Journal of Helminthology, page 1 of 5
q Cambridge University Press 2013
doi:10.1017/S0022149X13000126
Isotopic discrimination of stable isotopes
of nitrogen (d15N) and carbon (d13C) in a
host-specific holocephalan tapeworm
J. Navarro1*, M. Albo-Puigserver1, M. Coll1, R. Saez1,
M.G. Forero2 and R. Kutcha3
1
Institut de Ciències del Mar (ICM-CSIC), Passeig Marı́tim de la
Barceloneta 37-49, 08003 Barcelona, Spain: 2Estación Biológica de Doñana
(EBD-CSIC), C/Américo Vespucio s/n, 41092, Sevilla, Spain: 3Institute of
Parasitology, Biology Centre of the Academy of Sciences of the Czech
Republic, Branišovská 31, 370 05 Ceské Budejovice, Czech Republic
(Received 27 September 2012; Accepted 29 January 2013)
Abstract
During the past decade, parasites have been considered important
components of their ecosystems since they can modify food-web structures
and functioning. One constraint to the inclusion of parasites in food-web models
is the scarcity of available information on their feeding habits and host – parasite
relationships. The stable isotope approach is suggested as a useful methodology
to determine the trophic position and feeding habits of parasites. However,
the isotopic approach is limited by the lack of information on the isotopic
discrimination (ID) values of parasites, which is pivotal to avoiding the biased
interpretation of isotopic results. In the present study we aimed to provide
the first ID values of d15N and d13C between the gyrocotylidean tapeworm
Gyrocotyle urna and its definitive host, the holocephalan Chimaera monstrosa. We
also test the effect of host body size (body length and body mass) and sex of the
host on the ID values. Finally, we illustrate how the trophic relationships of the
fish host C. monstrosa and the tapeworm G. urna could vary relative to ID values.
Similar to other studies with parasites, the ID values of the parasite –host system
were negative for both isotopic values of N (Dd15N ¼ 2 3.33 ^ 0.63‰) and
C (Dd13C ¼ 2 1.32 ^ 0.65‰), independent of the sex and size of the host. By
comparing the specific ID obtained here with ID from other studies, we illustrate
the importance of using specific ID in parasite – host systems to avoid potential
errors in the interpretation of the results when surrogate values from similar
systems or organisms are used.
Introduction
The consideration of parasites as important components of their ecosystems has seen a progressive
increase during the past decade (Hatcher et al., 2012; Kéfi
et al., 2012). Parasites, as consumers, modify food-web
structures by increasing food-chain length and the
*Fax: þ 34 932309555
E-mail: [email protected]
number of links, which influence the structure and
stability of food webs (Kuris et al., 2008; Lafferty et al.,
2008). One of the major limitations of including parasites
in models of food webs is the scarcity of accurate
information on their feeding habits, mainly due to the
difficulty of applying conventional methodologies such
as the analysis of the digestive tract. This aspect is
especially limiting for tapeworms, as they are organisms
without digestive tracts and feed by absorbing the
nutrients directly from the host. For tapeworms, the use
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J. Navarro et al.
of intrinsic markers such as the stable isotopes of nitrogen
(15N/14N, denoted as d15N) and carbon (13C/12C, denoted
as d13C) is an alternative and useful methodology to
determine trophic position, trophic habits and host –
parasite relationships (Doucett et al., 1999; Butterworth
et al., 2004).
Stable isotope analysis, which is especially advantageous when investigating the feeding ecology of
parasites, provides information on assimilated food (not
just ingested food), as well as time-integrated information
(Layman et al., 2012). This approach is based on d15N and
d13C values being transformed from dietary sources to
consumers in a predictable manner (Layman et al., 2012).
d15N values are indicators of trophic positions as
consumers are predictably enriched in d15N relative to
their food (Post, 2002). d13C values show little change
with trophic transfers, but are useful indicators of the
dietary source of carbon (De Niro & Epstein, 1981).
However, the use of stable isotope analysis to
accurately investigate trophic relationships is limited
by the correct knowledge and application of the isotopic
discrimination values (ID) specific for the studied
consumer – prey relationships. ID is the amount of change
in isotope ratios as isotopes are incorporated from prey
into the consumers’ tissue, usually 3– 4‰ and 0 – 1‰ for
d15N and d13C, respectively. The importance of using a
correct ID is crucial to avoid a biased interpretation of the
isotopic results (Wolf et al., 2009).
