Diet and Physiological Responses of Spondyliosoma Cantharus (Linnaeus, 1758) to the Caulerpa Racemosa Var

Journal of Experimental Marine Biology and Ecology 380 (2009) 11–19

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Journal of Experimental Marine Biology and Ecology

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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 b Instituto Español de Oceanograﬁa. Centro Oceanográﬁco de Baleares. P.O. Box 29107015, Palma de Mallorca, Spain c 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 article info abstract

Article history: Marine invasions are a worldwide problem that involves changes in communities and the acclimation of Received 30 January 2009 organisms to them. The invasive Chlorophyte Caulerpa racemosa var. cylindracea is widespread in the Received in revised form 11 August 2009 Mediterranean and colonises large areas from 0 to 70 m in depth. The omnivorous fish Spondyliosoma cantharus Accepted 12 August 2009 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 Keywords: between invaded and non-invaded sites for δ13C(−16.67‰±0.09 and −17.67‰±0.08, respectively), δ15N Antioxidant enzymes ‰ ‰ Balearic Islands (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). Caulerpa racemosa Despite the high frequency of occurrence of C. racemosa in the stomach contents of S. cantharus and its important 13 15 Spondyliosoma cantharus contribution to the δ C source (20.7%±16.2), the contribution of C. racemosa to the δ NinS. cantharus food Stable isotopes 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 δ15NcontributionvaluesofC. 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 Boudouresque (Verlaque et al., 2003). Distance between south-west Australia and the Mediterranean points to Ship trafficandtheaquaria Around 100 macrophytes species are thought to have been trade as possible introduction vectors. Only 17 years after its first introduced into the Mediterranean Sea (Ribera, 2002). The invasive observation, C. racemosa var cylindracea (hereafter C. racemosa)has variety of Caulerpa racemosa (Forsskal) J. Agardh (Chlorophyta, Bryopsi- colonised 12 countries (Italy, Greece, Albania, Cyprus, France, Turkey, dales, Caulerpaceae) was observed for the first time in the Mediterra- Malta, Spain, Tunisia, Croatia, Algeria and Libya) (Klein and Verlaque, nean Sea in Libya in 1990 (Nizamuddin, 1991). Morphological and 2008). C. racemosa spreads in sheltered and exposed areas, colonising all molecular studies indicate that the invasive variety of C. racemosa in the kinds of substrates ranging from 0 to 70 m in depth (Argyrou et al., 1999; Mediterranean is similar to the south-west Australian endemic C. Piazzi and Cinelli, 1999; Zuljevic et al., 2003; Klein and Verlaque, 2008), racemosa var. laetevirens f. cylindracea (Sonder) Weber-van Bosse spreading over coralligenous bottoms and colonising important areas, therefore, the invasive form of C. racemosa in the Mediterranean was constituting an important threat to such communities (Piazzi et al., named as C. racemosa var. cylindracea (Sonder) Verlaque, Huisman et 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 ⁎ Corresponding author. Tel.: +34 971 17 33 52; fax: +34 971 17 31 84. over seagrass beds, especially in the case of juvenile individuals, and E-mail address: [email protected] (A. Box). rocky and sandy bottoms to about 300 m depth (Bauchot and Hureau,

1990), Isolated rocks and reefs produce a concentration of larger contaminants in marine pollution monitoring (Sarkar et al., 2006). S. cantharus individuals. Previous studies about S. cantharus diet found Altogether, the detoxification process and the presence of the toxins that this species is an omnivorous fish with a wide spectrum of prey in the environment could lead to an increased production of reactive such as mysidacea, crustacean, polychaete, (Bell and Harmelin-Vivien, oxygen species (ROS). Thus, detoxification and antioxidant enzymes 1983), algae, copepods, amphipods and hydrozoans (Quéro, 1984; play a crucial role in maintaining cell homeostasis. These enzymes Goncalves and Erzini, 1998; Pita et al., 2002; Dulčić et al., 2006). have been proposed as biomarkers of contaminant-mediated oxida- According to these previous studies S. cantharus is considered as an tive stress in a variety of marine organisms, and their induction opportunist fish. The colonisation of the S. cantharus habitat, such as reflects a specific response to pollutants or toxins (Cossu et al., 1997). coralligenous bottoms, by invasive species could lead to changes in the The aim of the present work was to evaluate the importance of C. feeding strategies of this omnivorous species (Goncalves and Erzini, racemosa in the S. cantharus diet and the response of the detoxification 1998). The presence of Caulerpa species also leads to the presence of and antioxidant systems in the liver of individuals of this species toxic compounds inside the invaded area (Amade et al., 1994; Jung et al., living and feeding on C. racemosa-invaded areas. 2002; Box, 2008) and thus, the activation of the antioxidant defence systems we evidenced in the fish Coris julis (Linnaeus, 1758) living in 2. Materials and methods C. racemosa mats (Sureda et al., 2006) and in the invertebrate Bittium reticulatum (da Costa, 1778) introduced in aquariums containing 2.1. Study areas C. racemosa (Sureda et al., 2009). The use of stable isotope analyses in marine ecosystems is increasing, S. cantharus individuals were captured in Mallorca (SanTelmo, particularly for the measurement of carbon and nitrogen isotope Palma Bay, Cap Vermell and Alcudia Bay), Ibiza (Santa Eulalia and assimilation by organisms from food sources in trophic webs (Pinnegar Tagomago) and Formentera in the proximity of rocky areas at 36– and Polunin, 1999) and for the detection of the impact of fish farming 70 m depth. Once captured the stomach content was removed and the waste on adjacent environments (Sarà et al., 2004; Vizzini and Mazzola, presence or absence of C. racemosa in the stomach was recorded. 