Journal of Experimental Marine Biology and Ecology 380 (2009) 11–19
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Journal of Experimental Marine Biology and Ecology
j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / j e m b e
Diet and physiological responses of Spondyliosoma cantharus (Linnaeus, 1758) to the
Caulerpa racemosa var. cylindracea invasion
Antonio Box a,⁎, Salud Deudero a,b, Antoni Sureda c, Andreu Blanco a, Josep Alòs d, Jorge Terrados d,
Antoni Maria Grau e, Francisco Riera e
a
Laboratorio de Biología Marina Universidad de las Islas Baleares, Ctra Valldemossa Km 7.5 CP: 07122 Balearic Islands, Spain
Instituto Español de Oceanografia. Centro Oceanográfico de Baleares. P.O. Box 29107015, Palma de Mallorca, Spain
Sciences of the Physical Activity Laboratory, Fundamental Biology and Healthy Sciences Department, University of the Balearic Islands, Ctra. Valldemossa Km 7.5, E-07122 — Palma de
Mallorca, Balearic Islands, Spain
d
Mediterranean Institute for Advanced Studies, IMEDEA (CSIC-UIB), C/ Miquel Marqués 21 E-07190, Esporles, Balearic Islands, Spain
e
Direcció General de Pesca, Govern de les Illes Balears, C/ Foners 10, 07006 Palma de Mallorca, Balearic Islands, Spain
b
c
a r t i c l e
i n f o
Article history:
Received 30 January 2009
Received in revised form 11 August 2009
Accepted 12 August 2009
Keywords:
Antioxidant enzymes
Balearic Islands
Caulerpa racemosa
Spondyliosoma cantharus
Stable isotopes
a b s t r a c t
Marine invasions are a worldwide problem that involves changes in communities and the acclimation of
organisms to them. The invasive Chlorophyte Caulerpa racemosa var. cylindracea is widespread in the
Mediterranean and colonises large areas from 0 to 70 m in depth. The omnivorous fish Spondyliosoma cantharus
presents a high frequency of occurrence of C. racemosa in the stomach contents at invaded areas (76.3%) while no
presence of C. racemosa was detected in control areas. The isotopic composition of muscle differed significantly
between invaded and non-invaded sites for δ13C (−16.67‰ ± 0.09 and −17.67‰ ± 0.08, respectively), δ15N
(10.22‰ ± 0.22 and 9.32‰ ± 0.18, respectively) and the C:N ratio (2.01 ± 0.0002 and 1.96 ± 0.009, respectively).
Despite the high frequency of occurrence of C. racemosa in the stomach contents of S. cantharus and its important
contribution to the δ13C source (20.7% ± 16.2), the contribution of C. racemosa to the δ15N in S. cantharus food
sources was very low (6.6% ± 5.8). Other invertebrate prey such as decapods and polychaetes were more
important contributors to the δ15N source at both invaded and non-invaded sites. Activation of enzymatic
pathways (catalase, superoxide dismutase, glutathione-s-tranferase, 7-ethoxy resorufin O-de-ethylase) but not a
significant increase in lipid peroxidation MDA (0.49 ± 0.01 nmol/mg prot at non-invaded and 0.53± 0.01 nmol/
mg prot at invaded sites) was observed in S. cantharus individuals living in C. racemosa-invaded sites compared
with control specimens. The low δ15N contribution values of C. racemosa by S. cantharus together with the toxicity
demonstrated by the activation of the antioxidant defences and the important contribution of invertebrate prey
to the δ15N could mean that the ingestion of C. racemosa by S. cantharus might be unintentional during the
predation of invertebrate preys living underneath the entanglement of the C. racemosa fronds and stolons mats.
© 2009 Elsevier B.V. All rights reserved.
1. Introduction
Around 100 macrophytes species are thought to have been
introduced into the Mediterranean Sea (Ribera, 2002). The invasive
variety of Caulerpa racemosa (Forsskal) J. Agardh (Chlorophyta, Bryopsidales, Caulerpaceae) was observed for the first time in the Mediterranean Sea in Libya in 1990 (Nizamuddin, 1991). Morphological and
molecular studies indicate that the invasive variety of C. racemosa in the
Mediterranean is similar to the south-west Australian endemic C.
racemosa var. laetevirens f. cylindracea (Sonder) Weber-van Bosse
therefore, the invasive form of C. racemosa in the Mediterranean was
named as C. racemosa var. cylindracea (Sonder) Verlaque, Huisman et
⁎ Corresponding author. Tel.: +34 971 17 33 52; fax: +34 971 17 31 84.