Although much work has been done to determine ID
values of different groups of organisms over the past
decade, and to study the factors that could influence their
variation within and among species (Caut et al., 2009),
information on ID in parasite – host systems is still very
scarce (Dubois et al., 2009). General findings suggest that,
in comparison to the conventional positive enrichment in
consumers (Caut et al., 2009), endoparasites apparently
have depleted N and C isotopic values in comparison to
those of their hosts, resulting in negative ID values for this
group of parasites (Pinnegar et al., 2001; Persson et al.,
2007; Dubois et al., 2009; but see Doucett et al., 1999).
The Gyrocotylidea is one of the most basal groups of
tapeworms, with a unique monozoic body. The group is
poorly studied and is specific to holocephalan chimaeroid
fish, with high prevalence of infection being recorded
(Bandoni & Brooks, 1987; Williams et al., 1987; Bristow,
1992). As in other tapeworms, gyrocotylids lack an
intestine and feed directly on nutrients of the host by
using their surfaces, which are covered by microtiches
(Poddubnaya et al., 2006). Hence, the determination of
intestinal contents is not possible using conventional
methodology. Therefore this host– parasite system is a
most suitable model for using stable isotopes to study
feeding ecology. To our knowledge, no data are available
on values of stable isotopes in this gyrocotylean
tapeworm, nor on the ID between the species Gyrocotyle
urna and its fish host.
In the present study we provide the first estimation of
ID values between a gyrocotylidean tapeworm (G. urna)
and its main host, the holocephalan Chimaera monstrosa.
ID values may be influenced by aspects directly related to
the quality of the resources consumed by the consumer
(i.e. Pearson et al., 2003; Robbins et al., 2005; Dubois et al.,
2009) which, in this case, is directly determined by the
nutritional state of the host. For this reason, we also tested
the effect of three important parameters directly related to
the nutritional state of the host (host size, host sex and
parasite intensity) on ID values, and we illustrate how the
trophic relationships of C. monstrosa and G. urna could
vary by applying different ID values.
Materials and methods
Sampling procedures and isotopic analysis
In July 2011, 15 specimens of C. monstrosa were
collected at depths ranging from 511 to 571 m by Spanish
bottom trawlers fishing in the Gulf of Lions, NW
Mediterranean (428360 N, 38390 E). Following capture,
each C. monstrosa was immediately frozen and stored
at 2208C.
In the laboratory, the body weight (kg), length (cm) and
sex of each fish was recorded. Following dissection of the
stomach and spiral valve, the number of tapeworms was
recorded to determine the prevalence and intensity of
infection. From each host, a single tapeworm and a small
portion of host muscle were collected for the determination of stable isotope values. All tapeworms collected
were identified as G. urna (Williams et al., 1987).
All samples (G. urna and muscle from C. monstrosa) were
freeze-dried, powdered and 0.9 – 1.0 mg of each sample
was packed into tin capsules. Stable isotope analyses were
performed at the Laboratory of Stable Isotopes at the
Estación Biológica de Doñana (www.ebd.csic.es/lie/
index.html). All samples were combusted at 10208C using
a continuous-flow isotope-ratio mass spectrometry system
(Thermo Electron, Thermo Fisher Scientific, Bremen,
Germany), Flasj HT elemental analyser (Thermo Fisher
Scientific) and Delta V Advantage mass spectrometer
(Thermo Fisher Scientific). All isotope abundances are
expressed in d-notation as parts per thousand (‰)
deviation from the International Atomic Energy Agency
(IAEA) standards air (AIR; d15N) and Vienna pee dee
belemnite standard (VPDB; d13C). Based on laboratory
standards, the measurement error was ^ 0.2 and ^ 0.1 for
d15N and d13C, respectively. IDs (Dd15N and Dd13C) were
calculated as the difference between d15N and d13C values
of each individual parasite and its host.
Data analysis
We used non-independent Student’s t-tests to examine
if the values of d15N and d13C differed between C.
monstrosa and G. urna. The effects of host body weight and
length on ID values were analysed using Pearson
correlation tests. The potential effects of the host sex
and parasite intensity on the ID were also tested using
Student’s t-tests. All analyses were conducted in SPSS
18.0 software (SPSS, Inc., Chicago, Illinois, USA) and
results were expressed as means ^ standard deviation
and significant levels of differences set at P # 0.05.
