Thomas Lacoue-Labarthe

Michel Warnau

Laureen Beaugeard

Pierre-Yves Pascal

Trophic transfer of radioisotopes in Mediterranean sponges through bacteria consumption

Keywords:

Numerous field studies highlighted the capacities of marine sponges to bioaccumulate trace elements and assessed their potential as biomonitors of the marine environment. Experimental works demonstrated that dissolved metals and radionuclides can be taken up directly by sponge tissues but, to the best of our knowledge, little is known on the contribution of the dietary pathway through the consumption of contaminated bacteria considered as one of the trophic source in sponge diet. Objectives of this work are to study trophic transfer of radiotracers 110m Ag, 241 Am, 109 Cd, 57 Co, 134 Cs, 54 Mn and 65 Zn from the marine bacteria Pseudomonas stutzeri to the Mediterranean sponges Aplysina cavernicola and Ircinia oros. P. stutzeri efficiently bioaccumulated trace elements in our culture experimental conditions with CF comprised between 10 5 and 10 7 after 48 hours of growth in radiolabeled medium. When fed with these radiolabelled bacteria, A. cavernicola took up around 60% of radiotracers accumulated in trophic source except 134 Cs for which only 8% has been transferred from bacteria to sponge. Contrasting to this, I. oros retained only 7% of 110m Ag, 109 Cd and 65 Zn counted in bacteria, but retained 2-fold longer accumulated metals in its tissues. The sponge inter-specific differences of accumulation and depuration following a trophic exposure are discussed with respect to the structure and the clearance capacities of each species.

Introduction

Over the last 50 years, industrial and urban discharges considerably increased the input of foreign chemicals such as metals in coastal environment [START_REF] Zhou | Biomonitoring: An appealing tool for assessment of metal pollution in the aquatic ecosystem[END_REF]. Heavy metals form complexes with organic compounds and lead to malfunction or death of affected cells. Consideration of this toxicity is essential in the management of coastal areas and the evaluation of heavy metal concentrations in marine environment is consequently a widespread concern. Measuring dissolved heavy metal presents analytical problems due to detection limits and contamination biases (Rainbow, 1995a). Water contamination is difficult to infer from sediment as benthic accumulation is strongly linked with sediment characteristics such as grain size and organic content [START_REF] Luoma | Processes affecting metal concentrations in estuarine and coastal marine sediments[END_REF]. Analysis of heavy metal accumulated by marine organisms overcomes these disadvantages as tissues could be highly concentrated at rates proportional to environmental concentrations. Furthermore, only the bioavailable fraction of metal presenting ecotoxicological relevance is measured and reflects metal availability over longtime period. For those different reasons, biota concentrations are routinely used to monitor environmental contamination and there is a continuous need for new marine biomonitors [START_REF] Phillips | Biomonitoring of Trace Aquatic Contaminants[END_REF][START_REF] Conti | Marine organisms as biomonitors[END_REF]. Ideal biomonitor should be sedentary, abundant, easy to identify, widely dispersed, long lived and should accumulate heavy metal in his tissue at rate proportional to environmental contamination (Rainbow, 1995b). Mussels have been the group most commonly used in heavy metal biomonitoring however sponge have been proposed as an additional group since they can dominate benthic community and are adapted to various ecological niches extending world widely from intertidal zones to deep sea [START_REF] Patel | Sponge "sentinel" of heavy metals[END_REF]Pérez et al., 2005a;Cebrian et al., 2007a). By their filtering activity, sponges present high capacity to collect and concentrate heavy metals from dissolved and suspended phases [START_REF] Hansen | Accumulation of copper, zinc, cadmium and chromium by the marine sponge Halichondria paniciea Pallas and the implications for biomonitoring[END_REF].

Integration of heavy metal in sponge varies according to species [START_REF] Patel | Sponge "sentinel" of heavy metals[END_REF] due to different amount of skeleton fibers and mesohyl in their tissues [START_REF] Verdenal | Pollutant levels in Mediterranean commercial sponges[END_REF]). Short and long life cycle sponge integrate contamination at different time scale and can be used simultaneously for a more effective monitoring of heavy metal pollution [START_REF] Batista | Marine sponges with contrasting life histories can be complementary biomonitors of heavy metal pollution in coastal ecoystems[END_REF].

As a result, knowledge about accumulation strategies of particular metals is clearly a prerequisite to use sponge as biomonitor.

Dissolved fraction of metal can be taken up directly by sponge tissues but also indirectly by their symbiotic bacteria that contribute to their nutrition and health and can represent 40% of sponge volume [START_REF] Hentschel | Marine sponges as microbial fermenters[END_REF]. Sponge species densely packed with symbiotic bacteria are known to extract considerable amount of Dissolved Organic Mater (DOM) from the water [START_REF] Reiswig | Partial carbon and energy budgets of the bacteriosponge Verongia fistularis (Porifera, Demospongiae) in Barbados[END_REF][START_REF] Yahel | In situ feeding and element removal in the symbiont-bearing sponge Theonella swinhoei: bulk DOC is the major source for carbon[END_REF][START_REF] De Goeij | Major bulk dissolved organic carbon (DOC) removal by encrusting coral reef cavity sponges[END_REF] and this flux can be significant enough to structure marine environment at ecosystem scale [START_REF] De Goeij | Surviving in a Marine desert: the sponge loop retains resources within coral reefs[END_REF]. Assimilation of heavy metal associated with DOM [START_REF] Iyer | Biosorption of heavy metals by a marine bacterium[END_REF] could consequently be an important way of contamination for sponges [START_REF] Webster | The effect of copper on the microbial community of a coral reef sponge[END_REF].