E-mail address: [email protected] (A. Box).
0022-0981/$ – see front matter © 2009 Elsevier B.V. All rights reserved.
doi:10.1016/j.jembe.2009.08.010
Boudouresque (Verlaque et al., 2003). Distance between south-west
Australia and the Mediterranean points to Ship traffic and the aquaria
trade as possible introduction vectors. Only 17 years after its first
observation, C. racemosa var cylindracea (hereafter C. racemosa) has
colonised 12 countries (Italy, Greece, Albania, Cyprus, France, Turkey,
Malta, Spain, Tunisia, Croatia, Algeria and Libya) (Klein and Verlaque,
2008). C. racemosa spreads in sheltered and exposed areas, colonising all
kinds of substrates ranging from 0 to 70 m in depth (Argyrou et al., 1999;
Piazzi and Cinelli, 1999; Zuljevic et al., 2003; Klein and Verlaque, 2008),
spreading over coralligenous bottoms and colonising important areas,
constituting an important threat to such communities (Piazzi et al.,
2007; Klein and Verlaque, 2008).
The fish Spondyliosoma cantharus (Linnaeus, 1978) (Black seabream)
is a common species in the western Mediterranean and can be found
over seagrass beds, especially in the case of juvenile individuals, and
rocky and sandy bottoms to about 300 m depth (Bauchot and Hureau,
12
A. Box et al. / Journal of Experimental Marine Biology and Ecology 380 (2009) 11–19
1990), Isolated rocks and reefs produce a concentration of larger
S. cantharus individuals. Previous studies about S. cantharus diet found
that this species is an omnivorous fish with a wide spectrum of prey
such as mysidacea, crustacean, polychaete, (Bell and Harmelin-Vivien,
1983), algae, copepods, amphipods and hydrozoans (Quéro, 1984;
Goncalves and Erzini, 1998; Pita et al., 2002; Dulčić et al., 2006).
According to these previous studies S. cantharus is considered as an
opportunist fish. The colonisation of the S. cantharus habitat, such as
coralligenous bottoms, by invasive species could lead to changes in the
feeding strategies of this omnivorous species (Goncalves and Erzini,
1998). The presence of Caulerpa species also leads to the presence of
toxic compounds inside the invaded area (Amade et al., 1994; Jung et al.,
2002; Box, 2008) and thus, the activation of the antioxidant defence
systems we evidenced in the fish Coris julis (Linnaeus, 1758) living in
C. racemosa mats (Sureda et al., 2006) and in the invertebrate Bittium
reticulatum (da Costa, 1778) introduced in aquariums containing
C. racemosa (Sureda et al., 2009).
The use of stable isotope analyses in marine ecosystems is increasing,
particularly for the measurement of carbon and nitrogen isotope
assimilation by organisms from food sources in trophic webs (Pinnegar
and Polunin, 1999) and for the detection of the impact of fish farming
waste on adjacent environments (Sarà et al., 2004; Vizzini and Mazzola,
2006; Dolenec et al., 2007). In comparison to stomach content analysis,
which provides information on recently ingested food sources, carbon
and nitrogen stable isotope analyses are a powerful tool for deciphering
the cycling and contribution of multiple organic sources in food web
dynamics (Deniro and Epstein, 1978; Fry and Sherr, 1984; Cabana and
Rasmussen, 1996; Pinnegar and Polunin, 2000; Fisher et al., 2001;
Lorrain et al., 2002; Carabel et al., 2006; Marín Leal et al., 2008) providing
information on the long-term diets of organisms (Dubois et al., 2007).
The discrimination of organic matter sources has been made
possible by the addition of C:N ratios to 13C determinations (Meyers,
1994). The use of δ13C values as evidence of dietary and trophic
differences among species was validated by the examination of the
relationship between biomass δ13C and biomass C:N (Dunton, 2001).
The application of IsoSource mixing models to the main isotopic
contribution source allowed trophic relationships between organisms
and their food sources to be established, showing the potential
contributions of each source to the consumer (Phillips and Gregg, 2003).
C. racemosa produces secondary metabolites, such as caulerpenyne, that may be involved in chemical defence against herbivores
and in competition with other species (Jung et al., 2002). The
antiproliferative and apoptotic effects of C. racemosa crude extracts
and caulerpenyne have been shown in chemoresistant and chemosensitive SHSY5Y cell lines (Cavas et al., 2006). Despite the secondary
metabolites produced by C. racemosa, several potential herbivores,
such as Ascobulla fragilis (Jeffreys, 1856) and Bittium reticulatum, are
encountered in C. racemosa meadows (Box, 2008). The fish species
that have been observed to graze on C. racemosa include Boops boops
(Linnaeus, 1758), Sarpa salpa (Linnaeus, 1758) (Nizamuddin, 1991;
Ruitton et al., 2006) and Diplodus vulgaris (Linnaeus, 1758) and Diplodus sargus (Linnaeus, 1758) (A. Grau personal observations).