To ensure the accuracy of ID values we estimated and
represented the isotopic space of the parasite (G. urna)
and the host (C. monstrosa) at different potential ID values:
ID-standard (Dd15N ¼ 3.5‰ and Dd13C ¼ 1‰), ID from
previous parasite studies (Dd15N ¼ 21.9‰ and
Dd13C ¼ 2 0.9‰; Pinnegar et al., 2001) and the ID
Isotopic discrimination of a holocephalan tapeworm
Table 1. Mean ^ SD (and range) of d15N and d13C and isotopic
discrimination (ID) values of the host Chimaera monstrosa and the
tapeworm Gyrocotyle urna.
Chimaera
monstrosa
Gyrocotyle
urna
ID (D)
n
d15N (‰)
d13C (‰)
15
11.62 ^ 0.38 (1.05)
216.26 ^ 0.44 (1.60)
15
8.29 ^ 0.58 (1.95)
217.58 ^ 0.51 (2.01)
15
t-test
23.33 ^ 0.63 (2.12)
t ¼ 20.43*
21.32 ^ 0.65 (2.4)
t ¼ 7.74*
* Significant values at P , 0.0001.
estimated in the present study (see table 1). The isotopic
space of the host was represented by a convex-hull
polygon for all C. monstrosa individuals. A convex-hull
polygon is the total area encompassed by the smallest
convex polygon containing these individuals in d15N and
d13C niche dimensions (Layman et al., 2007). The convexhull areas were calculated using the Animal Movement
Extension in ArcView GIS 3.3 (ESRI, Redlands, California,
USA). Additional information on the application of
convex hulls in ecological studies is given by Cornwell
et al. (2006) and Layman et al. (2007).
Results and discussion
The tapeworm G. urna was recorded in the spiral valve
of all 15 C. monstrosa individuals examined, with
intensities of 2.0 and 1.0 individual in 81.25 and 18.75%
of fish, respectively. These values are the first data of
intensity and prevalence of G. urna reported for the
Mediterranean Sea, and show values similar to those
reported previously in other marine systems of the North
Sea coast of Norway (Dienske, 1968; Halvorsen &
Williams, 1968; Williams et al., 1987; Bristow, 1992). The
gyrocotylideans tend to occur in pairs, are protoandric,
and are believed to act as a reproductive unit. The
prevalence of infection tends to increase rapidly to 100%
in fish with body lengths in the range of 35 –50 cm, but
then decreasing to 70% in larger-sized fishes (Dienske,
1968; Halvorsen & Williams, 1968).
Gyrocotyle urna showed significantly lower d15N and
d13C values than C. monstrosa, presenting a negative ID
(mean and standard deviation) of 23.33 ^ 0.63‰ for
d15N and 2 1.32 ^ 0.65‰ for d13C (fig. 1, table 1).
Furthermore, neither fish body size, sex nor parasite
prevalence had a significant effect on the ID values. Both
male and female C. monstrosa showed similar IDs for d15N
(males: n ¼ 8, Dd15N ¼ 23.29 ^ 0.63‰; females: n ¼ 7,
Dd15N ¼ 23.37 ^ 0.67‰; t ¼ 0.21, P ¼ 0.83) and d13C
(males: n ¼ 8, Dd13C ¼ 2 1.17 ^ 0.43‰; females: n ¼ 7,
Dd13C ¼ 2 1.47 ^ 0.85‰; t ¼ 0.86, P ¼ 0.41). Similarly,
host parasite intensity did not affect the IDs for d15N
(C. monstrosa with parasite intensity ¼ 1; n ¼ 3, Dd15N ¼
23.22 ^ 0.83‰; C. monstrosa with parasite intensity ¼ 2:
n ¼ 12, Dd15N ¼ 23.35 ^ 0.61‰; t ¼ 0.29, P ¼ 0.75) and
d13C (C. monstrosa with parasite intensity ¼ 1; n ¼ 3,
Dd13C ¼ 2 0.92 ^ 0.46‰; C. monstrosa with parasite
3
intensity ¼ 2: n ¼ 12, Dd13C ¼ 2 1.41 ^ 0.67‰; t ¼ 1.17,
P ¼ 0.26). Neither body length (mean ¼ 22.67 ^ 2.31 cm,
n ¼ 15) nor body weight (mean ¼ 0.85 ^ 0.25 kg, n ¼ 15)
of C. monstrosa influenced the ID values of d15N (body
length: r ¼ 0.42, P ¼ 0.12; body weight: r ¼ 0.32, P ¼ 0.24)
or of d13C (body length: r ¼ 0.35, P ¼ 0.19; body weight:
r ¼ 0.07, P ¼ 0.81).
