s HSD, P < 0.05), with the exception
being between Poikilolaimus sp. (6.31 ± 0.32h) and
Panagrolaimus sp. (6.13 ± 0.54h). In nematodes fed the
Matsuebacter sp. diet, the mean δ 13 C values of the four
nematode species showed only slight differences (oneway ANOVA, F3,12 = 0.82, P = 0.51) whereas the
differences in the δ 15 N values were significant (one-way
ANOVA, F3,12 = 13.60, P < 0.001), with the highest
value in Poikilolaimus sp. and the lowest in C. elegans
(Table 2).
The comparison of SI values in nematodes fed two
different food sources showed that nematodes that fed
on E. coli were substantially more depleted in δ 13 C
(average −22.46 ± 0.84h) than those fed Matsuebacter
sp. (average −19.71 ± 0.45h; t-test, t(30) = −11.59,
n = 16, P < 0.001). The δ 13 C values of each nematode
5
T.K. Estifanos et al.
Table 2. Nematode SI values, δ 13 C and δ 15 N, (n = 4; mean ± SD in h) of Caenorhabditis elegans, Acrobeloides tricornis,
Poikilolaimus sp. and Panagrolaimus sp., cultured in two different bacterial food sources, Escherichia coli (EC) and Matsuebacter
sp. (MAT) under laboratory conditions.
δ 13 C
C. elegans
A. tricornis
Poikilolaimus sp.
Panagrolaimus sp.
δ 15 N
EC
P
MAT
EC
P
MAT
−21.19 ± 0.12
−23.33 ± 0.13
−22.92 ± 0.08
−22.37 ± 0.03
***
−19.45 ± 0.16
−19.71 ± 0.31
−19.95 ± 0.47
−19.72 ± 0.7
8.15 ± 0.08
4.85 ± 0.56
6.31 ± 0.32
6.13 ± 0.54
***
−5.34 ± 0.2
−4.65 ± 0.53
−2.83 ± 0.56
−3.30 ± 0.98
***
***
**
***
***
***
Asterisks represent results from t-tests between the two bacterial food sources for each of the four nematode species for C and N;
significance level: ** P < 0.01, *** P < 0.001.
Table 3. Nematodes’ carbon and nitrogen fractionation, δ 13 C and δ 15 N (n = 4; mean ± SD in h) of Caenorhabditis elegans,
Acrobeloides tricornis, Poikilolaimus sp. and Panagrolaimus sp., cultured in two different bacterial food sources, Escherichia coli (EC)
and Matsuebacter sp. (MAT) under laboratory conditions.
δ 13 C
Nematode
EC
C. elegans
A. tricornis
Poikilolaimus sp.
Panagrolaimus sp.
−0.94 ± 0.12
−3.08 ± 0.13
−2.67 ± 0.08
−2.13 ± 0.03
δ 15 N
P
MAT
EC
P
MAT
ns
−0.96 ± 0.16
−1.23 ± 0.31
−1.47 ± 0.47
−1.24 ± 0.70
4.39 ± 0.08
1.09 ± 0.56
2.55 ± 0.32
2.37 ± 0.54
***
2.42 ± 0.2
3.11 ± 0.53
4.97 ± 0.56
4.48 ± 0.98
***
**
*
**
***
**
Asterisks represent results from t-tests between the two bacterial food sources for each of the four nematode species for C and N;
significance level: * P < 0.05, ** P < 0.01, *** P < 0.001. ns, not significant (P > 0.05).
species fed the E. coli diet varied significantly from
those of the same nematode species that were fed on
Matsuebacter sp. (Table 2). By contrast, the δ 15 N values
of the nematode species were highly variable, ranging
from −5.34 to 8.15h, and were significantly higher in
the E. coli diet (average 6.36 ± 1.36h) than in the
Matsuebacter sp. diet (average −4.03 ± 1.16h) (ttest, t(30) = 23.9, P < 0.001). The δ 15 N value of
each nematode species fed the E. coli diet was always
significantly higher than those of the same nematode
species that fed on Matsuebacter sp. (Table 2).
a lower shift was obtained when Matsuebacter sp. was
the food source (−1.23h). C. elegans had the lowest
trophic shift, with similar results obtained for the two food
sources (E. coli 0.94h and Matsuebacter sp. 0.97h).