A key function of the liver is to metabolise xenobiotics. This
generally involves the biotransformation of lipophilic substances into
more water-soluble metabolites prior to excretion. P450-associated
enzymes catalyse the oxidative conversion of lipophilic xenobiotics
into entities (phase I reactions) that are more water soluble and thus
can be readily excreted and detoxified (Stegeman and Hahn, 1994).
Phase II reactions are biosynthetic reactions in which the foreign
compound or a phase I-derived metabolite are covalently linked to an
endogenous molecule (Sipes and Gandolfi, 1986). Glutathione-stransferases (GST) (phase II enzymes) are essential components of the
cellular antioxidant defence system, as they catalyse the conjugation
of glutathione (GSH) to several dangerous compounds (Rahman et al.,
1999). EROD (7-ethoxy resorufin-O-deethylase) activity has been
used successfully as a potential biomarker of exposure to xenobiotic
contaminants in marine pollution monitoring (Sarkar et al., 2006).
Altogether, the detoxification process and the presence of the toxins
in the environment could lead to an increased production of reactive
oxygen species (ROS). Thus, detoxification and antioxidant enzymes
play a crucial role in maintaining cell homeostasis. These enzymes
have been proposed as biomarkers of contaminant-mediated oxidative stress in a variety of marine organisms, and their induction
reflects a specific response to pollutants or toxins (Cossu et al., 1997).
The aim of the present work was to evaluate the importance of C.
racemosa in the S. cantharus diet and the response of the detoxification
and antioxidant systems in the liver of individuals of this species
living and feeding on C. racemosa-invaded areas.
2. Materials and methods
2.1. Study areas
S. cantharus individuals were captured in Mallorca (SanTelmo,
Palma Bay, Cap Vermell and Alcudia Bay), Ibiza (Santa Eulalia and
Tagomago) and Formentera in the proximity of rocky areas at 36–
70 m depth. Once captured the stomach content was removed and the
presence or absence of C. racemosa in the stomach was recorded.
Having located individuals with/without C. racemosa in the stomach
contents, two invaded/non-invaded sites, i.e. colonised/non-colonised
by C. racemosa, were checked visually by SCUBA (when possible until
40 m depth). Areas invaded by C. racemosa were located in Palma Bay,
on the Mallorcan south coast, where C. racemosa is widespread
(Gamundi-Boyeras et al., 2006) and S. cantharus is very common. Two
different sites inside the bay (separated by more than 8 nautical
miles) with a similar depth ranging from 25 to 34 m with coordinates
39°29'55.24 N 2°42’33.85’’ E and 39°27'11.96" N 2°32'25.76"E respectively were selected as invaded areas. A non-invaded area was selected
on the Mallorcan north coast and two sampling sites separated by more
than 10 nautical miles (Cap Vermell 39°34'42.48" N 3°24'40.12" E and
Alcudia Bay 39°47'21.70" N and 3°18'35.40" E) were selected for capture
of S. cantharus individuals (Fig. 1).
2.2. Analysis of the stomach contents
Once sites were selected, S. cantharus individuals (n = 40 per site)
were captured alive by hook between March and May 2007. Fish were
measured (total length, TL) at the lower 1 cm. Digestive contents were
removed from each individual and stored in 70% ethanol. The stomach
contents were analysed by stereomicroscope to check the frequency
of occurrence of C. racemosa in the digestive tract and the other most
frequent prey (over 10% frequency of occurrence).
Frequency of occurrence =
Number of stomach with the prey considered
Total number of stomachs
The total number of preys in the stomach contents for invaded and
non-invaded sites was also calculated and the relative abundance
(%N) of individuals of a prey category to the total number of prey
individuals in tracts was also calculated (not possible for algae).
2.3. Invertebrate and algae sampling methodology
The benthic fauna was sampled means trawls of 20 m long with a
hand net of 40 × 20, accumulating the sample in a 250 µm mesh bag
following the sampling technique followed by Sanchez-Jerez et al.
(1999). Most abundant prey observed in the S. cantharus stomach
contents were sorted and prepared for stable isotope analysis.
Rodophyta macroalgae (from genus Peyssonnelia, Trichleocarpa, Platoma, Ceramium among others) and C. racemosa were collected
manually by scuba diving.