Sponge can also take up metals from the suspended particles collected during feeding. This filter feeding activity is based on pumping ambient water through an intricate system of aquiferous canals to choanocyte chambers where suspended particules are retained by sieving and then phagocyted (Ulrick [START_REF] Riisgård | Particle capture mechanisms in suspension-feeding invertebrates[END_REF]. Sponges can retains diatom, flagellate and ciliate [START_REF] Coma | The ultimate opportunits: consumers of seston[END_REF]) however this filtering system is rather specialized in retaining smaller preys (size range 0.1 to 2 µm) such as cyanobacteria, bacteria (Ribes et al., 1999b;[START_REF] Trussell | Depth-specific differences in growth of the reef sponge Callyspongia vaginalis: role of bottom-up effects[END_REF][START_REF] Yahel | In situ feeding and metabolism of glass sponges (Hexactinellida, Porifera) studied in a deep temperate fjord with a remotely operated submersible[END_REF] and viruses [START_REF] Hadas | Virus predation by sponges is a new nutrient-flow pathway in coral reef food webs[END_REF]. The fact that sponges are able to retain smaller particles than most filter feeders is an adaptative advantage, particularly in oligotrophic environment (Ribes et al., 1999a). Bacteria have been considered as one of the primary source of energy in sponge diet [START_REF] Pile | In situ grazing on plankton < 10 µm by the boreal sponge Mycale lingua[END_REF][START_REF] Kowalke | Ecology and energetics of two Antarctic sponges[END_REF].

Bacteria can take up heavy metals at rates high enough to be considered for remediation in polluted environments [START_REF] Valls | Exploiting the genetic and biochemical capacities of bacteria for the remediation of heavy metal pollution[END_REF][START_REF] Malik | Metal bioremediation through growing cells[END_REF]. The cell surface of bacteria carries a net negative charge and can passively adsorb appreciable quantities of positively charged cationic metals [START_REF] Brierley | Bioremediation of metal-contaminated surface and ground-water[END_REF][START_REF] Scott | Sites of cadmium uptake in bacteria used for biosorption[END_REF].

Metal can also actively pass across the cell membrane through the cell metabolic cycle and bind to intracellular compounds [START_REF] Malik | Metal bioremediation through growing cells[END_REF]. Both modes of metal uptake give to bacteria good bioaccumulation properties.

The aim of the present study is to enrich bacteria Pseudomonas stutzeri with different metals and radionuclides (Mn, Co, Zn, Ag, Cd, 137 Cs, 241 Am) in order to feed two Mediterranean sponges species (Ircinia oros and Aplysina cavernicola) and to be able to evaluate their accumulation capacities of each element.

Materials and Methods

Organisms

Specimens of Aplysina cavernicola and Ircinia variabilis were collected off the Fontvieille harbour at St Nicolas rocks in Monaco by SCUBA in March 2012 (lat: 43° 43' 36.768";long: 7° 25' 25.248"). Immediately after collection, sponges were kept in seawater at constant temperature during their transfer to the aquaria within 30 min. At the IAEA premises, sponges were placed in a 20-L glass aquarium containing 1 µm filtered and UV sterilized natural seawater (seawater flux: 40 L h -1 ; temperature 17°C; 38 p.s.u.; light/dark cycle 12h/12h) and acclimated for one week before experiment and fed daily with live Isochrysis galbana.

Aplysina cavernicola (Class: Demospongiae, Order: Verongida, Family: Aplysinidae) is a yellow digitate-shaped sponge, organized in one or more chimney-like structures. I. oros harboured abundant and phylogenetically diverse symbiont consortia including Gammaproteobacteria [START_REF] Erwin | Phylogenetic diversity, host-specificity and community profiling of sponge-associated bacteria in the Northern Gulf of Mexico[END_REF]. Numerous inhalant oscula pump water to the central atrium structure and then to the apical exhalent osculum [START_REF] Pfannkuchen | In situ pumping activity of the sponge Aplysina aerophoba, Nardo 1886[END_REF]. The close species Aplysina aerophoba displays a clearance rate of 3-18 ml.g -1 .min -1 [START_REF] Hoffmann | Oxygen dynamics and transport in the Mediterranean sponge Aplysina aerophoba[END_REF].

Ircinia variabilis (Class: Demospongiae, Order: Dictyoceratida, Family: Irciniidae) is a massive sponge. Among the large amounts of symbiotic bacteria hosted by A. cavernicola, gamma-proteobacteria are the second most abundant [START_REF] Friedrich | Microbial diversity in the marine sponge Aplysina cavernicola (formerly Verongia cavernicola) analyzed by fluorescence in situ hybridization (FISH)[END_REF]. Oscules are large and located at the top of the conical lobes. This species does not have mineral spicules but a dense fiber skeleton structurally diverse from classical spongins [START_REF] Junqua | Biochemical and morphological studies on collagens of horny sponges. Ircinia filaments compared to spongines[END_REF]. The clearance rate for this species has been estimated experimentally (data not shown) to ca. 0.8 ml.g -1 .min -1 .