2006; Dolenec et al., 2007). In comparison to stomach content analysis, Having located individuals with/without C. racemosa in the stomach which provides information on recently ingested food sources, carbon contents, two invaded/non-invaded sites, i.e. colonised/non-colonised and nitrogen stable isotope analyses are a powerful tool for deciphering by C. racemosa, were checked visually by SCUBA (when possible until the cycling and contribution of multiple organic sources in food web 40 m depth). Areas invaded by C. racemosa were located in Palma Bay, dynamics (Deniro and Epstein, 1978; Fry and Sherr, 1984; Cabana and on the Mallorcan south coast, where C. racemosa is widespread Rasmussen, 1996; Pinnegar and Polunin, 2000; Fisher et al., 2001; (Gamundi-Boyeras et al., 2006) and S. cantharus is very common. Two Lorrain et al., 2002; Carabel et al., 2006; Marín Leal et al., 2008) providing different sites inside the bay (separated by more than 8 nautical information on the long-term diets of organisms (Dubois et al., 2007). miles) with a similar depth ranging from 25 to 34 m with coordinates The discrimination of organic matter sources has been made 39°29'55.24 N 2°42’33.85’’ E and 39°27'11.96" N 2°32'25.76"E respec- possible by the addition of C:N ratios to 13C determinations (Meyers, tively were selected as invaded areas. A non-invaded area was selected 1994). The use of δ13C values as evidence of dietary and trophic on the Mallorcan north coast and two sampling sites separated by more differences among species was validated by the examination of the than 10 nautical miles (Cap Vermell 39°34'42.48" N 3°24'40.12" E and relationship between biomass δ13C and biomass C:N (Dunton, 2001). Alcudia Bay 39°47'21.70" N and 3°18'35.40" E) were selected for capture The application of IsoSource mixing models to the main isotopic of S. cantharus individuals (Fig. 1). contribution source allowed trophic relationships between organisms and their food sources to be established, showing the potential 2.2. Analysis of the stomach contents contributions of each source to the consumer (Phillips and Gregg, 2003). C. racemosa produces secondary metabolites, such as caulerpe- Once sites were selected, S. cantharus individuals (n=40 per site) nyne, that may be involved in chemical defence against herbivores were captured alive by hook between March and May 2007. Fish were and in competition with other species (Jung et al., 2002). The measured (total length, TL) at the lower 1 cm. Digestive contents were antiproliferative and apoptotic effects of C. racemosa crude extracts removed from each individual and stored in 70% ethanol. The stomach and caulerpenyne have been shown in chemoresistant and chemo- contents were analysed by stereomicroscope to check the frequency sensitive SHSY5Y cell lines (Cavas et al., 2006). Despite the secondary of occurrence of C. racemosa in the digestive tract and the other most metabolites produced by C. racemosa, several potential herbivores, frequent prey (over 10% frequency of occurrence). such as Ascobulla fragilis (Jeffreys, 1856) and Bittium reticulatum, are encountered in C. racemosa meadows (Box, 2008). The fish species Number of stomach with the prey considered that have been observed to graze on C. racemosa include Boops boops Frequency of occurrence = Total number of stomachs (Linnaeus, 1758), Sarpa salpa (Linnaeus, 1758) (Nizamuddin, 1991; Ruitton et al., 2006) and Diplodus vulgaris (Linnaeus, 1758) and Di- plodus sargus (Linnaeus, 1758) (A. Grau personal observations). The total number of preys in the stomach contents for invaded and A key function of the liver is to metabolise xenobiotics. This non-invaded sites was also calculated and the relative abundance generally involves the biotransformation of lipophilic substances into (%N) of individuals of a prey category to the total number of prey more water-soluble metabolites prior to excretion. P450-associated individuals in tracts was also calculated (not possible for algae). enzymes catalyse the oxidative conversion of lipophilic xenobiotics into entities (phase I reactions) that are more water soluble and thus 2.3. Invertebrate and algae sampling methodology can be readily excreted and detoxified (Stegeman and Hahn, 1994). Phase II reactions are biosynthetic reactions in which the foreign The benthic fauna was sampled means trawls of 20 m long with a compound or a phase I-derived metabolite are covalently linked to an hand net of 40×20, accumulating the sample in a 250 µm mesh bag endogenous molecule (Sipes and Gandolfi, 1986). Glutathione-s- following the sampling technique followed by Sanchez-Jerez et al. transferases (GST) (phase II enzymes) are essential components of the (1999). Most abundant prey observed in the S. cantharus stomach cellular antioxidant defence system, as they catalyse the conjugation contents were sorted and prepared for stable isotope analysis. of glutathione (GSH) to several dangerous compounds (Rahman et al., Rodophyta macroalgae (from genus Peyssonnelia, Trichleocarpa, Pla- 1999). EROD (7-ethoxy resorufin-O-deethylase) activity has been toma, Ceramium among others) and C. racemosa were collected used successfully as a potential biomarker of exposure to xenobiotic 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.