Negative values of ID of d15N and d13C are in
agreement with values previously reported for other
parasites of fish, including trematodes (Iken et al., 2001),
nematodes (Iken et al., 2001) and cestodes (Pinnegar et al.,
2001; Power & Klein, 2004), but in contrast to the common
positive ID values reported between predators and prey
(Caut et al., 2009). Different explanations related to
parasite physiology have been proposed to explain the
depletion in the isotopic values between endoparasites
and their hosts. For example, one potential explanation of
depleted N is the ability of some endoparasites to use
ammonia excreted from host tissues (which contains a
higher proportion of the light isotope 14N) for amino acid
synthesis, via a reversal of the glutamate dehydrogenase
reaction (Barrett, 1981; Pinnegar et al., 2001). Alternative
explanations include a differential selection of isotopicdepleted amino acids from the host (Hare et al., 1991).
Large excesses of substrates and products sometimes
result in the formation of complexes, which inhibit
transamination (Boyer et al., 1962), the process responsible
for d15N trophic enrichment (Macko et al., 1986). The
negative ID of d13C between G. urna and C. monstrosa
could be related to the lipid content of G. urna (Berland
et al., 1990) since lipid-rich tissues are known to be 13C
depleted (Focken & Becker, 1998), and thereby show
lower values of d13C compared to the less lipid-rich
muscle of C. monstrosa.
The need to use adequate ID in food-web studies is
pivotal to a correct interpretation of food-web results
(Wolf et al., 2009). This was apparent when we compared
the specific ID values versus different surrogates of ID in
our parasite – host system (fig. 2). By using the ID values
estimated in the present study, the isotopic space of
Fig. 1. Individual and mean ^ SD d15N and d13C values of the
parasite G. urna and its host C. monstrosa. The numbers indicate
each parasite and its host at the individual level.
4
J. Navarro et al.
Fig. 2. Isotopic space (convex-hull polygon) of the parasite G. urna
(grey area) and host C. monstrosa (white area) with different ID
values (ID-standard: Dd15N ¼ 3.5‰ and Dd13C ¼ 1‰; ID-other
parasites: Dd15N ¼ 21.9‰ and Dd13C ¼ 20.9‰; ID-present
study: Dd15N ¼ 23.33‰ and Dd13C ¼ 21.32‰).
C. monstrosa clearly overlapped with that occupied by
G. urna, indicating that the parasite mainly consumes the
host tissue (fig. 2). However, when standard ID of
predator – prey systems (Dd15N ¼ 3.5‰ and Dd13C ¼ 1‰)
were used, and ID values for other parasite– host systems
(Pinnegar et al., 2001), results indicated that the isotopic
space covered by C. monstrosa did not match that of
G. urna, suggesting that the parasite is not feeding on its
host. Therefore the correct estimation of ID values for
specific host– parasite systems is essential in correctly
interpreting food-web dynamics that involve parasites.
In conclusion, we report the first data on ID values of
d15N and d13C between a gyrocotylid tapeworm (G. urna)
and its specific host C. monstrosa. As in other studies, the
ID was negative for both isotopic values of N and
C. Moreover, by comparing the specific ID obtained here
with ID from other studies we have clearly demonstrated
the importance of using specific ID in host – parasite
systems to avoid potential errors in the interpretation of
the results when surrogate values from similar systems or
parasites are used.
Acknowledgements
We thank Isabel Palomera for encouraging this study.
Ricardo Álvarez helped in the stable isotope analysis.
Claudio Barria provided the Chimaera picture of fig. 2.
J.N. and M.C. were supported by research contracts of the
Juan de la Cierva program and Ramon y Cajal (Spanish
Ministry of Economy and Competitiveness), respectively.
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