Among the nematode species, C. elegans had the highest
enrichment in δ 15 N when fed an E. coli diet (by 4.39h)
and the lowest when fed a Matsuebacter sp. diet (by
2.42h), which were significantly different. Poikilolaimus
sp., however, were notably more enriched in δ 15 N when
fed Matsuebacter sp. rather than E. coli (4.97 vs 2.55h,
respectively) (Table 3).
S TABLE ISOTOPE FRACTIONATION IN NEMATODES
C ONTRIBUTION OF BACTERIAL FOOD SOURCES TO
The trophic shift in δ 13 C was significantly higher
in nematodes on the E. coli diet (average −2.20 ±
0.93h) than in those fed Matsuebacter sp. (average
−1.23 ± 0.20h) (t-test; t(30) = −4.13, P < 0.001).
Differences in the trophic shift of the four nematode
species between the food sources are given in Table 3. The
highest and significantly different trophic shift in δ 13 C
was determined in A. tricornis fed E. coli (−3.08h) and
6
THE NEMATODE DIET
The δ 13 C and δ 15 N values of nematodes fed mixed bacterial food sources of E. coli and Matsuebacter sp. were
determined (Fig. 1). The δ 13 C values were significantly
different for C. elegans fed a mixed diet (mean −20.68 ±
0.23h), an E. coli diet (mean −21.19 ± 0.12h), or a
Matsuebacter sp. diet (mean −19.45 ± 0.16h) (one-way
ANOVA, F2,9 = 104.15, P < 0.001; Tukey’s HSD test,
Nematology
Stable isotope analysis of food sources of free-living nematodes
F OOD SOURCES OF PERIPHYTIC NEMATODES IN THE
C LABELLING EXPERIMENT
13
Fig. 1. Carbon (δ 13 C) and nitrogen (δ 15 N) stable isotope values
(h; mean ± SD, n = 4) of Caenorhabditis elegans and
Acrobeloides tricornis in mixed (Mix) and single Escherichia
coli (EC) and Matsuebacter sp. (MAT) food sources. The
nematode species stable isotope values in the single food source
were adopted from the single food source experiment.
P < 0.05). In the mixed food source, E. coli and Matsuebacter sp. contributed 71 and 29% of carbon, respectively, to C. elegans. The δ 13 C value of A. tricornis fed
a mixed diet (mean −19.90 ± 0.89h) was significantly
different from that determined when the food source consisted only of E. coli (one-way ANOVA, F2,9 = 55.38,
P < 0.001; Tukey’s HSD test, P < 0.05). However, this
was not the case for A. tricornis fed only Matsuebacter sp.
(mean −19.71 ± 0.31h). Consequently, for A. tricornis
cultured in the mixed food source, Matsuebacter sp. contributed 94% of the carbon while E. coli contributed only
6%.
Significantly different δ 15 N values were obtained for C.
elegans fed a mixed diet (mean −1.59 ± 0.29h), an E.
coli diet (mean 8.15 ± 0.08h), or a Matsuebacter sp. diet
(mean −5.34 ± 0.2h) (one-way ANOVA, F2,9 = 4381.3,
P < 0.001; Tukey’s HSD test, P < 0.05). Similarly, the
δ 15 N values of A. tricornis on a mixed (−3.22 ± 0.33h),
Matsuebacter sp. (−4.64 ± 0.53h), or E. coli (4.85 ±
0.56h) diet were also significantly different (one-way
ANOVA, F2,9 = 443.94, P < 0.001; Tukey’s HSD test,
P < 0.05).