13 15 13 12 15 14 2.4. Stable isotope analyses where X is Cor N and R is the corresponding C/ Cor N/ N ratio. Once in the laboratory, the fish were dissected in order to sample fi free-bonemuscletissue(13 sh per site). Muscle was taken from the left 2.5. Enzymatic activities and lipid peroxidation markers: tissue fi fi side of each sh back to the dorsal n. Most frequent preys in the preparation S. cantharus were dissected removing all carbonated tissues. C. racemosa and rodophyta macroalgae were cleaned from epiphytes. Water A total of 12 individuals per site with a similar fish total length fi samples (10 l) for POM determination were ltered through pre- (15–20 cm) were selected for antioxidant biochemical determination. fi fi combusted berglass lters (Whatman GF/C) at 450 °C for 4 h. Whole Specimens were immediately killed by decapitation and the liver was samples were then rinsed thoroughly with 3" distilled water. Muscle carefully removed and washed with 0.9% NaCl to prevent enzyme tissue, POM, invertebrate preys, rodophyta macroalgae and C. racemosa activities in the blood. Finally, samples were frozen in liquid nitrogen fi were dried at 60 °C for 24 h and then ground to a ne powder using a and maintained at −70 °C until biochemical analysis. 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 13Cand15N stable isotope composition by continuous flow isotope 2.6. Biochemical analyses ratio mass spectrometry (CF-IRMS) using a THERMO DELTA X-PLUS mass spectrometer. The ISODAT software calculates delta values with Aliquots of liver were homogenised in a 1:5 w/v ratio in 100 mM – reference to a working standard placed in the dual inlet bellows Tris HCl buffer pH 7.5, and subsequently centrifuged at 9000 g for 13 15 min at 4 °C. The resulting supernatant was collected for (OzTech). The global standard was CO2 for δ C and atmospheric nitrogen for δ15N. In addition, an internal reference material was spectrophotometric determination of activities of GST and antioxidant analysed after every eight samples in order to calibrate the system and enzymes. Total protein contents were also determined to normalise to compensate for drift over time. Reference material used for carbon enzyme activities (Biorad Protein Assay). These enzyme activities and nitrogen stables isotopes analysis was Bovine Liver Standard were determined with a Shimadzu UV-2100 spectrophotometer at (1577b) (U.S. Department of Commerce, National Institute of Standards 20 °C. and Technology, Gaithersburg, MD 20899). The analytical precision was Catalase (CAT) activity was determined according to the method of based on the standard deviation of the standard replicates; this Aebi (1984) by the decrease in H2O2 at 240 nm in 50 mM phosphate deviation was 0.11‰ for δ13C and 0.1‰ for δ15N. buffer. Stable isotope abundances were measured by comparing the ratio Superoxide dismutase (SOD) activity was measured by an of the most abundant isotopes (13C/12C and 15N/14N) in the sample adaptation of the method of McCord and Fridovich (1969). The with the international isotopic standards. Carbon and nitrogen stable xanthine/xanthine oxidase system was used to generate the super- isotopic ratios were expressed in δ notation in terms of parts per oxide anion. This anion caused the reduction of cytochrome c, which thousand (‰) deviations from the standards according to the was monitored at 550 nm. The superoxide dismutase in the sample following equation: removed the superoxide anion and inhibited reduction. GST activity was measured by the method developed by Habig et al. hi (1974) using reduced GSH and 1-chloro-2,4-dinitrobenzene (CDNB) as δX = R = R − 1 ×103 sample reference 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 the invaded area ranged from 12 to 24 cm. No size differences were buffer (50 mM Tris–HCl, KCl 150 mM, pH 7.4) and centrifuged at observed in the prey frequency of occurrence for each locality. No 10,000 g for 20 min at 4 °C. The supernatant was then centrifuged for differences in the frequency of occurrence of C. racemosa in the 60 min at 100,000 g at 4 °C. Precipitate was recovered and stomach contents were observed between the two invaded sites. In resuspended in 100 mM phosphate buffer, 20% glycerol, pH 8.0. The both invaded and non-invaded areas the most frequent prey in the EROD assay was performed using a Bio-Tek Fluorescence Microplate stomach contents were shrimps (Caridea) (frequency of occurrence Reader (BioTek Instruments). The fluorescence of coumarin was 57.89% and 78.32% respectively), squat lobsters (Anomura) (frequen- determined by measuring the excitation at emission wavelengths of cy of occurrence 10.53% and 30.21% respectively) and polychaetes 360 and 460 nm. (Aciculata) (frequency of occurrence 15.79% and 7.10% respectively). Malondialdehyde (MDA), a marker of lipid peroxidation, was Other macroalgae (mainly rodophyta) were also present in the analysed by a MDA-specific colorimetric assay kit (Calbiochem, San stomach contents both in invaded and non invaded sites (frequency of Diego, CA, USA) following the manufacturer's instructions. Briefly, liver occurrence 18.42% and 10.00% respectively). homogenates or standard were placed in glass tubes containing n- A total of 550 invertebrate preys had been observed in the S. methyl-2-phenylindole (10.3 mM) in acetonitril:methanol (3:1). HCl cantharus stomach contents; 330 invertebrates prey in C. racemosa 12 N was added and samples were incubated for 1 h at 45 °C (Ince et al., invaded sites and 220 in the control sites. No size differences were 2007). Absorbance was measured at 586 nm. The MDA concentration observed in the prey relative abundance for each treatment. Caridea was calculated using a standard curve of known concentration. invertebrate preys showed the highest relative abundance both in invaded and non invaded sites (54.64% and 57.07% respectively). 