trophic relationships and uv-absorbing compounds in a mediterranean medio-littoral rocky shore...

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Trophic relationships and UV-absorbing compounds in a Mediterranean medio-littoral rocky shore community Sarah Nahon a, , Christian Nozais b, c , Jérôme Delamare-Deboutteville a , Karine Escoubeyrou d , Martin Desmalades e , Audrey M. Pruski a , Ulf Karsten f , François Charles e, a UPMC Univ Paris 06, FRE 3350, LECOB, Observatoire Océanologique, F-66651, Banyuls/mer, France b Département de biologie et centre d'études nordiques, Université du Québec à Rimouski, 300 Allée des Ursulines, Rimouski, QC, Canada G5L 3A1 c Department of Zoology, Miami University, 212 Pearson Hall, Oxford, Ohio 45056, USA d CNRS, UMS 2348, Observatoire Océanologique, F-66651 Banyuls/Mer, France e CNRS, FRE 3350, LECOB, Observatoire Océanologique, F-66651 Banyuls/mer, France f University of Rostock, Institute of Biological Sciences, Albert-Einstein-Strasse 3, D-18059 Rostock, Germany abstract article info Article history: Received 9 January 2012 Received in revised form 10 May 2012 Accepted 12 May 2012 Available online 5 June 2012 Keywords: Benthic invertebrates MAAs Mediterranean Sea Rocky shore Stable isotopes UV radiation Organisms inhabiting shallow coastal ecosystems are potentially exposed to damaging levels of solar UV radiation. UV-sunscreen compounds such as mycosporine like amino-acids (MAAs) are a widespread bio- chemical strategy among many marine organisms to counteract UV stress. These molecules are produced through a metabolic pathway restricted to cyanobacteria, various algal groups and fungi. This study was designed to search for UV sunscreen compounds in benthic invertebrates of the Catalan medio-littoral rocky shore of the north-western Mediterranean Sea. The relationship between food sources and consumers was investigated using the stable isotope approach in order to elucidate the potential pathways of MAA transfer. The food web associated with the littoral Catalan rocky shore is characterized by a high level of func- tional complexity. The variability of δ 13 C and δ 15 N among and between trophic guilds provides evidence of the diversity of coexisting trophic levels and trophic pathways in a single habitat type. MAAs were present in all sampled metazoan species. We observed a higher MAA diversity in animals than in food sources. This may be explained by the incorporation of MAAs from several trophic resources, by the conversion of the MAAs acquired from the food or by translocation from a symbiont. The amount of MAAs in sea urchins was fairly low compared to other trophic groups. This cannot be explained by depletion of MAAs in their diets, and hence other UV-protective strategies may be more important. Complementary experimental studies should be performed to determine the ability of primary and secondary consumers to assimilate MAAs and convert these compounds. © 2012 Elsevier B.V. All rights reserved. 1. Introduction Light is a key factor in controlling the structure and the distribution of benthic communities (Jokiel, 1980; Karsten et al., 1998; Nozais et al., 1999; Reizopoulou et al., 2000). Organisms living in shallow environ- ments are potentially exposed to damaging radiation and there is a gen- eral consensus that solar ultraviolet (UV) radiation and especially UVB radiation can negatively affect aquatic species (De Mora et al., 2000). In the Mediterranean Sea, water transparency enables deep UVB pene- tration meaning that littoral environments are particularly exposed to damaging levels of UV radiation (Kouwenberg and Lantoine, 2007). To counteract deleterious effects of UV radiation, marine organisms have developed several strategies such as avoidance, protection and repair (Cockell and Knowland, 1999). Cover protections, like external shells, spines and tests, are widespread among adult benthic invertebrates. However, gametes and early life stages, as well as some species such as cnidarians, have no external protection that could shield them from UV radiation. For these forms and more generally for organisms directly exposed to solar radiation, cellular sunscreen compounds represent another strategy to mitigate the direct effect of UV radiation. Mycosporine like amino-acids (MAAs) are the most common class of sunscreen compounds in marine organisms from cyanobacteria to sh. These compounds apparently arose early in evolution (Banaszak et al., 1998; Carreto et al., 1990; Chioccara et al., 1980, 1986; Dunlap and Shick, 1998; Karentz et al., 1991, 1992; Shiabata, 1969; Shick et al., 1992). More than 20 structurally distinct MAAs have already been identied. Due to their high molar absorptivity within the 309 to 360 nm wavelength range, they provide efcient protection from bio- logically damaging UV radiation (Carreto et al., 2005; Dunlap and Shick, 1998). Moreover, MAA concentrations within tissues have been shown to be correlated with UV exposition (Dunlap and Chalker, Journal of Experimental Marine Biology and Ecology 424425 (2012) 5965 Corresponding authors. Tel.: +33 4 68 88 73 35; fax: +33 4 68 88 73. E-mail addresses: [email protected] (S. Nahon), [email protected] (F. Charles). 0022-0981/$ see front matter © 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.jembe.2012.05.009 Contents lists available at SciVerse ScienceDirect Journal of Experimental Marine Biology and Ecology journal homepage: www.elsevier.com/locate/jembe

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Journal of Experimental Marine Biology and Ecology 424–425 (2012) 59–65