Vol. 15(1), 2013
The δ 13 C values of nematodes subjected to different
treatments were compared (Fig. 2). Except for samples
comprising nematodes fed 13 C6 -enriched E. coli, field
nematodes were significantly enriched in δ 13 C compared
to the control (natural background, δ 13 C, −21.67 ± 1.1h)
(one-way ANOVA, F5,12 = 37.64, P < 0.001). Label
uptake by nematodes in 13 C-enriched NaH13 CO3 (δ 13 C,
1462h) and 13 C6 -enriched glucose (δ 13 C, 3104h) was
significantly higher than that by nematodes provided
only with 13 C6 -enriched E. coli (δ 13 C, 4.8h) or with
13
C6 -enriched E. coli combined with 15 N-enriched nitrate
(δ 13 C, 1.1h), which in neither case differed notably
from the control (Tukey’s HSD test, P > 0.05).
Similarly, nematode uptake of 15 N-labelled organic
matter in the 15 N-enriched samples (δ 15 N, 1667 ±
177h in 15 N-enriched nitrate and 1797 ± 329h in
15
N-enriched nitrate mixed with 13 C6 -enriched E. coli,
which showed slight but non-significant differences) was
significantly different and higher as determined based on
the difference with the nematodes’ natural background
δ 15 N value of 6.9 ± 0.17h (one-way ANOVA, F2,6 =
64.66, P < 0.001). Label uptake by the nematodes was
proportional to that by the periphyton community (Fig. 2),
in which algae and diatom were the primary components.
The natural nematode community at the time of sampling
in the study area was dominated by epistrate feeders
(84%), represented by Punctodora ratzeburgensis (82%)
and Chromadorina viridis (1.7%; Table 4). Besides their
dependence on bacteria, these species feed on diatoms and
microalgae as their buccal cavity is equipped with a small
tooth.
Periphytic samples from enriched inorganic (13 Cenriched NaH13 CO3 , 15 N-enriched Na15 NO3 ) and organic (13 C6 -enriched glucose) substrates were significantly highly enriched compared to the natural background (one-way ANOVA, F5,12 = 137.14, P < 0.001
for δ 13 C; and F2,6 = 51.24, P < 0.001 for δ 15 N). 13 C uptake was the highest in 13 C6 -enriched glucose treatment
(δ 13 C, 18 725 ± 2261h), followed by the 13 C-enriched
NaH13 CO3 treatment (δ 13 C, 3261 ± 1256h). Uptake
was the lowest in the 13 C6 -enriched E. coli treatment
(11h, in 13 C6 -enriched E. coli mixed with 15 N-enriched
nitrate; Fig. 2).
7
T.K. Estifanos et al.
Fig. 2. Mean (± SD, n = 3) δ 13 C and δ 15 N values of periphytic nematodes and the periphyton fraction < 20 μm in the enrichment
experiment using different single enrichment (13 C-SoBiCarb, sodium bicarbonate (13 C); 13 C-Gluc, glucose (13 C); 13 C-Ecoli,
Escherichia coli (13 C); 15 N-SoNitr, sodium nitrate (15 N)), mixed enrichment treatments (13 C-Ecoli + 13 C-SoBiCarb (13 C); 13 C-Ecoli +
15 N-SoNitr (13 C + 15 N)) and a non-enriched control (control).
Discussion
NATURAL STABLE ISOTOPE COMPOSITION OF
NEMATODES FED BACTERIAL FOOD SOURCES
Our study clearly shows that food uptake by nematodes can be detected with the stable isotope method.