2.7. Statistical analysis Respect to the others category preys in invaded and non invaded sites, the relative abundance of polychaetes was 7.49% and 5.00% (respec- Differences in antioxidant response and isotopic composition at tively), for squad lobsters 10.49% and 15.80% (respectively). Amphi- invaded and non-invaded sites were compared by nested multifac- pods also presented an important relative abundance in invaded sites torial ANOVA (STATISTICA, 7.0). The factors considered were Invasion 3.33% and non-invaded sites 2.00%. The relative abundance in the (fixed, two levels: invaded sites and non-invaded sites) and Site stomach contents of nematodes was also remarkable with 2.12% in (random, nested in “Invasion”, two levels, sites). Size related changes invaded sites and 3.01% in control sites. Other category preys in the prey frequency of occurrence, preys relative abundance and observed in the stomach contents were amphipods, crabs, sipuncu- isotopic signatures were tested to establish size classes ranges if lids, isopods, mysids, copepods, ostracods and chaetognaths. necessary. To determine which of the most frequent and abundant prey in the 3.2. Stable isotope analyses stomach contents (i.e. C. racemosa, shrimps, squat lobsters and polychaetes, among others) were assimilated by S. cantharus at each S. cantharus from the same locality did not present size differences site (invaded/non-invaded), we estimated the potential contributions in the isotopic signatures. According to this result and that no for each source by isotope mixing models using IsoSource version differences in prey frequency of occurrence were observed, no size 1.3.1 software (Phillips and Gregg, 2003). The model was used to intervals were considered. There were differences in the δ13C, δ15N estimate the potential contributions of most frequent preys observed and the C:N ratio of muscle between invaded and non-invaded sites. in S. cantharus stomach contents. For each prey, the mean, 1st to 99th The δ13C and C:N ratio also differed between sites of the same percentiles and range of probability contributions to the consumer at invasion status (Table 1). δ13C values varied between treatments, increments of 1% were determined (Decottignies et al., 2007; Ince being enriched at invaded sites (−16.67‰±0.09) compared with et al., 2007; Pitt et al., 2008). Tolerance was calculated as half this non-invaded sites (−17.67‰±0.08). Likewise the δ15N signatures amount (0.5*increment*maximum differences between sources) varied with mean values of 10.22‰±0.22 for invaded localities and (Phillips and Gregg, 2001; Decottignies et al., 2007). In the absence low values for the non-invaded ones (9.32‰ ±0.18) (Fig. 2). of consumer-specific isotope discrimination factors an assumed The isotopic signature at the invaded sites ranged from −17.29‰ to discrimination of 1.3‰ was applied for carbon (McCutchan et al., −15.44‰ for δ13C, and from 9.12‰ to 14.45‰ for δ15N. Conversely, 2003). Trophic fractionation is much larger for δ15N than for δ13C values for the non-invaded sites ranged between −18.01‰ and (McCutchan et al., 2003; Behringer and Butler, 2006), so this must be −16.68‰ for 13C isotopic signatures and between 5.75‰ and 10.37‰ corrected based on a reported average fractionation increase of 2.2‰ for 15N. The C:N ratios exhibited a narrow range of variability between per trophic level; the precise value depends on the diet as it can be S. cantharus in invaded and non-invaded sites, being 1.97–2.12 and higher when feeding on invertebrates or high-protein diets 1.92–2.1, respectively. The mean C:N values (±SE) obtained (McCutchan et al., 2003; Li and Dudgeon, 2008) which provide at invaded sites was 2.01±0.0002 while at non-invaded sites it was much better information in the IsoSource analyses. 1.96±0.009. Analysis of the contributions of food sources using the IsoSource 3. Results routine showed differences in carbon and nitrogen sources in S. cantharus related to their origin from invaded/control sites. C. racemosa 3.1. Presence of C. racemosa in the stomach contents 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 In the preliminary survey to locate sampling areas, C. racemosa was main sources of carbon and nitrogen for S. cantharus at invaded and 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 Table 1 δ 13 δ 15 C. racemosa in the stomach contents. Due to the low number of captures Nested multifactorial analysis of variance of isotopic composition ( C , N and C:N ratio). in SanTelmo two invaded sites were located in Palma Bay. The two non- invaded sites were located on the north coast of Mallorca. df δC13 δN15 C:N Sampling in Palma Bay and Mallorca North coast involved the Invaded 1 14.55*** 11.91*** 0.045*** capture of 160 individuals and the analyses their stomach content. C. Site (invaded) 2 0.86** 2.32 0.012** racemosa was present in the stomach of a total of 76.3% of the Error 48 0.12 0.89 0.001 individuals captured at invaded sites. The size (TL) of S. cantharus in 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 individuals living in invaded sites (ANOVA p<0.05) (Table 2). MDA lobsters and polychaetes but also macroalgae contribute to the isotopic levels did not differ significantly between treatments (0.49±. signature in both sites (Fig. 3). In both invaded and non invaded, 0.01 nmol/mg prot non-invaded and 0.53±0.01 nmol/mg prot considering only nitrogen contributions, the importance of invertebrate invaded) (Table 2). preys was more clear being shrimps the most important contributor in invaded sites (48.8±8.6%) and polychaetes and shrimps in non- 4. Discussion invaded sites (28.6±19.5% and 27.2±13.3% respectively). The current invasion of C. racemosa in the Mediterranean is 3.3. Biochemical analysis changing ecosystem functioning, habitat structure and invertebrate communities (Box, 2008). The extent of the change does not only The activity of hepatic antioxidant