Bacteria radiolabelling

The marine bacteria Pseudomonas stutzeri, belong to the class of gamma-proteobacteria and is distributed widely in the water column and in sediment [START_REF] Lalucat | Biology of Pseudomonas stutzeri[END_REF]. Gammaproteobacteria is one of the most abundant bacterial taxa symbiotic of sponges [START_REF] Webster | Marine sponges and their microbial symbionts: love and other relationships[END_REF] and P. stutzeri has been previously identified in intertidal marine sponge [START_REF] Zhang | Purification and characterization of a dehalogenase from Pseudomonas stutzeri DEH130 isolated from the marine sponge Hymeniacidon perlevis[END_REF]. In the present study, P. stutzeri have been cultured in a medium radiolabelled with 110m Ag, 109 Cd (Amersham, UK) and 241 Am, 57 Co, 134 Cs, 54 Mn and 65 Zn (Isotope Product Laboratory, USA) and then used as trophic source of radioisotope to sponges specimen.

In detail, P. stutzeri isolates have been first grown for 48 h by adding bacteria in 5 ml of previously sterilized autoclaved bacterial liquid culture medium (Marine Broth 2216, Difco TM ) and kept in the dark at 4°C. Then, the 5 ml culture was transferred in 300 ml of culture medium radiolabelled with 13 kBq.l -1 of 110m Ag, 7 kBq.l -1 of 241 Am, 39 kBq.l -1 of 109 Cd, 11 kBq.l -1 of 57 Co, 20 kBq.l -1 of 134 Cs, 11 kBq.l -1 of 54 Mn and 11 kBq.l -1 of 65 Zn and kept in the dark at 25°C for 48 h. The culture media has been performed in triplicate.

At the end of the incubation period, bacteria were concentrated by centrifugation (3000 rpm during 10 min at 25°C) in order to remove the radiolabelled culture media, without causing destruction of bacteria. The supernatant was discarded and the bacteria were resuspended in 0.2 µm-filtered seawater and centrifugated again. The operation was repeated three times until no significant radioisotopes activities were detected in the supernatants. Finally, the pellet was resuspended in 50 ml of filtered seawater giving a suspension of living bacteria with a concentration of 2.10 7 ± 3.10 5 cell.ml -1 . One aliquot of this bacterial solution was radiocounted in order to assess the bioaccumulation of radiotracer in P. stutzeri.

Bacterial concentration assessment

The concentrations of radiolabelled cells in the bacterial suspension and in the experimental tanks were assessed through the optical density. Measured absorbance values (A 680nm ) have been transformed in bacteria concentration (cell.ml -1 ) thanks to a calibration curve (Figure 1) realized from successive dilutions of the bacterial suspension. The concentration of bacteria in diluted samples has been determined by cytometry. Briefly, bacterial suspensions were stained with Sybr Green I (1/10000 final concentration, Invitrogen) and incubated for 15 minutes in the dark. Samples were analyzed with a FACS CantoII (BDBiosciences) equipped with an air-cooled, 20 mW solid state blue laser (488nm) at low speed (~8 µL/min) during 1 minute. Stained cells were discriminated according to green fluorescence (FL1) from Sybr Green I staining and side scatter properties (SSC) in log mode. Accurate cell concentrations were performed using TruCount beads (BDBiosceinces).

Feeding procedure: the dietary uptake of metals in sponges has been assessed using the pulsechase feeding method (organisms were dietary exposed to radiotracers only once, in order to analyse metal assimilation and depuration from one ration; Warnau et al., 1996). For each sponge species, three individuals were placed individually in 450 ml plastic box (seawater flux: 20 L h -1 ; temperature: 17°C; salinity 38 p.s.u.; light/dark cycle 12h/12h) 24 h before the start of the experiment. Then, the water flow renewal has been closed and the radiolabelled bacteria have been resuspended in the seawater until reaching a cell density of 3.10 7 cell.ml -1 .

Sponges were left for one hour in closed-circuit tanks allowing them to feed with radiolabelled bacteria. After 20 and 60 min, the retention of bacteria by sponges has been assessed by measuring the bacteria concentration in seawater (A 680nm ).

Following this one-hour exposure period, sponges were sampled, rinsed in clean seawater, and immediately counted. Then, sponges were placed in a 20-l tank with clean seawater (seawater flux: 40 L h -1 ; temperature 17°C; 38 p.s.u.; light/dark cycle 12h/12h) and the radiotracers loss in sponges has been followed by counting individuals every days for the first week and every third days during the next four weeks.

Radioanalyses and data treatment

Radioactivities were measured using a high-resolution γ-spectrometry system consisting of four coaxial Germanium (N-or P-type) detectors (EGNC 33-195-R, Canberra ® and Eurysis ® ) connected to a multi-channel analyzer and a computer equipped with a spectra analysis software (Interwinner ® 6). The detectors were calibrated with an appropriate standard for each counting geometry used and measurements were corrected for background and physical decay of the radiotracers.

The bioaccumulation of each radiotracer in bacteria was expressed in concentration factors, which is the ratio between radiotracer activity in the bacteria -Bq g -1 -and the timeintegrated activity in Marine Broth culture medium -Bq g -1 . The concentration of radiotracers expressed in Bq cell -1 has been transformed using the bacteria mass value of Pseudomonas putida of 5.10 -14 g dw.cell -1 [START_REF] Troussellier | Variation of carbon content among bacterial species undere starvation condition[END_REF].

The radiotracer depuration kinetics in sponges was expressed in terms of change in percentage of remaining activity and was best by either a single-or a double-exponential equation (see [START_REF] Lacoue-Labarthe | Bioaccumulation of inorganic Hg by the juvenile cuttlefish Sepia officinalis exposed to 203 Hg radiolabelled seawater and food[END_REF] for detailed procedure).

Constants (and their statistics) of the best fitting uptake and depuration kinetic equations (decision based on ANOVA tables for two fitted model objects) were estimated by iterative adjustment of the models using the nls curve-fitting routine in R freeware [START_REF] Core | R: A language and environment for statistical computing[END_REF]. The level of significance for statistical analyses was always set at α = 0.05.