A. Box et al. / Journal of Experimental Marine Biology and Ecology 380 (2009) 11–19
13
Fig. 1. Distribution of sampling sites around the Balearic Islands. The map indicates areas in which Caulerpa racemosa was detected.
2.4. Stable isotope analyses
Once in the laboratory, the fish were dissected in order to sample
free-bone muscle tissue (13 fish per site). Muscle was taken from the left
side of each fish back to the dorsal fin. Most frequent preys in the
S. cantharus were dissected removing all carbonated tissues. C. racemosa
and rodophyta macroalgae were cleaned from epiphytes. Water
samples (10 l) for POM determination were filtered through precombusted fiberglass filters (Whatman GF/C) at 450 °C for 4 h. Whole
samples were then rinsed thoroughly with 3" distilled water. Muscle
tissue, POM, invertebrate preys, rodophyta macroalgae and C. racemosa
were dried at 60 °C for 24 h and then ground to a fine powder using a
mortar and pestle. Homogeneous dried powder (2 mg± 0.1) of each
sample was placed into cadmium tin cups and then combusted to study
the 13C and 15N stable isotope composition by continuous flow isotope
ratio mass spectrometry (CF-IRMS) using a THERMO DELTA X-PLUS
mass spectrometer. The ISODAT software calculates delta values with
reference to a working standard placed in the dual inlet bellows
(OzTech). The global standard was CO2 for δ13C and atmospheric
nitrogen for δ15N. In addition, an internal reference material was
analysed after every eight samples in order to calibrate the system and
to compensate for drift over time. Reference material used for carbon
and nitrogen stables isotopes analysis was Bovine Liver Standard
(1577b) (U.S. Department of Commerce, National Institute of Standards
and Technology, Gaithersburg, MD 20899). The analytical precision was
based on the standard deviation of the standard replicates; this
deviation was 0.11‰ for δ13C and 0.1‰ for δ15N.
Stable isotope abundances were measured by comparing the ratio
of the most abundant isotopes (13C/12C and 15N/14N) in the sample
with the international isotopic standards. Carbon and nitrogen stable
isotopic ratios were expressed in δ notation in terms of parts per
thousand (‰) deviations from the standards according to the
following equation:
δX =
h
i
3
Rsample = Rreference − 1 × 10
where X is
ratio.
13
C or
15
N and R is the corresponding
13
C/12C or
15
N/14N
2.5. Enzymatic activities and lipid peroxidation markers: tissue
preparation
A total of 12 individuals per site with a similar fish total length
(15–20 cm) were selected for antioxidant biochemical determination.
Specimens were immediately killed by decapitation and the liver was
carefully removed and washed with 0.9% NaCl to prevent enzyme
activities in the blood. Finally, samples were frozen in liquid nitrogen
and maintained at −70 °C until biochemical analysis.
2.6. Biochemical analyses
Aliquots of liver were homogenised in a 1:5 w/v ratio in 100 mM
Tris–HCl buffer pH 7.5, and subsequently centrifuged at 9000 g for
15 min at 4 °C. The resulting supernatant was collected for
spectrophotometric determination of activities of GST and antioxidant
enzymes. Total protein contents were also determined to normalise
enzyme activities (Biorad Protein Assay). These enzyme activities
were determined with a Shimadzu UV-2100 spectrophotometer at
20 °C.
Catalase (CAT) activity was determined according to the method of
Aebi (1984) by the decrease in H2O2 at 240 nm in 50 mM phosphate
buffer.
Superoxide dismutase (SOD) activity was measured by an
adaptation of the method of McCord and Fridovich (1969). The
xanthine/xanthine oxidase system was used to generate the superoxide anion. This anion caused the reduction of cytochrome c, which
was monitored at 550 nm. The superoxide dismutase in the sample
removed the superoxide anion and inhibited reduction.
GST activity was measured by the method developed by Habig et al.
(1974) using reduced GSH and 1-chloro-2,4-dinitrobenzene (CDNB) as
substrates.
14
A. Box et al. / Journal of Experimental Marine Biology and Ecology 380 (2009) 11–19
For the EROD assay livers were homogenised in homogenisation
buffer (50 mM Tris–HCl, KCl 150 mM, pH 7.4) and centrifuged at
10,000 g for 20 min at 4 °C. The supernatant was then centrifuged for
60 min at 100,000 g at 4 °C. Precipitate was recovered and
resuspended in 100 mM phosphate buffer, 20% glycerol, pH 8.0. The
EROD assay was performed using a Bio-Tek Fluorescence Microplate
Reader (BioTek Instruments). The fluorescence of coumarin was
determined by measuring the excitation at emission wavelengths of
360 and 460 nm.