enzymes in S. cantharus feeding affect invertebrates but it can also affect fish such as S. cantharus. This among C. racemosa at invaded sites and those feeding at control sites omnivorous opportunistic feeder (Goncalves and Erzini, 1998) seems showed significant differences related to the ingestion of the toxic to be feeding among C. racemosa, as shown by the high frequency of invasive macroalga. CAT activity (expressed as mK/ mg prot) occurrence of the invasive macroalga in the stomach contents of this increased from 125.4±12.3 at non-invaded sites to 227.0±28.0 at species. Previous studies in the Mediterranean showed feeding on this the invaded sites; SOD activity (expressed as pKat/mg prot) also invasive macroalga by other organisms such as the fish Boops boops increased from 2.18±0.03 at the non-invaded sites to 2.29±0.05 at and Sarpa salpa, the sea-urchin Paracentrotus lividus (de Lamarck, the invaded sites. EROD (RFU/mg prot) and GST (nKat/mg prot) 1816) (Ruitton et al., 2006), the opisthobranchs Lobiger serradifalci showed the same pattern, with increases from 464.00±15.83 (Calcara, 1840), Oxynoe olivacea Rafinesque (1814) (Cavas et al., and 364.72±23.44 at the non-invaded sites to 544.61±14.77 and 2005) and the opisthobranch Ascobulla fragilis (Box, 2008). 532.26±34.23 at invaded sites respectively. All enzymatic activities Caulerpa species share a common thallus architecture composed of of CAT, SOD, GST and EROD were significantly higher in the liver of the 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 mechanical barrier to the invertebrate feeders. The presence of the depending on the species (Bold and Wynne, 1978). The architecture of invasive Caulerpa taxifolia (M.Vahl) C. Agardh, also in the Mediterra- Caulerpa, in particular the stolons and rhizoids, results in a high nean, has caused a decrease in the abundance of Mullus surmuletus capacity to retain ﬁne sand fractions according to Caulerpa highest (Linnaeus, 1758) due to the resulting barrier that prevents ﬁsh biomasses (Sanchez-Moyano et al., 2001). This means a complex accessing their food sources (Longepierre et al., 2005). In the Balearic structure over substrate of invasive macroalga which could form a 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 17

Table 2 detoxification and stress tolerance has been reported by several Nested multifactorial analysis of variance of antioxidant enzyme activities and MDA authors (Porte et al., 2002; Cavas and Yurdakoc, 2005). CAT is concentration in Spondyliosoma cantharus. involved in H2O2 detoxification and SOD uses the superoxide anion to df CAT (MS) SOD (MS) EROD (MS) GST (MS) MDA (MS) produce H2O2. GST participates in the detoxification of lipid hydro- Invaded 1 95,284*** 0.1989* 98,918*** 312767*** 0.01606a peroxides using GSH as a substrate. EROD is used as a potential Site (invaded) 2 9388 0.0073 1660 29076 0.00013 biomarker of the presence of xenobiotics in the aquatic medium Error 44 135,677 0.0473 5657 19597 0.00402 (Fatima and Ahmad, 2005). The antioxidant response observed in MS means square, ***p<0.001 **p<0.01 *p<0.05. a means p=0.052. 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 20 to 40 m (over coralligenous bottoms and rocky reefs) which could results imply that the ingestion of C. racemosa produces a detoxifying also act as a barrier against predators of invertebrates (authors response in S. cantharus. To evaluate whether toxicity is negatively personal observations). affecting this species we used MDA as a useful marker of lipid Isotopic values provide information related to shifts in food peroxidation. MDA concentration did not differ significantly between sources (Pinnegar and Polunin, 2000; Deudero et al., 2004)of treatments, but the higher MDA values obtained for invaded sites S. cantharus. The isotopic composition of the fish was similar between were only just outside the limit of significance. sites with equal treatments but differed between invaded/non- This study has presented a combination of methodologies such as invaded sites. The main food sources for S. cantharus were stomach contents analysis, stable isotope determinations and antiox- invertebrates such as squad lobsters (Galathea spp.), shrimps and idant response quantifications to allow us to understand the polychaetes. The contribution of C. racemosa as a carbon and nitrogen contribution of the ingestion of C. racemosa in the diet of S. cantharus. food source was low, despite the high frequency of occurrence in the This fish feeds on C. racemosa but does not assimilate important levels stomach contents. The contribution of δ13CbyC. racemosa was in the of this food source. Previous work in this field demonstrated the same range as other invertebrate food sources (shrimps and toxicological effects of Caulerpa on invertebrate and fish species polychaetes), but the δ15N contribution of C. racemosa was very low (Sureda et al., 2006, 2009; Box et al., 2008b). The same effect was compared with invertebrate food sources. For δ15N the contribution of observed in S. cantharus with increased antioxidant levels in invertebrate preys, especially shrimps and polychaetes, was similar individuals living in C. racemosa. The toxicity of the alga altogether between invaded and non-invaded sites, which reflect its importance with its low assimilation by the fish, in contrast with the high in the S. cantharus diet. assimilation of invertebrates, indicates that the ingestion of δ13C has been used to track animal movements between areas with C. racemosa is at least in part accidental while the fish are attempting different food sources (Kurle and Worthy, 2001) and to evaluate the to access the invertebrates that live under the fronds and stolon net importance of different food sources (Pinnegar and Polunin, 2000). In formed by C. racemosa. The invertebrate community in C. racemosa is this case, the contribution of C. racemosa