Contents lists available at SciVerse ScienceDirect

Journal of Experimental Marine Biology and Ecology

j ourna l homepage: www.e lsev ie r .com/ locate / jembe

Trophic relationships and UV-absorbing compounds in a Mediterraneanmedio-littoral rocky shore community

Sarah Nahon a,⁎, Christian Nozais b,c, Jérôme Delamare-Deboutteville a, Karine Escoubeyrou d,Martin Desmalades e, Audrey M. Pruski a, Ulf Karsten f, François Charles e,⁎a UPMC Univ Paris 06, FRE 3350, LECOB, Observatoire Océanologique, F-66651, Banyuls/mer, Franceb Département de biologie et centre d'études nordiques, Université du Québec à Rimouski, 300 Allée des Ursulines, Rimouski, QC, Canada G5L 3A1c Department of Zoology, Miami University, 212 Pearson Hall, Oxford, Ohio 45056, USAd CNRS, UMS 2348, Observatoire Océanologique, F-66651 Banyuls/Mer, Francee CNRS, FRE 3350, LECOB, Observatoire Océanologique, F-66651 Banyuls/mer, Francef University of Rostock, Institute of Biological Sciences, Albert-Einstein-Strasse 3, D-18059 Rostock, Germany

⁎ Corresponding authors. Tel.: +33 4 68 88 73 35; faE-mail addresses: [email protected] (S. Nahon

(F. Charles).

0022-0981/$ – see front matter © 2012 Elsevier B.V. Alldoi:10.1016/j.jembe.2012.05.009

a b s t r a c t

a r t i c l e i n f o

Article history:Received 9 January 2012Received in revised form 10 May 2012Accepted 12 May 2012Available online 5 June 2012

Keywords:Benthic invertebratesMAAsMediterranean SeaRocky shoreStable isotopesUV radiation

Organisms inhabiting shallow coastal ecosystems are potentially exposed to damaging levels of solar UVradiation. UV-sunscreen compounds such as mycosporine like amino-acids (MAAs) are a widespread bio-chemical strategy among many marine organisms to counteract UV stress. These molecules are producedthrough a metabolic pathway restricted to cyanobacteria, various algal groups and fungi. This study wasdesigned to search for UV sunscreen compounds in benthic invertebrates of the Catalan medio-littoralrocky shore of the north-western Mediterranean Sea. The relationship between food sources and consumerswas investigated using the stable isotope approach in order to elucidate the potential pathways of MAAtransfer. The food web associated with the littoral Catalan rocky shore is characterized by a high level of func-tional complexity. The variability of δ13C and δ15N among and between trophic guilds provides evidence ofthe diversity of coexisting trophic levels and trophic pathways in a single habitat type. MAAs were presentin all sampled metazoan species. We observed a higher MAA diversity in animals than in food sources. Thismay be explained by the incorporation of MAAs from several trophic resources, by the conversion of theMAAs acquired from the food or by translocation from a symbiont. The amount of MAAs in sea urchinswas fairly low compared to other trophic groups. This cannot be explained by depletion of MAAs in theirdiets, and hence other UV-protective strategies may be more important. Complementary experimentalstudies should be performed to determine the ability of primary and secondary consumers to assimilateMAAs and convert these compounds.

© 2012 Elsevier B.V. All rights reserved.

1. Introduction

Light is a key factor in controlling the structure and the distributionof benthic communities (Jokiel, 1980; Karsten et al., 1998; Nozais et al.,1999; Reizopoulou et al., 2000). Organisms living in shallow environ-ments are potentially exposed to damaging radiation and there is a gen-eral consensus that solar ultraviolet (UV) radiation and especially UVBradiation can negatively affect aquatic species (De Mora et al., 2000).In the Mediterranean Sea, water transparency enables deep UVB pene-tration meaning that littoral environments are particularly exposed todamaging levels of UV radiation (Kouwenberg and Lantoine, 2007). Tocounteract deleterious effects of UV radiation, marine organisms havedeveloped several strategies such as avoidance, protection and repair

x: +33 4 68 88 73.), [email protected]

rights reserved.

(Cockell and Knowland, 1999). Cover protections, like external shells,spines and tests, are widespread among adult benthic invertebrates.However, gametes and early life stages, as well as some species suchas cnidarians, have no external protection that could shield them fromUV radiation. For these forms andmore generally for organisms directlyexposed to solar radiation, cellular sunscreen compounds representanother strategy to mitigate the direct effect of UV radiation.