Malondialdehyde (MDA), a marker of lipid peroxidation, was
analysed by a MDA-specific colorimetric assay kit (Calbiochem, San
Diego, CA, USA) following the manufacturer's instructions. Briefly, liver
homogenates or standard were placed in glass tubes containing nmethyl-2-phenylindole (10.3 mM) in acetonitril:methanol (3:1). HCl
12 N was added and samples were incubated for 1 h at 45 °C (Ince et al.,
2007). Absorbance was measured at 586 nm. The MDA concentration
was calculated using a standard curve of known concentration.
2.7. Statistical analysis
Differences in antioxidant response and isotopic composition at
invaded and non-invaded sites were compared by nested multifactorial ANOVA (STATISTICA, 7.0). The factors considered were Invasion
(fixed, two levels: invaded sites and non-invaded sites) and Site
(random, nested in “Invasion”, two levels, sites). Size related changes
in the prey frequency of occurrence, preys relative abundance and
isotopic signatures were tested to establish size classes ranges if
necessary.
To determine which of the most frequent and abundant prey in the
stomach contents (i.e. C. racemosa, shrimps, squat lobsters and
polychaetes, among others) were assimilated by S. cantharus at each
site (invaded/non-invaded), we estimated the potential contributions
for each source by isotope mixing models using IsoSource version
1.3.1 software (Phillips and Gregg, 2003). The model was used to
estimate the potential contributions of most frequent preys observed
in S. cantharus stomach contents. For each prey, the mean, 1st to 99th
percentiles and range of probability contributions to the consumer at
increments of 1% were determined (Decottignies et al., 2007; Ince
et al., 2007; Pitt et al., 2008). Tolerance was calculated as half this
amount (0.5 * increment * maximum differences between sources)
(Phillips and Gregg, 2001; Decottignies et al., 2007). In the absence
of consumer-specific isotope discrimination factors an assumed
discrimination of 1.3‰ was applied for carbon (McCutchan et al.,
2003). Trophic fractionation is much larger for δ15N than for δ13C
(McCutchan et al., 2003; Behringer and Butler, 2006), so this must be
corrected based on a reported average fractionation increase of 2.2‰
per trophic level; the precise value depends on the diet as it can be
higher when feeding on invertebrates or high-protein diets
(McCutchan et al., 2003; Li and Dudgeon, 2008) which provide
much better information in the IsoSource analyses.
3. Results
3.1. Presence of C. racemosa in the stomach contents
In the preliminary survey to locate sampling areas, C. racemosa was
absent from the stomach contents of individuals from Ibiza (n = 10),
Formentera (n = 12) and Mallorca (north coast) (n = 25) while
individuals from Palma Bay (n = 30) and SanTelmo (n = 5) contained
C. racemosa in the stomach contents. Due to the low number of captures
in SanTelmo two invaded sites were located in Palma Bay. The two noninvaded sites were located on the north coast of Mallorca.
Sampling in Palma Bay and Mallorca North coast involved the
capture of 160 individuals and the analyses their stomach content. C.
racemosa was present in the stomach of a total of 76.3% of the
individuals captured at invaded sites. The size (TL) of S. cantharus in
the invaded area ranged from 12 to 24 cm. No size differences were
observed in the prey frequency of occurrence for each locality. No
differences in the frequency of occurrence of C. racemosa in the
stomach contents were observed between the two invaded sites. In
both invaded and non-invaded areas the most frequent prey in the
stomach contents were shrimps (Caridea) (frequency of occurrence
57.89% and 78.32% respectively), squat lobsters (Anomura) (frequency of occurrence 10.53% and 30.21% respectively) and polychaetes
(Aciculata) (frequency of occurrence 15.79% and 7.10% respectively).
Other macroalgae (mainly rodophyta) were also present in the
stomach contents both in invaded and non invaded sites (frequency of
occurrence 18.42% and 10.00% respectively).
A total of 550 invertebrate preys had been observed in the S.
cantharus stomach contents; 330 invertebrates prey in C. racemosa
invaded sites and 220 in the control sites. No size differences were
observed in the prey relative abundance for each treatment. Caridea
invertebrate preys showed the highest relative abundance both in
invaded and non invaded sites (54.64% and 57.07% respectively).