Results

Radiolabelling of Pseudomonas stutzeri

The culture of the bacteria P. stutzeri in a bacterial culture medium radiolabelled with dissolved radiotracers allowed a significant bioaccumulation in bacteria cells of 110m Ag, 241 Am, 109 Cd, 57 Co, 134 Cs, 54 Mn and 65 Zn after 48 hours of growth (Figure 2). It is noteworthy that bioaccumulation efficiency varied among elements with the highest CF values observed for the non-essential metals 110m Ag and 109 Cd (1.10 7 ± 4.10 5 and 1.10 7 ± 5.10 5 , respectively) and the radionuclide 241 Am (5.10 6 ± 3.10 5 ). Contrasting to this, the essential elements 65 Zn, 57 Co and 54 Mn have been taken up 3, 20 and 30-fold lower, respectively, in bacteria than 110m Ag and 109 Cd. Caesium reached a CF value of 2.10 6 ± 6.10 4 .

Radiotracer trophic transfer from bacteria to sponges

During the one-hour pulse-chase feeding experiment, the monitoring of the absorbance (680 nm) of tank seawater allowed at determining the bacterial retention by both species. Thus, after one hour, the three individuals of Aplysina cavernicola removed 20, 45 and 65 % of bacteria suspended in seawater, whereas no significant decrease of bacterial concentration was observed in Ircinia oros tanks, suggesting a lower filtration activity of this species in our experimental condition.

At the end of trophic exposure, the activities of radiotracers accumulated by Aplysina cavernicola (Bq.g -1 dw) were ranked as followed: 109 Cd >> 110m Ag > 241 Am ≈ 65 Zn >> 57 Co > 134 Cs > 54 Mn (Figure 3A), whereas only 109 Cd >> 110m Ag > 65 Zn were significantly detected in Ircinia oros. This pattern of bioaccumulation is congruent with the radioactivity levels measured in bacteria for each element, showing that A. cavernicola took up around 60% of radiotracers (Figure 3B) accumulated in bacteria except 134 Cs for which only 8% has been transferred from bacteria to sponge. Contrasting to this, I. oros retained only 7% of 110m Ag, 109 Cd and 65 Zn counted in bacteria.

Following the 1-h pulse chase feeding, the whole-body depuration for 110m Ag, 241 Am, 109 Cd, 57 Co, 54 Mn and 65 Zn was followed for 35 days in both sponge species. The 134 Cs loss kinetic was not shown as this radionuclide activity has been found below the detection limit in sponge placed in clean seawater after 24 hours, suggesting a rapid depuration of this element.

The depuration kinetics in Aplysina cavernicola were best fitted by a two-compartment model for all element except 54 Mn loss better fitted by a single-compartment model (Figure 4 and5; Table 1). Both 110m Ag and 241 Am were the most rapidly depurated by sponge with 88 ± 3 and 79 ± 8% lost with a biological half-life (T b½ ) of 1 and 1.5 d, respectively. The essential elements displayed the highest assimilation efficiency for the long lived components (A 0l = 55, 95 and 62% for 57 Co, 54 Mn and 65 Zn, respectively, with relatively short Tb 1/2 comprised between 12 and 18 d. Contrasting to this, 33% of 110m Ag, 57% of 109 Cd and 63 % of 65 Zn significantly accumulated by Ircinia oros displayed longer T b½ (41, 57 and 33 d, respectively), implying a better efficient and longer retention of these elements by sponge tissues or within their aquifer system.

Discussion

The paper first reports that bacteria P. stutzeri efficiently bioaccumulated trace elements in our culture experimental conditions with CF comprised between 10 5 and 10 7 after 48 hours of growth in radiolabeled medium. Similar high bioaccumulation efficiencies have been already reported in picoplankton Synechococcus sp [START_REF] Fisher | Accumulation of metals by marine picoplankton[END_REF], under similar exposure conditions.

The small size of bacteria and the subsequent high surface/volume ratio are argued as the main explanation fostering the uptake capacities. Basically, two main processes drive the trace element uptake in microorganisms, i.e. the adsorption of metal on the particle walls and external materials such as the exopolysaccharides, and the absorption in cells through ionic transporters [START_REF] Gadd | Heavy metal accumulation by bacteria and other microorganisms[END_REF].

The gram-negative wall of P. stutzeri and its associated negative charges might favour the binding of metallic cations [START_REF] Gadd | Silver accumulation in Pseudomonas stutzeri AG259[END_REF]. For each element, the adsorption on bacterial surface could be estimated through the partition coefficient between particles and seawater (K d ) reported in the literature. According to available data on particles or phytoplankton, the particle reactivity among studied elements decreases according to 241 Am > Mn > Zn > Ag > Co > Cd > Cs [START_REF] Wang | Kinetic determination of trace element bioaccumulation in the mussel Mytilus edulis[END_REF][START_REF] Keung | Influences of metal-ligand complexes on the cadmium and zinc biokinetics in the marine bacterium, Bacillus firmus[END_REF]Metian et al., 2008a;[START_REF] Metian | Delineation of heavy metal uptake pathways (seawater and food) in the variegated scallop Chlamys varia, using radiotracer techniques[END_REF]. This ranking is not congruent with the CF values observed in this study, especially in the case of Ag and Cd supposed to be less bound on bacterial surface, but here highly accumulated on P. stutzeri.