In the laboratory study, the nematodes were depleted in
δ 13 C, on average by −1.72 ± 0.57h (mean across all
nematode species and bacterial resources; range: −0.94
to −3.08). This value was highly variable compared with
the mean trophic shift of most consumers, δ 13 C, 0.8h
(range −1.5 to +2.7h) (DeNiro & Epstein, 1978). However, this deviation was inevitable as stable isotope values depend on the consumer’s metabolism and the type
of diet (McCutchan et al., 2003). On the other hand, nematodes were enriched in δ 15 N, on average by 3.17 ±
1.27h (mean across all nematode species and bacterial
resources). This value is very close to the average enrich8
ment value of Diaptomus (3.2 ± 0.3h), a copepod from
arctic lakes (Kling et al., 1992). In general, δ 15 N enrichment in nematodes corresponds to the average value of
most consumers, δ 15 N, 3.4 ± 1.0h (DeNiro & Epstein,
1981; Minagawa & Wada, 1984; Peterson & Fry, 1987;
Post, 2002).
The stable isotope values of the nematode species
followed those of their bacterial diet, with the assumption,
based on the negligible trophic shift in δ 13 C, that negative
values were also common (DeNiro & Epstein, 1978). The
food sources E. coli and Matsuebacter sp. consistently
showed distinct stable isotope values, as reflected in
those of the nematodes. This is because the type of
diet primarily determines the stable isotope values of the
consumer (Webb et al., 1998; McCutchan et al., 2003).
In general, nematode species were depleted in δ 13 C but
the fractionation was not uniform. The δ 13 C value of
C. elegans differed depending on whether E. coli or
Nematology
Stable isotope analysis of food sources of free-living nematodes
Table 4. Nematode species identified in the periphyton community and their mean relative abundance (± SD, n = 5) at the
study site, Lake Erken. Feeding types (FT) are based on Traunspurger (1997): deposit feeders (D) and epistrate feeders (E).
Nematode taxon
FT
Relative
abundance (%)
1. Eumonhystera vulgaris
(de Man, 1880)
2. E. cf. vulgaris de Man, 1880
3. E. pseudobulbosa (Daday, 1896)
4. E. simplex (de Man, 1880)
5. Plectus aquatilis Andrássy, 1985
6. P. tenuis Bastian, 1865
7. Rhabdolaimus aquaticus
de Man, 1880
8. Chromadorina viridis
(von Linstow, 1876)
9. Punctodora ratzeburgensis
(von Linstow, 1876)
D
0.6 ± 1.4
D
D
D
D
D
D
6.7 ± 6.5
2.2 ± 3
1.5 ± 2.1
0.6 ± 1.4
3.9 ± 6.5
0.6 ± 1.4
E
1.7 ± 3.9
E
82.1 ± 11.6
Matsuebacter sp. was the food source, but the trophic
shifts were fairly similar. While a difference in trophic
shift was expected in C. elegans fed two different diets,
the differences in the relative proportions of the major
biochemical fractions from the diet that accounted for this
small variability may have been compromised by the bulk
(whole organism) carbon in the diet (DeNiro & Epstein,
1978). In the case of A. tricornis, δ 13 C depletion was
−1.85h greater in nematodes fed E. coli than in those
fed Matsuebacter sp.
The δ 15 N values were positive for nematodes fed on
the E. coli diet but with a lower and more variable
trophic shift than determined in nematodes fed on a diet
of Matsuebacter sp. The trophic shifts of the nematode
species were inconsistent with respect to food source.
Thus, C. elegans was highly enriched (and A. tricornis
the least enriched) in δ 15 N when fed an E. coli diet
but, compared with the other nematode species, the
least enriched when fed the Matsuebacter sp. diet. By
contrast, when Matsuebacter sp. was the food source,
Poikilolaimus sp. had the highest enrichment (δ 15 N,
4.97 ± 0.56h) followed by Panagrolaimus sp. This high
variability in the trophic shift may have been due to the
food quality (Post, 2002). For example, in the spider
Pardosalugubris (Araneae, Lycosidae) a greater increase
in δ 15 N content was obtained with high-quality vs lowquality prey (Oelbermann & Scheu, 2002), as was the
case in our E. coli-fed nematodes. Furthermore, in a
stable isotope study on birds by Pearson et al. (2003),
Vol. 15(1), 2013
trophic shifts increased with increasing dietary C and N
concentrations. On the other hand, in locusts (Webb et al.,
1998) and in Daphnia magna (Adams & Stenner, 2000),
higher stable isotope enrichment was measured with lowquality rather than high-quality food. In both cases, the
δ 15 N values were inversely related. By considering these
facts, food quality is also likely to play a role in the
formation of nematodes’ stable isotope signatures, but
this remains highly speculative as it was not measured in
our study Thus, further study of the correlation between
food quality and nematode stable isotope composition is
needed before rigorous conclusions can be reached.