Mycosporine like amino-acids (MAAs) are the most common classof sunscreen compounds in marine organisms from cyanobacteria tofish. These compounds apparently arose early in evolution (Banaszaket al., 1998; Carreto et al., 1990; Chioccara et al., 1980, 1986; Dunlapand Shick, 1998; Karentz et al., 1991, 1992; Shiabata, 1969; Shick etal., 1992). More than 20 structurally distinct MAAs have already beenidentified. Due to their high molar absorptivity within the 309 to360 nm wavelength range, they provide efficient protection from bio-logically damaging UV radiation (Carreto et al., 2005; Dunlap andShick, 1998). Moreover, MAA concentrations within tissues have beenshown to be correlated with UV exposition (Dunlap and Chalker,

60 S. Nahon et al. / Journal of Experimental Marine Biology and Ecology 424–425 (2012) 59–65

1986; Gleason and Wellington, 1993). These compounds were for longtime considered to beproduced froma branchof the shikimic acid path-way, a biochemical route restricted to bacteria, algae and fungi (Favre-Bonvin et al., 1987; Towers and Subba Rao, 1972; Yoshida, 1969).However, recent molecular biological evidence in cyanobacteria clearlyindicates that MAA biosynthesis branches out of central metabolismrather than out of shikimate pathway (Balskus and Walsh, 2010).These authors proved with a set of elegant methods that sedoheptulose7-phosphate from central metabolism is converted to 4-deoxygadusolwhich is the direct precursor of mycosporine-glycine. Nevertheless, inmarine vertebrates and invertebrates, MAAs are still considered to bederived from the diet (Carefoot et al., 1998; Carroll and Shick, 1996;Gao and Garcia-Pichel, 2011), or via translocation from algal symbionts(Shick et al., 1999). Primary consumers may thus ensure their own pro-tection and the protection of their offspring by accumulating highamounts of MAAs in their tissues and their eggs.

In sea urchin embryos, MAAs are known to reduce the incidence ofUV-induced abnormalities (Adams and Shick, 2001). There is a nega-tive correlation between the concentration of UV-absorbing com-pounds in the eggs and the embryo susceptibility to UV radiation(Lesser et al., 2006). However, a recent study has revealed that theelevated sensitivity of embryos of the Mediterranean Sea urchinSphaerechinus granularis to UV radiation is probably correlated witha lack of significant amount of sunscreen compounds in the eggs(Nahon et al., 2009).

The aim of this study was to search for the occurrence of MAAs inthe tissues of Mediterranean benthic invertebrates and to define thetrophic relationships between the autotrophic producers of MAAsand their heterotrophic consumers. The dominant species of benthicinvertebrates and their potential sources of organic matter were sam-pled in a shallow rocky shore located in the Bay of Banyuls-sur-mer(NW Mediterranean Sea, France). MAAs were separated and quanti-fied by reverse phase HPLC. The food web structure was definedusing stable isotope ratios (δ13C and δ15N) as this method is a usefultool in delineating carbon flows and tropic relationships in a largevariety of continental and marine ecosystems (Fry and Sherr, 1984;Peterson and Fry, 1987).

2. Materials and methods

2.1. Sample collection and preparation

Sampling was performed in the Bay of Banyuls-sur-mer (North-Western Mediterranean Sea, France) on the rocky walls of the islet,l'Ile Grosse, down to 2 m depth in May and June 2007. Water sampleswere collected twice in triplicate with a bucket (10 L) and pre-filteredthrough a 200 μm mesh to remove large zooplankton and detritus.Suspended particulate organic matter (SPOM) was then recoveredby filtration on pre-combusted Whatman GF/F filters. The tendominant macrophytes (listed in the Table 2) were collected byhand. Epibionts were removed from the samples, and these werethen rinsed with distilled water, freeze-dried and ground to powderusing a mortar and a pestle. The biofilm resulting in the formationof a complex community known as biofouling of solid surface(Abarzua and Jakubowski, 1995) was sampled by scraping the surfaceof two PVC panels (10×15 cm) moored at depth of 1 m in the area15 days before. Scraped material was concentrated on pre-combusted Whatman GF/F filters. Nine benthic invertebrate speciesrepresentative of the area and belonging to a variety of feedingmodes (i.e., suspension feeders, predators, grazers, herbivores) werealso collected manually. The invertebrates were kept alive for 48 hin the laboratory to allow evacuation of the gut contents and, ifnecessary, were then dissected to separate flesh from the shells. Ani-mal tissues were rinsed with distilled water, freeze-dried and groundto a fine powder. The list of all species collected is given in Table 2.

2.2. MAA extraction and analysis

Extraction of mycosporine-like amino acids was carried out onthree replicates per sample as described in Nahon et al. (2009). Brief-ly, 10 to 20 mg dry weight (DW)were extracted three times in screw-capped centrifuge vials filled with 1 mL of methanol 25% (HPLCgrade) for 1.5 h at 45 °C (Tartarotti and Sommaruga, 2002). Sampleswere then centrifuged at 5000 g for 5 min and the supernatantswere pooled. UV-absorption spectra were recorded from 210 to410 nm on a Shimadzu UV-1605 scanning spectrophotometer in1 cm-quartz cuvettes. For HPLC analysis of MAAs, aliquots of thesupernatants (800 μL) were evaporated to dryness under vacuum(Speed Vac UVS400A, Savant). The dried extracts were re-dissolvedin 800 μL distilled water (Karsten et al., 2009), vortexed for 30 s andcentrifuged at 5000 g for 5 min. The extracts were partially cleanedby filtration onto 0.2 μm pore-sized membrane filters (filters UptidiscRC 13 mm, Interchrom) before analysis on a Dionex Ultimate 3000®HPLC system (injection volume: 10 μL). Elution was performedisocratically at 25 °C on a stainless-steel Phenomenex Synergi FusionRP-18 column (4 μm, 250×3.0 mm I.D.) and a security guard car-tridge (20×4 mm I.D.) with a flow rate of 0.5 mL min−1. The mobilephase consisted of 2.5% methanol and 0.1% acetic acid in water. MAAswere detected online with a photodiode array detector (DIONEXUVD340) at 330 nm and the absorption spectra of each peak wererecorded and stored online in the 260–400 nm wavelength range.Identification was done by comparison of spectra, retention timeand by co-chromatography with biological standards of palythine,porphyra-334 and shinorine, palythene and mycosporine-glycineprovided by U. Karsten. Quantification was made using the molar ex-tinction coefficients given in Karsten et al. (1998). Concentrationswere normalized to nitrogen to allow comparison between samples.