In cells of Pseudomonas aeruginosa, high Hg uptake in strain has been attributed to cysteinerich transport proteins carrying metal from external medium to cytoplasm [START_REF] Chang | Biosorption of mercury by the inactivated cells of Pseudomonas aeruginosa PU21 (Rip64)[END_REF]. Similar mechanisms might be expected to contribute to the high accumulation of Ag, and to a lesser extent Cd, according to the affinity of these metals for sulfhydryl-rich group [START_REF] Bell | Structural chemistry and geochemistry of silver-sulfur compounds: critical review[END_REF]. Moreover, some bacteria strains are known for their high resistance to silver toxicity [START_REF] Haefeli | Plasmid-determined silver resistance in Pseudomonas stutzeri isolated from a silver mine[END_REF][START_REF] Li | Silver-resistant mutants of Escherichia coli display active efflux of Ag + and are deficient in porins[END_REF]. While this resistance seems to be associated with energy-dependent ion efflux [START_REF] Li | Silver-resistant mutants of Escherichia coli display active efflux of Ag + and are deficient in porins[END_REF] in most of studied bacteria (e.g.

Escherichia coli)

, it has been demonstrated that the strain Pseudomonas stutzeri AG259 accumulated Ag under a metal-based single crystal, precipitating then high Ag concentration under non-metabolically available form [START_REF] Klaus | Silver-based crystalline nanoparticles, microbially fabricated[END_REF].

Regarding Cd, [START_REF] Chang | Biosorption of lead, copper and cadmium by biomass of Pseudomonas aeruginosa PU21[END_REF] highlighted a rapid adsorption of the element on cells, but a difference of biosorption capacity between inactivated and free cells, suggesting that a metabolic-dependent absorption occurs in this strain. Furthermore, same authors showed that cells at exponential growth phase exhibited higher Cd adsorption capacities, suggesting that in our radiolabelling conditions, the culture of P. stutzeri in a 109 Cd radiolabelled medium could contribute to reach high CF value.

In the present study, it is noteworthy that essential elements (i.e. 57 Co, 54 Mn, and 65 Zn) displayed lower CF values than 110m Ag, 241 Am and 109 Cd. The microorganisms such as bacteria are known to synthesize secondary metabolites, called "siderophores", that play a key role in the essential metal homeostasis [START_REF] Lalucat | Biology of Pseudomonas stutzeri[END_REF]. Indeed, some bacteria strains produce low-molecular-weight chelators with a high affinity for Fe and other transition metals to bind them in the extracellular environment and transport them inside the cells [START_REF] Zawadzka | Identificiation of siderophores of Pseudomonas stutzeri[END_REF]. In Pseudomonas aerunginosa, salicylate-derived pyochelin seems to have a key role in supplying divalent metals such as Co or Ni, but displays a low affinity for Mn [START_REF] Visca | Metal regulation of siderophore synthesis in Pseudomonas aeruginosa and functional effects of siderophoremetal complexes[END_REF], suggesting that siderophore unlikely contribute to the uptake of this element. Some P. stutzeri strains produce deferrioxamine or PDTC (pyridine-2,6-bisthiocarboxylate) siderophores [START_REF] Lewis | Physiological and molecular genetic evaluation of the dechlorination agent, pyridine-2,6-bis(monothiocarboxylic acid) (PDTC) as a secondary siderophore of Pseudomonas[END_REF]) also known for their role in transition element nutrition as demonstrated for the Zn [START_REF] Leach | The role of the siderophore pyridine-2,6-bis (thiocarboxylic acid) (PDTC) in zinc utilization by Pseudomonas putida DSM 3601[END_REF]. However, information are lacking on the various affinity of siderophores for the essential transition metals to better assess their role in metal metabolism.

Additionally, it is important to remind that Pseudomonas stutzeri were cultured in a medium (Marine Broth 2216; [START_REF] Zobell | Studies on marine bacteria. I. The cultural requirements of heterotrophic aerobes[END_REF]), which contains essential compounds and nutrients for an optimal bacteria growth. Yeast extract supplies trace element and may contribute to lower the bioaccumulation efficiency of essential metals. According to [START_REF] Grant | Minor element composition of yeast extract[END_REF], Mn and Co concentrations are almost one order of magnitude lower than those of Zn (3 and 3.5 µg.g -1 vs. 75 µg.g -1 dw) but similar to the Cd concentration (~1.5 µg.g -1 dw). Considering that addition of stable element in culture medium may lowers concentration factor (Metian et al., 2008b), the yeast extract composition would contribute to explain surprising lower CF values observed for essential elements such as Co and Mn, compared to non-essential element or radionuclide, such as Cs, known to be poorly accumulated in marine invertebrates and microorganisms [START_REF] Wang | Kinetic determination of trace element bioaccumulation in the mussel Mytilus edulis[END_REF][START_REF] Pouil | Delineation of 134 Cs uptake pathways (seawater and food) in the variegated scallop Mimachlamys varia[END_REF].. Tissues of Mediterranean sponges fed with radiolabeled bacteria during one hour present an activity pattern of the seven radiotracers similar to bacterial pattern (Figure 3A). Aplysina cavernicola displayed around 60 % of the radioactivities (no significant difference among radiotracers except 134 Cs; U-test; p > 0.05) added through bacteria whereas Ircinia oros only accumulated around 7 %. This contrasting bacterial metal transfer between both species is likely due to the low clearance rate of the massive I. oros being 4-22 fold lower than this of the branching erect A. cavernicola [START_REF] Hoffmann | Oxygen dynamics and transport in the Mediterranean sponge Aplysina aerophoba[END_REF]. Thus, this estimation is congruent with the 9-fold difference of radiotracers activities counted in both species. As a result, only the elements highly accumulated in P. stutzeri, i.e. Ag, Cd and Zn, reached activities levels in sponge above the detection threshold, limiting the assessment of trophic transfer behaviour for the others radionuclides.