Individuals of different species fed the same diet may
differ significantly in their stable isotope values. The
four nematode species of this study showed quite surprising variations in δ 13 C when fed the same bacterial food
source, i.e., E. coli. This wide variation was associated
with differences in trophic fractionation, which ranged
from −0.94h for C. elegans to −3.08h for A. tricornis. In line with this result, Vander Zanden and Rasmussen
(2001) reported more variable trophic fractionations in invertebrates, and in laboratory experiments compared with
most field studies. In this regard, different species may
vary in their ability to assimilate the nutrients in food,
such that the relationship between ingested and assimilated food is unclear (Levey & Martinez del Rio, 2001).
In nematodes, species-specific differences may result in
differences in the isotopic discriminations of 13 C. These
variations potentially reflect the physiological characteristics of consumers and their feeding rates (Vander Zanden & Rasmussen, 2001; McCutchan et al., 2003) as well
as the metabolic fates of ingested C and N (Macko et al.,
1987). In C. elegans, increasing food availability is associated with a higher feeding rate (Nicholas et al., 1973;
Venette & Ferris, 1998) and with a high rate of respiration (Schiemer, 1983). In our study, these factors might
explain why, among the four species, C. elegans was the
least depleted in δ 13 C. Consistent with our results, a much
earlier study by DeNiro and Epstein (1978) showed that
C. elegans was depleted by −0.6h in δ 13 C when fed the
same E. coli diet.
In the case of Matsuebacter sp., by contrast, the four
nematode species raised on this food source did not
differ significantly either in their δ 13 C stable isotope
composition or their trophic shift – consistent with “you
are what you eat” (DeNiro & Epstein, 1978). The stable
isotope composition of consumers has been shown to
reflect the isotope value of their diet, albeit with slight
differences (Fry & Sherr, 1984; Peterson & Fry, 1987).
9
T.K. Estifanos et al.
Differences in the δ 15 N values of the nematodes species
were also prominent when E. coli was the food source.
The variability in the relationship between the δ 15 N values
of animals and those of their diets is greater for different
species raised on the same diet than for the same species
raised on different diets (DeNiro & Epstein, 1981). These
differences in enrichment may be due to fractionation
by the consumers, leading to a predominance of lighter
nitrogen (14 N) in excreted nitrogen (DeNiro & Epstein,
1981; Gannes et al., 1998).
C ONTRIBUTIONS OF DIFFERENT FOOD SOURCES TO
THE NEMATODE DIET
As discussed above, the distinct δ 13 C and δ 15 N values
of the two food sources, E. coli and Matsuebacter sp.,
enabled us to determine the relative contribution of each
one to the consumer nematodes. Under these experimental
conditions, the isotopic values of the samples may directly
reflect the amounts of the two sources (Fry & Sherr,
1984; Ruess et al., 2005). Moreover, the two bacterial
species were chosen for practical reasons as they are
preferentially fed upon by the consumer nematodes used
in our study. The selectivity test revealed that E. coli rather
than Matsuebacter sp. contributed most (more than twothirds) of the carbon to C. elegans. This may be attributed
to the food quality (discussed above) but also to the
nematodes’ feeding behaviour (Shtonda & Avery, 2005).
Additionally, preconditioning may positively contribute to
the selection process (Rodger et al., 2004); in the present
study, C. elegans was cultured using the same E. coli diet
later provided as the test diet, which may have accounted
for the nematode’s preferential consumption of this food
source over Matsuebacter sp., which was used only in the
experiments.