2.3. Stable isotope measurements

One subsample was analyzed for carbon isotopes after acidifica-tion with 1 N HCl to remove carbonates (DeNiro and Epstein, 1978)and a second subsample, without acidification, was analyzed fornitrogen isotope (Pinnegar and Polunin, 1999). Weighed massesfrom each treated sample batch were subsequently encapsulated.

Carbon and nitrogen isotopic analyses were carried out at theInstitut des Sciences de la Mer (ISMER, Rimouski, Québec, Canada)using a COSTECH ECS 4010 Elemental Analyser coupled with aDeltaPlus XP Isotope Ratio Mass Spectrometer (IRMS, Thermo ElectronCo). System control as well as acquisition and treatment of the datawas carried out using the Isodat 2 software. Isotopic values wereexpressed in parts per thousand as deviations from standards accordingto the equation:

δX ¼ Rsample

Rstandard−1

� �� 100

where X is 13C or 15N and R is the corresponding 13C/12C or 15N /14Nratios.

Standards used for themeasurement of 13C and 15Nwere anhydrouscaffeine (Sigma Chemical Co., St-Louis, USA), Mueller Hinton Broth(Becton Dickinson, USA) and Nannochloropsis. These homemade stan-dards were calibrated using standards from the National Institute ofStandards and Technology (NIST, USA). Replicate analyses of standardsgave analytical errors (SD) of ±0.30‰ for C and ±0.18‰ for N.

2.4. Statistical analysis

Average δ13C and δ15N values were compared by Kruskal–Wallisnon parametric one-way analysis of variance (ANOVA). To distinguishsignificant differences between groups, post-hoc multiple compari-sons were performed as proposed in Siegel and Castellan (1988).

Table1

MAAco

nten

tsin

prim

aryfood

sourcesan

dbe

nthicco

nsum

ers.λmax,m

axim

umab

sorban

cewav

elen

gthin

nm;n.d.,n

otde

term

ined

;mea

nMAAco

ncen

trationin

μgmgN

−1;tr,traces;

U,u

nkno

wn;

N,total

numbe

rof

MAAs;

andΣ,total

MAAco

ncen

trationin

μgmgN

−1.T

he*indicatesthemostab

unda

ntco

mpo

und.

Palythine

Asterina-33

0Mycospo

rine

-glycine

U-330

.5Sh

inorine

U-331

U-356

.6Po

rphy

ra-334

U-308

,4U-308

.3U-308

.7Pa

lythen

eN

ΣRe

tentiontime(m

in)

3.22

3.38

4.14

4.18

4.36

5.35

5.73

6.45

7.46

8.14

10.71

12.87

λmax

(nm)

319

331.3

308.7

330.5

332.2

331

356.6

332.9

308.4

308.3

308.7

359.2

Prim

aryfood

sources

SPOM

322

57.3

––

–1.2

––

123.8*

15.6

––

–4

197.9

Biofi

lm33

013

.4–

––

1.2

25.8

––

––

340

.4A.a

rmata

324

121.5*

––

–11

5.4

––

––

––

–2

236.9

R.verruc

ulosa

334

––

––

155.7*

––

––

––

–1

155.7

L.incrustans

334

––

––

26.2*

––

1.5

––

––

227

.7L.liche

noides

333

––

––

7.5*

––

2.5

––

––

210

.0C.

elon

gata

326

7.6

tr–

–18

.1*

––

1.5

––

––

427

.2P.

umbilicalis

334

13.4

21.1

1.9

3.4

2.1

3.4

2.6

221.6*

––

––

826

9.5

Benthicco

nsum

ers

P.aspe

ra33

01.8

0.9

––

21.6*

––

6.7

tr–

––

531

.1P.

rustica

332

1.2

0.9

––

3.7

––

24.9*

tr–

––

530

.7A.lixula

n.d.

trtr

––

2.1*

––

tr0.3

––

–5

2.4

P.liv

idus

n.d.

trtr

––

0.3*

––

tr0.1

––

–5

0.4

M.g

alloprov

incialis

324

4.4

0.9

––

19.1*

––

11.7

2.8

0.2

0.2

0.7

840

.1A.sulcata

331

0.3

0.1

–52

.4*

––

–tr

2.2

–0.8

–6

55.8

A.m

utab

ilis

310

1.5

0.2

––

3.2

––

tr4.8

3.4

2.7

–7

15.8

A.e

quina

331

tr–

––

20.8*

––

4.6

0.7

–0.6

–5

26.7

61S. Nahon et al. / Journal of Experimental Marine Biology and Ecology 424–425 (2012) 59–65