as one of the main food very diverse and some groups such as polychaetes (Box et al., in sources for S. cantharus is clear. It has been documented that fish press), molluscs and decapods are highly abundant and diverse (Box, usually produce C:N ratios ranging between 3.3 and 5.1 (McCon- 2008; Box et al., 2008a). naughey and McRoy, 1979). In the present work, the C:N of In conclusion, S. cantharus feeds on C. racemosa in invaded areas S. cantharus ranged from 1.92 to 2.12, and no differences were but does not use this source as its main carbon and nitrogen source. found between individuals at invaded and non-invaded sites which One possible reason is the toxicity of the algae which has a presented similar C:N values, indicating that none were experiencing physiological effect on the fish. Thus the faecal pellets contain high feeding stress. The low C:N ratios may be due to the high proportion of amounts of C. racemosa, which in most cases maintain the integrity of protein in the diet, which was of high quality (Waddington and fronds and stolons, which are not completely digested. Further work MacArthur, 2008). Moreover, the C:N ratio indicates diet quality and must be done to check the selection of prey of this fish in invaded explains the degree of fractionation among the individual compounds ecosystems. 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 Acknowledgements representative of the assimilation of the food sources since they are related to metabolic and physiological processes (Sweeting et al., This work was supported by the research projects “Macroalgas 2007). Metabolic fractionation differs between carnivores and marinas invasoras en las Islas Baleares: Evaluación de riesgos y efectos herbivores; while in carnivores metabolic fractionation is dominant en comunidades bentónicas” (CTM2005-01434/MAR) of the Minis- because animal-derived nitrogen is biochemically more homoge- terio de Educación y Ciencia and the Project “Avaluació I seguiment neous and dominated by proteins, in herbivores both assimilative and dels recursos marins de la CAIB, 2008” of the Direcció General de Pesca metabolic factors affect fractionation (Vander Zanden and Rasmussen, (Balearic Islands). We must agree the collaboration of the Mar-I-Pi II 2001). Herbivorous fish also have a very long alimentary tract to and Pedaç anglers during sampling in Ibiza and Majorca Islands. [SS] 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 References long alimentary tract like exclusive herbivores. Therefore, for this Aebi, H.E., 1984. Catalase. In: Bergmeyer, H.U. (Ed.), Methods in Enzymatic Analysis. species it may be necessary to ingest a large amount of Caulerpa to Methods in Enzymatic Analysis. Verlag Chemie, Basel, pp. 273–286. assimilate carbon and nitrogen. On the other hand, the assimilation Amade, P., Valls, R., Bouaiacha, R., Lemé, R., Artaud, J., 1994. Méthodes de dosage de la rates of invertebrates were very large in fish caught at the invaded caulerpényne produite par Caulerpa taxifolia. In: Boudouresque, C.F., Meinesz, A., Gravez, V. (Eds.), First International Workshop on Caulerpa taxifolia. GIS Posidonie sites, which could imply that animal-derived nitrogen was the best publ., Fr., pp. 163–167. assimilated. Argyrou, M., Demetropoulos, A., Hadjichristophorou, M., 1999. Expansion of the Cellular antioxidant status is widely used as a tool to evaluate the macroalga Caulerpa"racemosa and changes in softbottom macrofaunal assemblages – ability of organisms to resist environmental stress (Frenzilli et al., in Moni Bay, Cyprus. Oceanol. Acta 22 (5), 517 528. Bauchot, M.L., Hureau, J.C., 1990. Sparidae. In: Quero, J.C., Hureau, J.C., Karrer, C., Post, A., 2004) and the effects of invasive species on faunistic communities Saldanha, L. (Eds.), Check-list of the Fishes of the Eastern Tropical Atlantic. UNESCO, (Sureda et al., 2006, 2009; Box et al., 2008b). The efficiency of ROS Paris, p. 907. 18 A. Box et al. / Journal of Experimental Marine Biology and Ecology 380 (2009) 11–19

Behringer, D., Butler, M., 2006. Stable isotope analysis of production and trophic Li, A.O.Y., Dudgeon, D., 2008. Food resources of shredders and other benthic relationships in a tropical marine hard-bottom community. Oecologia 148 (2), macroinvertebrates in relation to shading conditions in tropical Hong Kong 334–341. streams. Freshw. Biol. 53, 2011–2025. Bell, J.D., Harmelin-Vivien, M.L., 1983. Fish fauna of French Mediterranean Posidonia Longepierre, S., Robert, A., Levi, F., Francour, P., 2005. How an invasive alga species (Caulerpa oceanica seagrass meadows. Feeding habits. Tethys 11, 1–14. taxifolia) induces changes in foraging strategies of the benthivorous fish Mullus"surmu- Bold, H.C., Wynne, M.J., 1978. Introduction to the algae. Structure and reproduction. letus incoastalMediterraneanecosystems.Biodivers.Conserv.14(2),365–376. Prentice-Hall, New Jersey. Lorrain, A., Paulet, Y.M., Chauvaud, L., Savoye, N., Donval, A., Saout, C., 2002. Differential Box, A., 2008. Ecología de Caulerpales: fauna y biomarcadores. Doctoral Thesis. Instituto δ13C and δ15N signatures among scallop tissues: implications for ecology and Mediterráneo de Estudios Avanzados, Palma. physiology. J. Exp. Mar. Biol. Ecol. 275 (1), 47–61. Box, A., Deudero, S., Martín, D., Sarriera, P., 2008a. Canvis en les comunitats de poliquets Marín Leal, J., Dubois, S., Orvain, F., Galois, R., Blin, J.L., Ropert, M., Bataillé, M.P., Ourry, A., del herbeis de Posidonia oceanica colonitzats per Caulerpa"racemosa var. cylindracea. Lefebvre, S., 2008. Stable isotopes (δ13C, δ15N) and modelling as tools to estimate the In: Pons, G.X. (Ed.), V Jornades de Medi Ambient de les Illes Balears. Societat d’Història trophic ecology of cultivated oysters in two contrasting environments. Mar. Biol. 153 Natural de les Illes Balears: Soc. Hist. Nat. Balears, pp. 39–40. Palma de Mallorca. (4), 