It is noteworthy that, in A. cavernicola, a low fraction of 134 Cs contained in bacteria, i.e. 8 ± 3 % vs. 43 ± 24 %, 74 ± 15 %, 63 ± 23 %, 63 ± 25 %, 51 ± 19 % and 57 ± 21 % for 54 Mn, 57 Co, 65 Zn, 110m Ag, 109 Cd, and 241 Am, respectively; U-test; p<0.05) has been transferred to the sponge, suggesting a rapid depuration of this element by bacteria and sponge during the one hour pulse feeding chase. Indeed, Cs is a soluble element, known as a mimetic of K [START_REF] Wang | Modeling radiocesium bioaccumulation in a marine food chain[END_REF], implying both rapid uptake in cells and then loss in seawater, following water movements between both compartments [START_REF] Lacoue-Labarthe | Contrasting accumulation biokinetics and distribution of 241 Am, Co, Cs, Mn and Zn during the whole development time of the eggs of the common cuttlefish, Sepia officinalis[END_REF].

Kinetic parameters following pulse-chase feeding (Figure 4 and 5; Table 1) suggest that depurations of all element except Mn were best fitted by a two-compartment models, implying that 1) a fraction is rapidly lost likely depending on the water renewal in the sponge aquifer system, and that 2) another fraction of metals remained longer associated to sponge tissue through retention of bacteria in the aquifer system or assimilation of elements in sponge tissues. Results clearly show that the longest T b½ is around 2 weeks in A. cavernicola whatever the metal, highlighting a poor retention of metals following particulate contamination. In comparison, T b½ of elements accumulated from waterborne pathway were between 2 and 10-fold longer (with 35 and 157 days for 241 Am and 109 Cd, respectively) in the branch erect sponge Axinella reniformis [START_REF] Genta-Jouve | Comparative bioaccumulation kinetics of trace elements in Mediterranean marine sponges[END_REF]. Moreover, calculated T b½ in this study for all elements exceeds one month in Ircina oros for Ag, Cd and Zn. Again, the contrasting clearance rates between both species could be one explanation of this difference, implying that the water renewal in the aquifer system drive the depuration rate for absorbed or assimilated trace elements in sponge. Thus, this work highlighted that the high pumping flow reported in erect sponge A. cavernicola contributed to a rapid accumulation of radiolabelled particles compared to the slow filter feeder massive sponge I. oros [START_REF] Turon | Clearance rates and aquiferous systems in two sponges with contrasting life-history strategies[END_REF], but foster also a rapid depuration of trace elements, suggesting a limited net accumulation of metals from trophic route for this species. This observation is congruent with the bioaccumulation capacities of dissolved metals in Mediterranean sponges showing that with massive sponges (Chondrosia reniformis, Agelas oroides and Ircina variabilis) display the highest uptake and retention capacities compared to erect species (Acanthella acuta, Axinella damicornis, Axinella verrucosa). These massive species possess higher choanocyte chamber volumes and consequently a much higher area to bind trace elements or retained contaminated particles as suggested by Cebrian and colleagues (2007b).

This study demonstrated experimentally that filtered marine bacteria by Mediterranean sponges could be considered as a biotic vector of metal transfer along the trophic web.

However, many questions merged from these results for enhancing our understanding of the observed metal transfer from microoganisms to sponges and the interspecific differences of bioaccumulation capacities among sponges. Further experiments including subcellular fractionation could help to describe the trace element bioaccumulation pathways in bacteria (adsorption vs. absorption) and thus provide some indications on the metal bioaccessibility for the next trophic level. Moreover, the bioaccumulation efficiencies through particulate pathway differs between the two studied species, likely linked to their massive vs. erect structure and their respective pumping and clearance capacities [START_REF] De Mestre | Sponges as sentinels: Patterns of spatial and intra-individual variation in trace metal concentration[END_REF].

Considering the potential use of sponges as "biomonitors", the massive I. oros requests a prolonged metal input to reveal a contamination of bacterial resources, but retains more longer this information compared to erect A. cavernicola. In other terms, the first species seems to be appropriate to detect chronic contamination compared to the erect sponge.

Nevertheless, beyond these characteristics, the role of symbiotic microorganisms in the particle retention, digestion and assimilation of nutrients and trace elements remains unknown. Both A. cavernicola and I. oros are considered as HMA (High Microbial Abundance) sponge species [START_REF] Taylor | Sponge-associated microorganisms: evolution, ecology, and biotechnological potential[END_REF][START_REF] Weisz | Do associated microbial abundances impact marine demosponge pumping rates and tissue densities?[END_REF][START_REF] Erwin | Phylogenetic diversity, host-specificity and community profiling of sponge-associated bacteria in the Northern Gulf of Mexico[END_REF]. Thus, the interspecific bioaccumulation differences cannot be inferred to microbial density but rather potentially to distinct microbial communities according to sponge species [START_REF] Selvin | Sponge-associated marine bacteria as indicators of heavy metal pollution[END_REF]. Additionally, only one bacteria strain has been used in this work, implying that only one particle size has been investigated as a potential vector for metal trophic transfer. Sponges

Figures captions

Figure 1. Calibration curve relating the bacteria concentrations (cell/ml) according to the absorbance at 680 nm.