3. Results

3.1. MAA contents

MAA contents of potential food sources and benthic invertebratesare presented in Table 1. UV-absorbing compounds have not beendetected in the tissues of the brown macrophytes Cytoseira compressa,Padina pavonica, Colpomenia sinuosa and of the green macrophyteUlva sp., even in trace amounts. In the other potential food sources(redmacroalgae, SPOMand biofilm)marked absorption peakswere ob-served related to the presence of MAAs as revealed by HPLC. A total ofnineUV-absorbingmoleculeswere found in the redmacroalgae, biofilmand SPOM, eight of them being found in Porphyra umbilicalis. Shinorinewas present in all primary food sources, especially in Coralina elongata,Litophyllum lichenoides, Litophyllum incrustans and Rissoella verruculosa(with a contribution ranging from 70 to 100% of the total MAA pool).Porphyra umbilicalis and SPOM were rich in porphyra-334 (83 and63%, respectively). The two MAAs occurring in Asparagopsis armata,namely palythine and shinorine, were present in nearly equal amounts(121.5 and 115.4 μg mgN−1, respectively). Other UV-absorbing com-poundswere found at low levels. The total amount ofMAAswas consid-erably higher in the non calcareous red algae (from 155.7 to269.5 μg mgN−1) than in the three calcareous algae that were tested(from 10.0 to 27.7 μg mgN−1).

All invertebrate species, except sea urchins, displayed significantlevels of MAAs (Table 1). Indeed, the absorption spectra of Arbacialixula and Paracentrotus lividus did not allow the discrimination ofany distinct peak in the UV wavelength band. UV absorption spectraof the other species exhibited absorption peaks ranging from 310 to324 nm corresponding to the combination of 5 to 8 different mole-cules (Table 1). Unknown-308.4, palythine, and porphyra-334 werepresent in all species. Asterina-330 and shinorine were present inall species except Anemonia sulcata. Shinorine was the most abundantMAA in Mytilus galloprovincialis (48%), Patella aspera (70%) andActinia equina (78%). Patella rustica was dominated by porphyra-334, which accounted for 81% of the MAAs. In A. sulcata, one com-pound with maximal absorption at 330.5 nm outclassed the pool ofUV-absorbing compounds with an estimated contribution of 94%.Among the seven molecules detected in Aiptasia mutabilis, therewas no clear pattern of dominance. Total amounts of MAAs were onaverage lower than in the food sources. Among the consumers,A. sulcata contained the highest amount of UV-absorbing compounds(55.8 μg mgN−1) followed by M. galloprovincialis, P. aspera, P. rustica,A. equina and A. mutabilis. Arbacia lixula and P. lividus contained lessthan 2.4 μg mgN−1.

3.2. δ13C and δ15N of organic matter sources and consumers

δ13C and δ15N values of the organic matter sources and the benthicinvertebrates are presented in Table 2. Significant differences wereobserved among potential food sources for δ13C (Kruskal Wallis test,df=11, pb0.001) with values ranging between −32.3 and −7.7‰.Macroalgae exhibited the largest range of values. Three differentgroups were distinguishable: A. armata was the most 13C-depleted,P. pavonica and C. sinuosa were the most 13C-enriched, and theother species had intermediary δ13C ranging between −21.8‰ and−14.5‰. SPOM was more 13C-depleted (−22.2‰) than the biofilm(−18.7‰). Regarding δ15N values, sources were well distinguishable(Kruskal Wallis test, df=11, pb0.001) ranging from 1.9‰ (biofilm)and 8.5‰ (C. compressa). Hence, the distinct δ13C and δ15N values ofthe different sources of organic matter allowed their use to infer tro-phic transfer to the associated benthic fauna.

Benthic invertebrates exhibited significant differences for δ13Cand δ15N (Kruskal Wallis tests, df=7, pb0.01 for δ13C and δ15N) asshown in Fig. 1. The range of δ13C values was narrower (from−19.4 to −11.3‰) than the one observed for the potential food

Table 2List of the selected species sampled from l'Ile Grosse. Stable carbon and nitrogen isotope ratios (means±SD), C/N ratio as well as the class or feeding mode to which the alga or theanimal belongs. n, number of replicates.

Code δ13C (‰) δ15N (‰) C/N n Class or feedingmodemean±SD mean±SD

Primary food sourcesSPOM S −22.2±2.0 4.2±1.0 8.8±1.7 3Biofilm B −18.7±1.3 1.9±0.2 17.0±3.4 3Macrophytes