673–688. Box, A., Sureda, A., Deudero, S., 2008b. Antioxidant response of the bivalve Pinna nobilis McConnaughey, T., McRoy, C.P., 1979. Food-web structure and the fractionation of colonised by invasive red macroalgae Lophocladia lallemandii. Comp. Biochem. carbon isotopes in the Bering Sea. Mar. Biol. 53 (3), 257–262. Physiol. C 149 (4), 456–460. McCord, J.M., Fridovich, I., 1969. Superoxide dismutase. An enzymic function for Box, A., Martín, D., Deudero, S., in press. Changes in seagrass polychaete assemblages erythrocuprein (hemocuprein). J. Biol. Chem. 244, 6049–6055. after invasion by Caulerpa racemosa var. cylindracea (Caulerpales, Chlorophyta): McCutchan, J.H., Lewis, W.M., Kendall, C., McGrath, C.C., 2003. Variation in trophic shift community structure, trophic guilds and taxonomic distinctness. Sci. Mar. for stable isotope ratios of carbon, nitrogen, and sulfur. Oikos 102, 378–390. Cabana, G., Rasmussen, J.B., 1996. Comparison of aquatic food chains using nitrogen Meyers, P.A., 1994. Preservation of elemental and isotopic source identification of isotopes. Proc. Natl. Acad. Sci. U. S. A. 93 (20), 10844–10847. sedimentary organic-matter. Chem. Geol. 114 (3–4), 289–302. Carabel, S., Godínez-Domínguez, E., Verísimo, P., Fernández, L., Freire, J., 2006. An Mill, A.C., Pinnegar, J.K., Polunin, N.V.C., 2007. Explaining isotope trophic-step fraction- assessment of sample processing methods for stable isotope analyses of marine ation: why herbivorous fish are different. Funct. Ecol. 21, 1137–1145. food webs. J. Exp. Mar. Biol. Ecol. 336 (2), 254–261. Nizamuddin, M., 1991. The Green Marine Algae of Lybia. Elga, Bern. Cavas, L., Yurdakoc, K., 2005. A comparative study: assessment of the antioxidant Phillips, D.L., Gregg, J.W., 2001. Uncertainty in source partitioning using stable isotopes. system in the invasive green alga Caulerpa racemosa and some macrophytes from Oecologia 127 (2), 171–179. the Mediterranean. J. Exp. Mar. Biol. Ecol. 321 (1), 35–41. Phillips, D.L., Gregg, J.W., 2003. Source partitioning using stable isotopes: coping with Cavas, L., Yurdakoc, K., Yokes, B., 2005. Antioxidant status of Lobiger serradifalci and Oxynoe too many sources. Oecologia 136 (2), 261–269. olivacea (Opisthobranchia, Mollusca). J. Exp. Mar. Biol. Ecol. 314 (2), 227–235. Piazzi, L., Cinelli, F., 1999. Development and seasonal dynamics of a population of the Cavas, L., Baskin, Y., Yurdakoc, K., Olgun, N., 2006. Antiproliferative and newly tropical alga Caulerpa racemosa (Forsskal) J. Agardh in the Mediterranean. attributed apoptotic activities from an invasive marine alga: Caulerpa racemosa var. Cryptogamie Algol. 20 (4), 295–300. cylindracea. J. Exp. Mar. Biol. Ecol. 339 (1), 111–119. Piazzi, L., Balata, D., Cinelli, F., 2007. Invasions of alien macroalgae in Mediterranean Cossu, C., Doyotte, A., Jacquin, M.C., Babut, M., Exinger, A., Vasseur, P., 1997. Glutathione coralligenous assemblages. Cryptogamie Algol. 28 (3), 289–301. reductase, selenium-dependent glutathione peroxidase, glutathione levels, and Pinnegar, J.K., Polunin, N.V.C., 1999. Differential fractionation of delta C-13 and delta N- lipid peroxidation in freshwater bivalves, Unio tumidus, as biomarkers of aquatic 15 among fish tissues: implications for the study of trophic interactions. Funct. Ecol. contamination in field studies. Ecotoxicol. Environ. Saf. 38, 122–131. 13 (2), 225–231. Decottignies, P., Beninger, P.G., Rince, Y., Riera, P., 2007. Trophic interactions between Pinnegar, J.K., Polunin, N.V.C., 2000. Contributions of stable-isotope data to elucidating two introduced suspension-feeders, Crepidula fornicata and Crassostrea gigas, are food webs of Mediterranean rocky littoral fishes. Oecologia 122 (3), 399–409. influenced by seasonal effects and qualitative selection capacity. J. Exp. Mar. Biol. Pita, C., Gamito, S., Erzini, K., 2002. Feeding habits of the gilthead seabream (Sparus Ecol. 342 (2), 231–241. aurata) from the Ria Formosa (southern Portugal) as compared to the black Deniro, M.J., Epstein, S., 1978. Influence of diet on distribution of carbon isotopes in seabream (Spondyliosoma"cantharus) and the annular seabream (Diplodus annu- animals. Geochim. Cosmochim. Acta 42 (5), 495–506. laris). J. Appl. Ichthyol. 18 (2), 81–86. Deudero, S., Pinnegar, J.K., Polunin, N.V.C., Morey, G., Morales-Nin, B., 2004. Spatial Pitt, K.A., Clement, A.L., Connolly, R.M., Thibault-Botha, D., 2008. Predation by jellyfish variation and ontogenic shifts in the isotopic composition of Mediterranean littoral on large and emergent zooplankton: implications for benthic–pelagic coupling. fishes. Mar. Biol. 145 (5), 971–981. Estuar. Coast. Shelf Sci. 76 (4), 827–833. Dolenec, T., Lojen, S., Kniewald, G., Dolenee, M., Rogan, N., 2007. Nitrogen stable isotope Porte, C., Escartin, E., de la Parra, L.M.G., Biosca, X., Albaiges, J., 2002. Assessment of composition as a tracer of fish farming in invertebrates Aplysina aerophoba, Balanus coastal pollution by combined determination of chemical and biochemical markers perforatus and Anemonia sulcata in central Adriatic. Aquaculture 262 (2–4), 237–249. in Mullus barbatus. Mar. Ecol. Prog. Ser. 235, 205–216. Dubois, S., Jean-Louis, B., Bedrtrand, B., Lefebvre, S., 2007. Isotope trophic step fraction of Quéro, J.C., 1984. Les Poissons de Mer Des Pêches Françaises. . Paris. suspension-feeding species: implications for food partitioning in coastal ecosystems. J. Rahman, I., Skwarska, E., Henry, M., Davis, M., O'Connor, C.M., FitzGerald, M.X., Greening, Exp. Mar. Biol. Ecol. 351, 121–128. A., MacNee, W., 1999. Systemic and pulmonary oxidative stress in idiopathic Dulčić, J., Lipej, L., Glamuzina, B., Bartulovic, V., 2006. Diet of Spondyliosoma"cantharus and pulmonary fibrosis. Free. Radic. Biol. Med. 27, 60–68. Diplodus puntazzo (Sparidae) in the eastern central Adriatic. Cybium 30, 115–122. Ribera, M.A., 2002. Review of non-native marine plants in the Mediterranean Sea. In: Dunton, K.H., 2001. Delta N-15 and delta C-13 measurements of Antarctic peninsula fauna: Leppäkoski, E., Gollasch, S., Olenin, S. (Eds.), Invasive Aquatic Species of Europe. trophic relationships and assimilation of benthic seaweeds. Am. Zool. 41 (1), 99–112. Distribution, Impacts and Management. Kluwer Academic Publishers, pp. 291–310. Fatima, R.A., Ahmad, M., 2005. Certain antioxidant enzymes of Allium cepa as biomarkers for Ruitton, S., Verlaque, M., Aubin, G., Boudouresque, C.F., 2006. Grazing on Caulerpa the detection of toxic heavy metals in wastewater. Sci. Total Environ. 