Figure 2. Concentration factors (CF) of radiotracers in the bacteria Pseudomonas stutzeri cultured during 48 h in Marine Broth medium radiolabelled with 54 Mn (11 Bq.ml -1 ), 57 Co (11 Bq.ml -1 ), 65 Zn (11 Bq.ml -1 ), 110m Ag (13 Bq.ml -1 ), 109 Cd (39 Bq.ml -1 ), 134 Cs (20 Bq.ml -1 ), 241 Am (7 Bq.ml -1 ).

Figure 3. (A) Radiotracers activities (Bq.g -1 dw; n = 3 per sponge) and (B) retained activities (%) measured in sponges Ircina oros (black histogram) and Aplysina cavernicola (grey histogram) after feeding with radiolabelled bacteria Pseudomonas stutzeri (2.9.10 7 bacteria.ml -1 ) for one hour. Please note the logarithmic scale for the figure (A) and that 54 Mn, 57 Co, 134 Cs and 241 Am activities were under the limit detection in I. oros.

Figure 4 .

 4 Figure 4. Ircina oros. Loss kinetics of 110m Ag, 109 Cd and 65 Zn (% remaining activity [RA]; mean ± SD) in the sponge Ircina oros (n=3) previously fed with radiolabelled bacteria for one hour. Parameters for the best fitting equations are given inTable 1.

Figure 5 .Figures

 5 Figure 5. Aplysina cavernicola. Loss kinetics of radiotracers (% remaining activity [RA]; mean ± SD) in the sponge Aplysina cavernicola (n=3) previously fed with radiolabelled bacteria for one hour. Parameters for the best fitting equations are given inTable 1.

  Figure 2.

Acknowledgement

The IAEA is grateful for the support provided to its Environment Laboratories by the Government of the Principality of Monaco. We thank Olivier Thomas from the University of Nice to provide with sponge specimen, and Sophie Sable from the University of La Rochelle to offer the bacteria strain of Pseudomonas stutzeri. We are grateful to the cytometry and imaging platform that provided organization and service of the UMR 7266 LIENSs, University of La Rochelle and CNRS. Authors thank the two anonymous reviewers for their comments and fruitful suggestions improving the manuscript.

however show adaptive interspecific variation with respect to clearance rate and particle size selection [START_REF] Turon | Clearance rates and aquiferous systems in two sponges with contrasting life-history strategies[END_REF]. Further studies investigating trophic bioaccumulation on a large range of particle size using other bacterial strains or picoplankton could be valuable to define the contribution of filtered particles in the bioaccumulation of metals in these biomonitors of growing interest [START_REF] Patel | The chemical form of cobalt-60 in the marine sponge Spirastrella cuspidifera[END_REF]Pérez et al., 2005b;Cebrian et al., 2007b). Finally, we hope that this study will revive some interest on the bacterivory as a pathway for metal integration in trophic web. Table 1. Parameters and statistics of the depuration kinetics of radiotracers in two species of sponge, Ircina oros and Aplysina cavernicola, fed for one hour with radiolabelled bacteria Pseudomonas stutzeri and kept for 36 days in depuration condition.

Tables


_________________________________________________________________________ BOOK REVIEW _________________________________________________________________________

Global Strategies in Retailing: Asian and European Experiences London, UK: Routledge (2014) pp. 252

Book Edited by John Dawson and Masao Mukoyama

This book, comprising a collection of articles edited by John Dawson and Masao Mukoyama, will appeal to a wide audience of both academics and practitioners. With contributions from international scholars, the volume offers a comprehensive overview of history, contemporary practices, and future directions of retail internationalization with particular focus on Asian and European companies. Within its eleven chapters it blends conceptual, empirical, and case studybased papers linked through the application of the global portfolio strategy paradigm. The reader is steered from discussion of the broader industry-wide context and historical evolution of international retailing to more textured analyses of present day retailer strategies.

In their introduction the authors explore the strengths and limitations of current theories pertaining to international retailing in accounting for the rapidly evolving multiformat and multicontinental contemporary practices of retailers. As intermediaries, retailers represent an eclectic group of businesses with an output comprising a mixture of physical goods, distribution, and consumer services. Once seen mostly as local resellers of manufacturers' products, today modern international retailers have become global megabrands in their own right with strong impacts upon patterns of consumption, supplier networks, and urban planning and development.

The first three chapters examine the evolution of international retailing from earlier entrepreneurial, cross-border pursuits of growth to present day, more organized global portfolio strategies. Here the authors explain the rapid international growth of retailers as a result of global political and economic changes resulting in ongoing trade liberalization. This, coupled with applications of new retail technologies, management learning, and sustained investment in international growth, has propelled once local retailers fully onto the international stage. The authors draw on structuration theory in highlighting international retailers' increasing endeavors to immerse themselves in the economic, sociocultural, and political environments in which they operate. Greater integration of internal and external resources (i.e., agency) with internal and external environments (i.e., structure) has facilitated the adoption of global portfolio strategies aimed at optimizing and managing effectively complex portfolios of retail brands, formats, and formulas.

In chapter 2 the authors distinguish between the terms retail format and formula. Whilst retail formats are business models described as a generalized set of relationships defined by particular cost structures, retail formulae identify the very places of transaction where retailerconsumer interactions take place. Further on they introduce the concept of the global portfolio strategy as a new way of leveraging synergies and co-ordination among multiple retail business units across multiple continents. The authors visualize this concept with a retail "GPS map": an analytical tool for understanding strategic shifts of international retailers over time. The next seven chapters (4-10) are case study-based, providing exemplars of the international strategies of Asian and European retailers. These include IKEA, Lotte Shopping, MUJI, Nitori, President Chain Store Corporation , and the Delhaize and Metro Groups.