Asparagopsis armata Aa −32.3±0.0 5.9±0.9 10.6±0.1 3 RhodophyceaeRissoella verruculosa Rv −19.2±1.8 6.7±0.1 11.9±0.0 3 RhodophyceaeLithophyllum incrustans Li −14.5±0.1 4.7±0.1 15.8±0.3 3 RhodophyceaeLithophyllum lichenoïdes Ll −15.7±0.3 4.7±0.1 13.2±0.3 3 RhodophyceaeCoralina elongata Ce −21.8±0.1 4.2±0.6 11.0±0.1 3 RhodophyceaePorphyra umbilicalis Pu −19.3±0.0 6.1±0.1 18.1±0.0 3 RhodophyceaeColpomenia sinuosa Cs −7.7±0.0 7.3±0.6 14.9±0.3 3 PhaeophyceaeCystoseira compressa Cc −18.3±2.2 8.5±0.3 32.4±0.2 3 PhaeophyceaePadina pavonica Pp −7.7±0.3 6.7±0.4 24.1±0.3 3 PhaeophyceaeUlva sp. Usp −20.9±0.0 5.7±0.2 20.6±0.2 3 Chlorophyceae

Benthic consumersPatella aspera Pa −11.3±2.5 5.9±0.2 4.1±0.1 3 Grazera

Patella rustica Pr −12.1±0.4 3.5±0.4 4.0±0.1 3 Grazera

Arbacia lixula Al −18.8±1.4 5.1±0.7 6.6±0.1 4 Grazerb

Paracentrotus lividus Pl −19.4±3.7 4.1±0.1 5.7±0.2 4 Grazerb

Mytilus galloprovincialis Mg −18.9±0.2 5.3±0.2 6.2±0.4 3 Suspension feederc

Anemonia sulcata As −17.3±0.8 5.7±0.5 5.6±0.4 3 Carnivored

Aiptasia mutabilis Am −17.2±0.1 4.9±0.1 6.8±0.1 3 Carnivored

Actinia equina Ae −18.2±0.2 7.3±0.3 4.3±0.1 3 Carnivored

a Della Santina et al., 1993.b Privitera et al., 2008.c Duggins et al., 1989.d Vanpraet, 1985.

62 S. Nahon et al. / Journal of Experimental Marine Biology and Ecology 424–425 (2012) 59–65

sources. In contrast to other consumers, both patellids were moreenriched in 13C. δ15N values were comprised between 3.5‰ for thelimpet P. rustica and 7.3‰ for the anemone A. equina, which departsfrom the two other anemones by a higher δ15N. Anemonia sulcataand A.mutabilis exhibited δ15N values close to the ones of the primaryconsumers M. galloprovincialis, P. lividus and A. lixula.

4. Discussion

The macroalgae and invertebrate species collected during the pre-sent study were selected for their dominance among the medio-littoral benthic communities of the Catalan rocky shores.

0

2

4

6

8

10

-35 -30 -25 -20 -15 -10 -5 0

δ13C

δ15N

Carnivore

Suspension feeder

Grazer

Primary food source

Pl

Al

Mg

Ae

As

Am

Pr

PaAa

Pp

Cs

Cc

SCe

Biofilm

LiLl

Rv

Pu

Usp

Fig. 1. δ13C vs δ15N (means) for the 20 primary food sources and consumers selected.Species' name coding is given in Table 2. Stable isotope signatures of primary organicsources not consumed by grazers (i.e., brown algae and A. armata) are isolated by thedotted lines.

4.1. MAA contents in organic matter sources and consumers

Many different methods have been described to extract and sepa-rate MAAs (see Carreto and Carignan (2011) for recent review). Inthis study, a proven protocol described by Karsten et al. (2009) formacroalgae has been used. However, for other types of tissues, thisprotocol might have been less efficient and too aggressive leading tohydrolytic cleavage of the most labile MAAs. Carreto et al. (2005) ad-vocates soaking dried samples with water in the dark at 4 °C andextracting with 100% methanol rather than 25% used in the presentstudy. However, these authors also specified that the extraction effi-ciency of the method should be determined for each type of tissuematrix to be analyzed. Considering animal tissues examined in thepresent study, one can be confident of the extraction efficiency(Carreto et al., 2005). Even if some transformation of labile MAAs can-not be totally excluded, we decided to apply the method that is mostwidely used for purposes of comparison.

The green and brownmacroalgae (Ulva sp., C. compressa, P. pavonicaand C. elongata) did not exhibit any detectable amount of MAAs. Con-versely, our results confirmed the presence of significant quantities ofMAAs in red macroalgae as previously reported for Mediterranean spe-cies (Banaszak et al., 1998; Karentz et al., 1991; Karsten et al., 1998) andprovided for this region the first qualitative and quantitative datarecorded for SPOM and the biofilm. All benthic invertebrates exhibitedsignificant concentrations of MAAs with the exception of the seaurchins.

Shinorine was found in all red macroalgae, SPOM and the biofilm.Shinorine is the most common MAA produced by diverse macro- andmicroalgae, and cyanobacteria (Banaszak et al., 1998, 2000; Carreto etal., 1990; Gao and Garcia-Pichel, 2011; Helbling et al., 1996; Karentz,1994; Karsten et al., 1998; Sinha et al., 1998). Franklin et al. (1999)showed that shinorine was the first MAA synthesized in the redmacroalgae Chondrus crispus. Moreover, shinorine provides a widespectral screen against UVR (Adams and Shick, 2001). The data onshinorine biosynthesis from seduheptulose 7-phosphate rather than