C ONSUMPTION OF ALGAE BY PERIPHYTIC
inated by green algae (51%) and diatoms (43%) (Peters, pers. commun.). The δ 13 C values of consumers
have been shown to match closely those of autotrophs
at the base of food webs (France & Peters, 1997). Previous studies from Lake Erken supported the conclusion
that epilithic/periphytic meiofaunal abundance is closely
linked to periphyton biomass, including a significant correlation with algal biomass (Peters & Traunspurger, 2005;
Peters et al., 2007). Consistent with this finding, the dominant species of epistrate feeders were shown to be more
abundant during prolonged undisturbed periods, in which
a high diatom biomass was determined in river epilithic
biofilms (Majdi et al., 2011). As previously reported from
the same lake (Peters & Traunspurger, 2005), we observed
that epistrate feeders of nematode species, with P. ratzeburgensis as the dominant species, were predominantly
abundant at the study site (Table 4). This nematode is
morphologically adapted to graze mainly on micro-algae
and diatoms (Taunspurger, 1997). Nematodes are known
to derive their assimilated carbon and nitrogen from algae by various means, either directly by grazing (Cahoon,
1999; Herman et al., 2000; Middelburg et al., 2000), or
indirectly by taking up the products of diatoms, extracellular polymeric substances (EPS), and by the consumption of bacteria that depend on EPS (Moens et al., 2002;
Evrard et al., 2010). From the dual labelling experiment
carried out in the laboratory, using 13 C6 -enriched E. coli
and 15 N-enriched Na15 NO3 , the former contributed hardly
or not at all to the nematodes’ food sources, in contrast to
the labelled nutrients from enriched inorganic substrates,
sodium bicarbonate and sodium nitrate, (derived indirectly through algae). Consequently, based on the dominance of morphologically classified algae-feeding nematodes, P. ratzeburgensis, and the preferred algae uptake
by nematodes in the labelling experiment, we assume that
periphytic nematodes rely on algae as their main food
source.
NEMATODES
Our labelling experiment with a natural nematode community notably illustrated the flow of carbon and nitrogen from food sources to consumer nematodes in Lake
Erken. Periphytic nematodes responded quickly, incorporating the enriched labels over 4 days. Similarly, the
rapid transfer of labelled nutrients has been reported in
other, comparable studies (Herman et al., 2000; Middelburg et al., 2000; Moens et al., 2002; Evrard et al.,
2010). The present study revealed that the labels were
largely traced into consumer nematodes from the periphyton, where the autotrophic algal community was dom10
Conclusions
Our study provides further evidence of the virtues of
controlled laboratory experiments to the application of
stable isotope techniques and as a complement to fieldbased studies of nematode feeding ecology. In addition
to indirect interpretation of nematode food sources from
the conventional feeding type schemes, the stable isotope
approach has the advantage that it allows a direct dietary
assessment. For example, it can be used to trace the
fate of the ingested diet by different nematode species,
Nematology
Stable isotope analysis of food sources of free-living nematodes
which is an important factor related to food source
contribution or selection in niche partitioning. The stable
isotope composition of the nematode species in this
study was found to depend on their diet. In general,
among nematodes, the stable isotope composition of
the same food source may (mainly due to speciesspecific differences in the trophic shift) or may not vary.
Furthermore, enrichment studies such as this one provide
new insights into the potential food sources of freshwater
nematodes in the natural community. Thus, given the
close association between the isotope values of nematodes
and periphyton, and the predetermined feeding type of the
dominant nematode, P. ratzeburgensis, the vast amount of
carbon assimilated by the nematode population in Lake
Erken was determined to have derived from the algal
community.
Acknowledgements
We thank Barbara Teichner for her help in weighing the
stable isotope samples. We are indebted to Prof. Kurt Pettersson from the Norr Malma Field Station (Erken Laboratory) who supported the study. This study was financially supported by the European Union Erasmus Mundus
programme (EUMAINE) and the German Research Foundation (DFG – PE 1522/2-2).
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