The first case (chapter 4) is that of Nitori. From being a small retailer in Hokkaido Nitori has grown to become the leading Japanese furniture retailer with a global sourcing network and international stores in Taiwan. Their uninterrupted growth has been attributed to a dual manufacturing-retailing model, combining an international production network with an expanding range of store formats and formulas for affordable, design-led furniture and home fashion accessories. Chapter 5 is another example of a global portfolio strategy based on business diversification. President Chain Store Corporation is a vertically integrated Taiwanese food conglomerate with a portfolio of franchises including 7-Eleven. Synchronizing vertical and horizontal diversification has allowed the company to grow internationally initially through a network of subsidiaries, joint ventures, and partnerships into the neighboring markets of China, Vietnam, and Indonesia and subsequently further afield in the United States (U.S.) and Russia.

Similarly, Lotte Shopping (discussed in chapter 6), with headquarters in Seoul, has grown by building an extensive portfolio of formats including department stores, hypermarkets, supermarkets, convenience stores, home shopping, and internet retailing. They have been successful in achieving vertical and horizontal synergies, whilst adapting their market entry strategies to the fast-growing retail markets of Asia.

In some contrast to the previous examples, chapter 7 examines IKEA's standardized model of growth on four continents based on its three elements: a single "big box" retail format, a catalogue, and an almost uniform range of 10,000 items comprising "flat-pack" furniture and housewares. The consistency of the IKEA concept across markets has been further reinforced by an in-house design department, an integrated global supply chain, and an international network of dedicated suppliers. Yet the concept provides for some market adaptations within the product range, advertising, store planning, and customer service areas. MUJI (in chapter 8) is another case of a largely standardized multicontinent strategy pursued by a single-format retailer. Although part of a larger group, MUJI remains a niche retailer of private label products. Its distinct concept is centered on selling high-quality, non-branded clothing and household products across Japan and the world. After an initial expansion in neighboring Singapore, Thailand, and Hong Kong, the company has grown further afield in the U.S. and Europe. Whilst its retail concept remains standardized, MUJI has been successful in codeveloping with local designers products rooted in the cultures of its international markets.

Yet another configuration of the GPS map is presented in chapter 9 through the case of the Delhaize Group. It eloquently charts out the international growth of the largest Belgian retailer. Belgium is a relatively small retail market that, coupled with retail legislation at the time restricting European growth of large grocery stores, has brought about change in Delhaize's growth strategy. The retailer withdrew altogether from European hypermarkets to concentrate on its growth in the U.S. where through acquisitions and investments Delhaize became an established, multibrand retailer. This strategy has been so successful that at present its chains in the U.S. account for over 60% of the group's overall revenues. Further expansion through acquisition in Southeast Europe and Asia paints a picture of dynamic international growth through multiple modes of market entry.

The case of the German Metro Group, in chapter 10, presents a multiformat, multicontinent portfolio strategy on a global scale. In this case Metro is more standardized than Delhaize, yet equally diversified. Metro is among the largest retailers with operations in 32 countries on three continents. Their global portfolio strategy has emerged through consecutive phases of international growth over time. The company defines standardized formats, which are then tested, rolled out internationally, and subsequently adapted locally. This case study pinpoints the variable international success of Metro's different retail formats with some, such as Metro Cash & Carry, proving to be more resilient and better adapted to economic fluctuations across different markets.

The concluding chapter 11 is a summary of key findings and reflections on the future directions of retailer internationalization. The authors point out that internationalization in retailing is evolving through various phases and that the global portfolio strategy is the latest but certainly not final one. Modern retailers are complex and geographically dispersed organizations in need of focus and flexibility. Many adopt portfolio strategies to develop synergies and maintain growth through scale-related cost economies.

Yet, such an approach is not without challenges. Some of these include retailers' managerial and technological capacity to devise workable implementation mechanisms and structures across multiple international formats and formulae. The authors emphasize the need for exploring these challenges and opportunities through broadening the theoretical bases of the research area and drawing on sociology, social science, and organization theory (most notably the theories of structuration, embeddedness, or by means of the resource-based perspective on internationalization) to do so. This volume is a welcome addition to a relatively short list of books dedicated to international retailing. Although for some time now the industry has been a major contributor to global gross domestic product as an innovator of products, services, and technologies, somehow retailing still receives less attention than it merits in the broader management literature. This volume presents research of a high standard, organized in a clear and logical way. The balance between conceptual and empirical papers with reference to Asian and European retailers has achieved important insights and depth that scholars and practitioners on the subject will find very useful and stimulating. Perhaps one area which could have been examined a little further would be innovation and globalization of nonstore retailing. We have recently seen rapid proliferation of e-commerce and multichannel-channel modes of retailing both in Asia and Europe. These have provided retailers with the capacity to grow abroad beyond simple exponential investment in store-based retailing.

As a whole this book works on different levels. Alongside its practical benefits, it offers a significant contribution to academic debates in international retailing. It is also refreshingly Australia. E-mail: jm.buchan@unsw.edu.au

  inclusive in extending the immediate theoretical domain of retailing into the broader domain of social science in search of new theories and ideas. Reviewed by Latchezar Hristov, DPhil, Audencia Nantes School of Management, 8 Route de la Jonelière -B.P. 31222, 44312 Nantes Cedex 3, France. E-mail: lhristov@audencia.com Note: If there are books you would like to see reviewed in this section, or you would like to submit a review, please contact the Book Review Editor, Jenny Buchan, PhD, Taxation & Business Law, Business School, University of New South Wales, Kensington, NSW 2052,