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from shikimate pathway has been convincingly documented at leastfor cyanobacteria (Balskus and Walsh, 2010), and hence it is reason-able to assume similar anabolic pathways also in eukaryotic algae.However, without genomic data this hypothesis cannot be properlyaddressed at the moment. Shinorine offers large spectral protectionalong with high photostability (Adams and Shick, 1996), and thesephysico-chemical properties may explain the ubiquitous accumula-tion of this MAA among marine organisms (Shick et al., 1992). Ac-cordingly, we found shinorine in all invertebrate species exceptA. sulcata. Regarding the total content of MAAs, a net decrease inthe total concentration of MAAs was observed between sources andconsumers. This indicates that unlike many other marine animals(Moeller et al., 2005; Orfeo et al., 2011; Whitehead et al., 2001), in-vertebrates from the considered Mediterranean medio-littoral shoreincorporate MAAs without biomagnification. In M. galloprovincialis,MAA diversity was greater than in SPOM. This may result from theconversion of MAAs acquired from the diet into other MAAs in animaltissues (Shick et al., 1999). Both species of limpets were characterizedby similar amounts and composition of MAAs. However, shinorinewas dominant in P. aspera whereas in P. rustica, porphyra-334 wasthe most abundant MAA. This could result from different trophicpathways. Both species of sea urchin exhibited extremely low con-tents in MAAs. Nahon (2009) already reported fairly low levels ofMAA in oocytes and ovaries of A. lixula and P. lividus. This was an un-expected result because other authors demonstrated that females ofanother urchin species Strongylocentrotus droebachensis, accumulateprincipally shinorine in their ovaries even when other MAAs wereavailable in high concentrations in their food (Adams and Shick,2001). This suggests a selective uptake of shinorine by specific MAAtransporters in the gut (Adams and Shick, 2001; Carreto andCarignan, 2011; Carroll and Shick, 1996; Shick and Dunlap, 2002). In-terestingly, it is worth mentioning that the two urchin species exam-ined in the present study contained mostly one MAA, shinorine,however, in A. lixula and P. lividus, intracellular defenses againstadverse UV effects are probably mediated by anti-oxidant moleculessuch as carotenoids and quinonoids (Koltsova et al., 1981; Lamareand Hoffman, 2004).

In A. equina, the major MAA was shinorine and secondarilyporphyra-334. Anemonia sulcata and A. mutabilis are also predatorsbut they contain symbiotic dinoflagellates in their tissues. Tissues ofA. sulcata exhibited the highest MAA concentration with one dominant,but still unknown compound characterized by a maximum absorptionat 330.5 nm. Mycosporine-glycines are the most frequently observedand the most concentrated MAAs among cnidarian species (Banaszaket al., 2006). As a consequence the unidentified compound consideredabove could be mycosporine-2-glycine, a MAA with a maximal molarabsorptivity around 330 nm. In comparison, A. mutabilis containedlower amounts of UV-absorbing compounds but exhibited a higherdiversity of MAAs. The differences may reflect a genotypic differenceamong symbiont and hosts (Banaszak et al., 2006). In symbiotic associ-ations, it has been inferred that the symbionts are responsible for thesynthesis of MAAs. MAA diversity in the host may result from simpletranslocation from the symbionts to the host or from secondary trans-formation by the host and conceivably by their associated bacteriaafter translocation (Dunlap and Shick, 1998; Shick, 2004). In cnidarians,however, it seems that MAAs are not host-modified (Banaszak et al.,2006; Shick et al., 2002). Anemones having symbionts can also accumu-late MAAs from their food, which may also explain the occurrence ofminor MAAs (Shick et al., 2002).

4.2. Food web mapping from stable isotope ratios

Carbon isotopic ratios were in the range of those available fororganic matter sources in the Mediterranean Sea literature (Carlieret al., 2007; Dauby, 1989; Dauby et al., 1998; Lepoint et al., 2000;Pinnegar and Polunin, 2000). Mytilus galloprovincialis was highly

13C-enriched in comparison with SPOM. This suggests that themussel, a filter-feeder, fractionates and consumes only one part ofthe SPOM (Duggins et al., 1989; Dupuy et al., 2000; Hsieh et al.,2000). SPOM is a complex source of organic matter including phyto-plankton, zooplankton, bacteria, protozoans and organic detritus. Ex-cept A. armata, which probably relies on CO2 rather than to HCO3

− ascarbon source for photosynthesis (Maberly et al., 1992), all macro-phytes were 13C-enriched in comparison with SPOM. The enriched13C- values of mussels thus also support the hypothesis that macro-algae, constituting the habitat of the mussel beds, are probably con-sumed through the detritic pathway (Riera et al., 2009).

Among invertebrate species, the two limpet species were themost13C-enriched suggesting that they mainly fed on the biofilm and onmacroalgae. However, trophic links are not straightforward as therange of 13C enrichment between sources and consumers is by fartoo different to be explained only by fractionation. The two patellidscoexist in close habitats, a few centimeters apart. However, thesehabitats are different enough to provide each species distinct trophicniches. According to Della Santina et al. (1993), P. rustica forages onfew species (mostly cyanobacteria) predominantly the low lying epi-lithic Entophysalis seusta and the endolithicMastigocoleus testarum. Incontrast, P. aspera feeds on a large number of species belonging to allmain algal classes and different forms including epilithics and epi-phytics. Limpets and sea urchins are strong candidates for omnivory,a trophic mode that could be prevalent across the whole rockyintertidal food web (Camus et al., 2008). The examination of the bio-film showed that it mainly consisted of detrital matter, benthic dia-toms, sprouts of red and brown macroalgae, protozoans Ephelotagemmipara, young recruits of molluscs, cnidarians, and serpulids.This assemblage of potential prey indicate that sea urchins and limpetsare omnivores at least as an opportunistic strategy.