346 (1–3), 256–273. racemosa var. cylindracea (Caulerpales, Chlorophyta) in the Mediterranean Sea by Fisher, S.J., Brown, M.L., Willis, D.W., 2001. Temporal food web variability in an upper herbivorous fishes and sea urchins. Vie Milieu 56 (1), 33–41. Missouri River backwater: energy origination points and transfer mechanisms. Sanchez-Moyano, J.E., Estacio, F.J., Garcia-Adiego, E.M., Garcia-Gomez, J.C., 2001. Effect Ecol. Freshw. Fish 10 (3), 154–167. of the vegetative cycle of Caulerpa prolifera on the spatio-temporal variation of Frenzilli, G., Bocchetti, R., Pagliarecci, M., Nigro, M., Annarumma, F., Scarcelli, V., invertebrate macrofauna. Aquat. Bot. 70 (2), 163–174. Fattorini, D., Regoli, F., 2004. Time-course evaluation of ROS-mediated toxicity in Sanchez-Jerez, P., Barbera-Cebrian, C., Ramos-Espla, A., 1999. Daily vertical migrations mussels, Mytilus galloprovincialis, during a field translocation experiment. Mar. in the epifauna associated with Posidonia oceanica meadows. J. Mar. Biol. Assoc. Environ. Res. 58 (2–5), 609–613. U. K. 79, 971–977. Fry, B., Sherr, E.B., 1984. δ13C measurements as indicators of carbon flow in marine and Sarà, G., Scilipoti, D., Mazzola, A., Modica, A., 2004. Effects of fish farming waste to fresh-water ecosystems. Contrib. Mar. Sci. 27, 13–47 (SEP). sedimentary and particulate organic matter in a southern Mediterranean area (Gulf of Gamundi-Boyeras, I., Terrados, J., Pérez, M., 2006. Relació entre la presència de l'alga Castellammare, Sicily): a multiple stable isotope study (d13Candd15N). Aquaculture Caulerpa racemosa var. cylindracea (Sonder) Verlaque, Huisman et Boudouresque i la 234 (1–4), 199–213. tipologia del substrat a la Badia de Palma (Mallorca). Boll. Soc. Hist. Nat. Balears 49, Sarkar, A., Ray, D., Shrivastava, A.N., Sarker, S., 2006. Molecular biomarkers: their significance 109–115. and application in marine pollution monitoring. Ecotoxicology 15 (4), 333–340. Goncalves, J.M.S., Erzini, K., 1998. Feeding habits of the two-banded sea bream Sipes, I.G., Gandolfi, A.J., 1986. Biotransformation of toxicants, In: Klaasen, C.D., (Diplodus vulgaris) and the black sea bream (Spondyliosoma"cantharus) (Sparidae) Amdurand, M.O., Doull, J. (Eds.), Casarett and Doull's Toxicology The Basic Science from the south-west coast of Portugal. Cybium 22 (3), 245–254. of Poisons, 3rd ed. McMillan, New York, pp. 64–98. Habig, W.H., Pabst, M.J., Jokoby, W.B., 1974. Glutathione S-transferase. The first Stegeman, J.J., Hahn, M.E., 1994. In: Malins, D.C., Ostrander, G.K. (Eds.), Biochemistry and enzymatic step in mercapturic acid formation. J. Biol. Chem. 249, 7130–7139. molecular biology of monooxygenases: current perspectives on forms, functions, and Ince, R., Hyndes, G.A., Lavery, P.S., Vanderklift, M.A., 2007. Marine macrophytes directly regulation of cytochrome P450 in aquatic species. Aquatic Publishers, Boca Raton, pp. enhance abundances of sandy beach fauna through provision of food and habitat. 87–203. Estuar. Coast. Shelf Sci. 74 (1–2), 77–86. Sureda, A., Box, A., Ensenat, M., Alou, E., Tauler, P., Deudero, S., Pons, A., 2006. Enzymatic Jung, V., Thibaut, T., Meinesz, A., Pohnert, G., 2002. Comparison of the wound-activated antioxidant response of a labrid fish (Coris julis) liver to environmental caulerpenyne. transformation of caulerpenyne by invasive and noninvasive Caulerpa species of the Comp. Biochem. Physiol. C 144 (2), 191–196. Mediterranean. J. Chem. Ecol. 28 (10), 2091–2105. Sureda, A., Box, A., Deudero, S., Pons, A., 2009. Reciprocal effects of caulerpenyne and Klein, J., Verlaque, M., 2008. The Caulerpa racemosa invasion: a critical review. Mar. intense herbivorism on the antioxidant response of Bittium reticulatum and Caulerpa Pollut. Bull. 56 (2), 205–225. taxifolia. Ecotoxicol. Environ. Saf. 72, 795–801. Kurle, C.M., Worthy, G.A.J., 2001. Stable isotope assessment of temporal and geographic Sweeting, C.J., Barry, J., Barnes, C., Polunin, N.V.C., Jennings, S., 2007. Effects of body size differences in feeding ecology of northern fur seals (Callorhinus"ursinus) and their and environment on diet-tissue delta N-15 fractionation in fishes. J. Exp. Mar. Biol. prey. Oecologia 126 (2), 254–265. Ecol. 340 (1), 1–10. A. Box et al. / Journal of Experimental Marine Biology and Ecology 380 (2009) 11–19 19

Vander Zanden, M.J., Rasmussen, J.B., 2001. Variation in delta N-15 and delta C-13 Waddington, K., MacArthur, L., 2008. Diet quality and muscle tissue location inﬂuence trophic fractionation: implications for aquatic food web studies. Limnol. Oceanogr. consumer-diet discrimination in captive-reared rock lobsters (Panulirus cygnus). 46 (8), 2061–2066. Mar. Biol. 154 (3), 569–576. Verlaque, M., Durand, C., Huisman, J.M., Boudouresque, C.F., Le Parco, Y., 2003. On the Zuljevic, A., Antolic, B., Onofri, V., 2003. First record of Caulerpa racemosa (Caulerpales: identity and origin of the Mediterranean invasive Caulerpa racemosa (Caulerpales, Chlorophyta) in the Adriatic Sea. J. Mar. Biol. Assoc. UK 83 (4), 711–712. Chlorophyta). Eur. J. Phycol. 38, 325–339. Vizzini, S., Mazzola, A., 2006. The effects of anthropogenic organic matter inputs on stable carbon and nitrogen isotopes in organisms from different trophic levels in a southern Mediterranean coastal area. Sci. Total Environ. 368 (2–3), 723–731.