Respect to the others category preys in invaded and non invaded sites,
the relative abundance of polychaetes was 7.49% and 5.00% (respectively), for squad lobsters 10.49% and 15.80% (respectively). Amphipods also presented an important relative abundance in invaded sites
3.33% and non-invaded sites 2.00%. The relative abundance in the
stomach contents of nematodes was also remarkable with 2.12% in
invaded sites and 3.01% in control sites. Other category preys
observed in the stomach contents were amphipods, crabs, sipunculids, isopods, mysids, copepods, ostracods and chaetognaths.
3.2. Stable isotope analyses
S. cantharus from the same locality did not present size differences
in the isotopic signatures. According to this result and that no
differences in prey frequency of occurrence were observed, no size
intervals were considered. There were differences in the δ13C, δ15N
and the C:N ratio of muscle between invaded and non-invaded sites.
The δ13C and C:N ratio also differed between sites of the same
invasion status (Table 1). δ13C values varied between treatments,
being enriched at invaded sites (−16.67‰ ± 0.09) compared with
non-invaded sites (−17.67‰ ± 0.08). Likewise the δ15N signatures
varied with mean values of 10.22‰ ± 0.22 for invaded localities and
low values for the non-invaded ones (9.32‰ ± 0.18) (Fig. 2).
The isotopic signature at the invaded sites ranged from − 17.29‰ to
−15.44‰ for δ13C, and from 9.12‰ to 14.45‰ for δ15N. Conversely,
values for the non-invaded sites ranged between − 18.01‰ and
−16.68‰ for 13C isotopic signatures and between 5.75‰ and 10.37‰
for 15N. The C:N ratios exhibited a narrow range of variability between
S. cantharus in invaded and non-invaded sites, being 1.97–2.12 and
1.92–2.1, respectively. The mean C:N values (± SE) obtained
at invaded sites was 2.01 ± 0.0002 while at non-invaded sites it was
1.96 ± 0.009.
Analysis of the contributions of food sources using the IsoSource
routine showed differences in carbon and nitrogen sources in S.
cantharus related to their origin from invaded/control sites. C. racemosa
mean contribution was 20.7 ± 16.2% and 6.6 ± 5.8% respectively to the
total carbon and nitrogen sources in the fish from invaded sites. The
main sources of carbon and nitrogen for S. cantharus at invaded and
Table 1
Nested multifactorial analysis of variance of isotopic composition (δ C13, δ N15 and C:N
ratio).
Invaded
Site (invaded)
Error
df
δC13
δN15
C:N
1
2
48
14.55***
0.86**
0.12
11.91***
2.32
0.89
0.045***
0.012**
0.001
MS means square, ***p < 0.001 **p < 0.01 *p < 0.05.
A. Box et al. / Journal of Experimental Marine Biology and Ecology 380 (2009) 11–19
15
Fig. 2. Relative contributions of carbon and nitrogen sources to Spondyliosoma cantharus captured at invaded (A) and non-invaded (B) sites based on mean stable isotope values
(± standard deviation). The most frequent prey observed in the stomach contents are shown (shrimps, squat lobsters, polychaetes, Rhodophyta and Caulerpa racemosa).
control sites were invertebrate preys such as decapoda shrimps, squat
lobsters and polychaetes but also macroalgae contribute to the isotopic
signature in both sites (Fig. 3). In both invaded and non invaded,
considering only nitrogen contributions, the importance of invertebrate
preys was more clear being shrimps the most important contributor in
invaded sites (48.8 ± 8.6%) and polychaetes and shrimps in noninvaded sites (28.6 ± 19.5% and 27.2 ± 13.3% respectively).
3.3. Biochemical analysis
The activity of hepatic antioxidant enzymes in S. cantharus feeding
among C. racemosa at invaded sites and those feeding at control sites
showed significant differences related to the ingestion of the toxic
invasive macroalga. CAT activity (expressed as mK/ mg prot)
increased from 125.4 ± 12.3 at non-invaded sites to 227.0 ± 28.0 at
the invaded sites; SOD activity (expressed as pKat/mg prot) also
increased from 2.18 ± 0.03 at the non-invaded sites to 2.29 ± 0.05 at
the invaded sites. EROD (RFU/mg prot) and GST (nKat/mg prot)
showed the same pattern, with increases from 464.00 ± 15.83
and 364.72 ± 23.44 at the non-invaded sites to 544.61 ± 14.77 and
532.26 ± 34.23 at invaded sites respectively. All enzymatic activities
of CAT, SOD, GST and EROD were significantly higher in the liver of the
individuals living in invaded sites (ANOVA p < 0.05) (Table 2). MDA
levels did not differ significantly between treatments (0.49 ± .