Both species of sea urchins (A. lixula and P. lividus) are known to con-sume the macroalgae present in their environment, which is in goodagreement with their δ13C values. However, feeding rates of generalistherbivore species are known to vary according to the presence of chemi-cal defenses in algae (Granado and Caballero, 2001). The red alga R. ver-ruculosa and the brown algae (C. compressa, C. sinuosa and P. pavonica)tend to be particularly rich in polyphenolic biologically active compounds(Frantzis et al., 1992). Field and laboratory observations confirmed thatthe sea urchins do not feed on thesemacroalgae (pers. obs). Arbacia lixulaand P. lividus feed on other algal species with a preference for calcareousalgae (Privitera et al., 2008). Arbacia lixula and especially P. lividus were15N-depleted as previously reported in sea urchins (Pinnegar andPolunin, 1999; Yatsuya and Nakahara, 2004). Nitrogen level in macro-algae is generally limiting for consumers in Mediterranean oligotrophicenvironments (Atkinson and Smith, 1983). To compensate for the lownutritional value of seaweeds, we may hypothesize that sea urchins con-sume the 15N-depleted biofilm present on rock surface or rely on dia-zotophic microorganisms and denitrifying bacteria present into their gutas has been shown for S. droebachiensis (Guerinot and Patriquin, 1981).

Predators were represented by the anemones considering themean δ15N and the feeding mode. Anemonia sulcata and A. mutabiliswere 15N-depleted compared to A. equina. Epifluorescence microsco-py observations of the tentacles revealed high abundances of photo-synthetic endosymbiotic dinoflagellates both in A. sulcata andA. mutabilis. The predatory feeding mode of these two cnidarian spe-cies associated with the presence of endosymbiontic dinoflagellatesgives these species a trophic status that is different from strict preda-tors. In laboratory experiments, Taylor (1969) showed that over 60%of the carbon fixed during photosynthesis is transferred to the hostas clearly reflected by isotopic values in this study.

4.3. MAA transfers and trophic pathways

MAA analysis confirmed the trophic relationships suggested bythe stable isotope approach. Carroll and Shick (1996) previously

64 S. Nahon et al. / Journal of Experimental Marine Biology and Ecology 424–425 (2012) 59–65

reported a concordance between the dietary MAA complement andthe MAA content of sea urchins. The isotopic signature of musselsshowed that only one part of the SPOM was fractionated by the mus-sels, and this was further confirmed by the fact that the main MAA(shinorine) in the consumer was different from the dominant onein the SPOM pool (porphyra-334). The same was true for limpets,when δ15N showed that food sources could be different for bothspecies, the main MAA (porphyra-334) in P. rustica being replacedby another MAA (shinorine) in P. aspera. A third example concerningthe anemones was that the three species can be discriminated basedon both their isotopic signatures and their MAA contents. The trophicposition of sea urchins and field observations suggest that they feedon red macroalgae. Their low MAA content could thus result fromthe utilization of calcareous species which displayed the lowest con-centration of MAAs among the red macroalgae.

5. Conclusion

Relationships between food sources and consumers have beeninvestigated in detail to elucidate the MAA transfers in a shallowMediterranean rocky shore community using stable isotope analysis.Trophic relationships are complex because multiple trophic pathwayscoexist. Although the rocky shore of l'Ile Grosse is dominated bymacroalgae, these ones do not constitute the major base of the asso-ciated food web. This has also been reported for Atlantic intertidalrocky shore communities (Riera et al., 2004, 2009). With the excep-tion of the brown and green macroalgae, MAAs were ubiquitousamong benthic organisms. Primary producers contained high concen-tration of MAAs in comparison with consumers and no significantamount of MAAs was found in sea urchins P. lividus and A. lixula.Our results also highlighted that MAAs were transferred from prima-ry producers to consumers and accumulated with low efficiency. Still,consumers contained a higher MAA diversity than producers. Thediversity of MAAs in macrobenthic invertebrate tissues may beexplained by the diversity of food sources exploited in the naturalenvironment and to some extent by the conversion of dietary MAA.Complementary experimental studies must be performed to deter-mine the ability of primary and secondary consumers to assimilateMAAs and convert these compounds.

Acknowledgments

S.N. was supported by a MENRT grant from the French Ministry ofEducation and Research. Dr P. Riera provided helpful comments onstable isotope analyses. We acknowledge Anne Kemp for thelanguage revision of the first draft of the manuscript and Alan Foster,Jan Ellis and Jennifer Coston-Guarini for the final checking of Englishspelling and grammar. This research was in part funded by the FrenchNational Program INSU “Ecosphères continentales et Côtières” underthe project ECOPOP. We wish to thank the anonymous reviewers fortheir very constructive criticisms. [ST]

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