0.01 nmol/mg prot non-invaded and 0.53 ± 0.01 nmol/mg prot
invaded) (Table 2).
4. Discussion
The current invasion of C. racemosa in the Mediterranean is
changing ecosystem functioning, habitat structure and invertebrate
communities (Box, 2008). The extent of the change does not only
affect invertebrates but it can also affect fish such as S. cantharus. This
omnivorous opportunistic feeder (Goncalves and Erzini, 1998) seems
to be feeding among C. racemosa, as shown by the high frequency of
occurrence of the invasive macroalga in the stomach contents of this
species. Previous studies in the Mediterranean showed feeding on this
invasive macroalga by other organisms such as the fish Boops boops
and Sarpa salpa, the sea-urchin Paracentrotus lividus (de Lamarck,
1816) (Ruitton et al., 2006), the opisthobranchs Lobiger serradifalci
(Calcara, 1840), Oxynoe olivacea Rafinesque (1814) (Cavas et al.,
2005) and the opisthobranch Ascobulla fragilis (Box, 2008).
Caulerpa species share a common thallus architecture composed of
a creeping portion, the stolon, which is attached to the substrate by
16
A. Box et al. / Journal of Experimental Marine Biology and Ecology 380 (2009) 11–19
Fig. 3. Distribution of potential carbon (left column) and nitrogen (right column) contributions (based on δ13C and δ15N) of the most frequent prey in Spondyliosoma cantharus
stomach contents at invaded (white bars) and non-invaded sites (black bars). Mean (% ± SD) carbon and nitrogen contributions are indicated.
rhizoids, and an erect portion, the fronds, with differing shapes
depending on the species (Bold and Wynne, 1978). The architecture of
Caulerpa, in particular the stolons and rhizoids, results in a high
capacity to retain fine sand fractions according to Caulerpa highest
biomasses (Sanchez-Moyano et al., 2001). This means a complex
structure over substrate of invasive macroalga which could form a
mechanical barrier to the invertebrate feeders. The presence of the
invasive Caulerpa taxifolia (M.Vahl) C. Agardh, also in the Mediterranean, has caused a decrease in the abundance of Mullus surmuletus
(Linnaeus, 1758) due to the resulting barrier that prevents fish
accessing their food sources (Longepierre et al., 2005). In the Balearic
Islands, C. racemosa forms very extensive mats at depths ranging from
A. Box et al. / Journal of Experimental Marine Biology and Ecology 380 (2009) 11–19
Table 2
Nested multifactorial analysis of variance of antioxidant enzyme activities and MDA
concentration in Spondyliosoma cantharus.
df
CAT (MS) SOD (MS) EROD (MS) GST (MS)
Invaded
1 95,284***
Site (invaded)
2 9388
Error
44 135,677
0.1989*
0.0073
0.0473
98,918***
1660
5657
MDA (MS)
312767*** 0.01606a
29076
0.00013
19597
0.00402
MS means square, ***p < 0.001 **p < 0.01 *p < 0.05. a means p = 0.052.
20 to 40 m (over coralligenous bottoms and rocky reefs) which could
also act as a barrier against predators of invertebrates (authors
personal observations).
Isotopic values provide information related to shifts in food
sources (Pinnegar and Polunin, 2000; Deudero et al., 2004) of
S. cantharus. The isotopic composition of the fish was similar between
sites with equal treatments but differed between invaded/noninvaded sites. The main food sources for S. cantharus were
invertebrates such as squad lobsters (Galathea spp.), shrimps and
polychaetes. The contribution of C. racemosa as a carbon and nitrogen
food source was low, despite the high frequency of occurrence in the
stomach contents. The contribution of δ13C by C. racemosa was in the
same range as other invertebrate food sources (shrimps and
polychaetes), but the δ15N contribution of C. racemosa was very low
compared with invertebrate food sources. For δ15N the contribution of
invertebrate preys, especially shrimps and polychaetes, was similar
between invaded and non-invaded sites, which reflect its importance
in the S. cantharus diet.
δ13C has been used to track animal movements between areas with
different food sources (Kurle and Worthy, 2001) and to evaluate the
importance of different food sources (Pinnegar and Polunin, 2000). In
this case, the contribution of C. racemosa as one of the main food
sources for S. cantharus is clear. It has been documented that fish
usually produce C:N ratios ranging between 3.3 and 5.1 (McConnaughey and McRoy, 1979). In the present work, the C:N of
S. cantharus ranged from 1.92 to 2.12, and no differences were
found between individuals at invaded and non-invaded sites which
presented similar C:N values, indicating that none were experiencing
feeding stress. The low C:N ratios may be due to the high proportion of
protein in the diet, which was of high quality (Waddington and
MacArthur, 2008). Moreover, the C:N ratio indicates diet quality and
explains the degree of fractionation among the individual compounds
of C:N within diets (Waddington and MacArthur, 2008). Thus, similar
values of C:N ratios imply that S. cantharus had a similar dietary
quality in invaded and non-invaded sites. The δ15N values are more
representative of the assimilation of the food sources since they are
related to metabolic and physiological processes (Sweeting et al.,
2007). Metabolic fractionation differs between carnivores and
herbivores; while in carnivores metabolic fractionation is dominant
because animal-derived nitrogen is biochemically more homogeneous and dominated by proteins, in herbivores both assimilative and
metabolic factors affect fractionation (Vander Zanden and Rasmussen,
2001). Herbivorous fish also have a very long alimentary tract to
increase absorption efficiency (Mill et al., 2007). S. cantharus is not
exclusively herbivorous and in some localities feeds mainly on
invertebrates (Goncalves and Erzini, 1998) and it does not have a
long alimentary tract like exclusive herbivores. Therefore, for this
species it may be necessary to ingest a large amount of Caulerpa to
assimilate carbon and nitrogen. On the other hand, the assimilation
rates of invertebrates were very large in fish caught at the invaded
sites, which could imply that animal-derived nitrogen was the best
assimilated.
Cellular antioxidant status is widely used as a tool to evaluate the
ability of organisms to resist environmental stress (Frenzilli et al.,
2004) and the effects of invasive species on faunistic communities
(Sureda et al., 2006, 2009; Box et al., 2008b). The efficiency of ROS
17
detoxification and stress tolerance has been reported by several
authors (Porte et al., 2002; Cavas and Yurdakoc, 2005). CAT is
involved in H2O2 detoxification and SOD uses the superoxide anion to
produce H2O2. GST participates in the detoxification of lipid hydroperoxides using GSH as a substrate. EROD is used as a potential
biomarker of the presence of xenobiotics in the aquatic medium
(Fatima and Ahmad, 2005). The antioxidant response observed in
S. cantharus feeding on C. racemosa mats involved increased
antioxidant and detoxifying activities (CAT, SOD, EROD and GST)
compared to those in fish feeding in non-invaded habitats. These
results imply that the ingestion of C. racemosa produces a detoxifying
response in S. cantharus. To evaluate whether toxicity is negatively
affecting this species we used MDA as a useful marker of lipid
peroxidation. MDA concentration did not differ significantly between
treatments, but the higher MDA values obtained for invaded sites
were only just outside the limit of significance.
This study has presented a combination of methodologies such as
stomach contents analysis, stable isotope determinations and antioxidant response quantifications to allow us to understand the
contribution of the ingestion of C. racemosa in the diet of S. cantharus.
This fish feeds on C. racemosa but does not assimilate important levels
of this food source. Previous work in this field demonstrated the
toxicological effects of Caulerpa on invertebrate and fish species
(Sureda et al., 2006, 2009; Box et al., 2008b). The same effect was
observed in S. cantharus with increased antioxidant levels in
individuals living in C. racemosa. The toxicity of the alga altogether
with its low assimilation by the fish, in contrast with the high
assimilation of invertebrates, indicates that the ingestion of
C. racemosa is at least in part accidental while the fish are attempting
to access the invertebrates that live under the fronds and stolon net
formed by C. racemosa. The invertebrate community in C. racemosa is
very diverse and some groups such as polychaetes (Box et al., in
press), molluscs and decapods are highly abundant and diverse (Box,
2008; Box et al., 2008a).
In conclusion, S. cantharus feeds on C. racemosa in invaded areas
but does not use this source as its main carbon and nitrogen source.
One possible reason is the toxicity of the algae which has a
physiological effect on the fish. Thus the faecal pellets contain high
amounts of C. racemosa, which in most cases maintain the integrity of
fronds and stolons, which are not completely digested. Further work
must be done to check the selection of prey of this fish in invaded
ecosystems.
Acknowledgements
This work was supported by the research projects “Macroalgas
marinas invasoras en las Islas Baleares: Evaluación de riesgos y efectos
en comunidades bentónicas” (CTM2005-01434/MAR) of the Ministerio de Educación y Ciencia and the Project “Avaluació I seguiment
dels recursos marins de la CAIB, 2008” of the Direcció General de Pesca
(Balearic Islands). We must agree the collaboration of the Mar-I-Pi II
and Pedaç anglers during sampling in Ibiza and Majorca Islands. [SS]
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