François Oberhänsli

Early development and molecular plasticity in the Mediterranean sea urchin Paracentrotus lividus exposed to CO2-driven acidification

SUMMARY Ocean acidification is predicted to have significant effects on benthic calcifying invertebrates, in particular on their early developmental stages. Echinoderm larvae could be particularly vulnerable to decreased pH, with major consequences for adult populations. The objective of this study was to understand how ocean acidification would affect the initial life stages of the sea urchin Paracentrotus lividus, a common species that is widely distributed in the Mediterranean Sea and the NE Atlantic. The effects of decreased pH (elevated PCO2) were investigated through physiological and molecular analyses on both embryonic and larval stages. Eggs and larvae were reared in Mediterranean seawater at six pH levels, i.e. pHT 8.1, 7.9, 7.7, 7.5, 7.25 and 7.0. Fertilization success, survival, growth and calcification rates were monitored over a 3 day period. The expression of genes coding for key proteins involved in development and biomineralization was also monitored. Paracentrotus lividus appears to be extremely resistant to low pH, with no effect on fertilization success or larval survival. Larval growth was slowed when exposed to low pH but with no direct impact on relative larval morphology or calcification down to pHT 7.25. Consequently, at a given time, larvae exposed to low pH were present at a normal but delayed larval stage. More surprisingly, candidate genes involved in development and biomineralization were upregulated by factors of up to 26 at low pH. Our results revealed plasticity at the gene expression level that allows a normal, but delayed, development under low pH conditions.

First experiments on the maternal transfer of metals in the cuttlefish Sepia officinalis

The aim of this study was to provide a first insight on the incorporation of eight metals in the eggs of the cuttlefish Sepia officinalis via maternal transfer, using radiotracer techniques ((110m)Ag, (241)Am, (109)Cd, (60)Co, (134)Cs, (54)Mn, (75)Se and (65)Zn). The cuttlefish was fed daily with radiolabelled crabs for two weeks; it then started to spawn every three days. Among the eight tracers, only (110m)Ag, (75)Se and (65)Zn were significantly transferred to the eggs. The radiotracer distribution among the egg compartments showed that (75)Se and (65)Zn were accumulated mainly in the vitellus whereas (110m)Ag was found in similar proportion in the vitellus and the eggshell. During the embryonic development, (75)Se and (65)Zn contained in the vitellus were progressively transferred to the embryo, likely to supply its metabolic needs in these essential elements. Although it has no known biological functions, Ag contained in both vitellus and eggshell was also transferred to the embryo. Overall, our results showed that transfer of Ag, Se, and Zn does actually occur from a female cuttlefish to its eggs, at least during the last two weeks before spawning.

Differential bioaccumulation behaviour of Ag and Cd during the early development of the cuttlefish Sepia officinalis

Cuttlefish eggs were exposed to background concentrations of dissolved Ag and Cd, using the radiotracers (110m)Ag and (109)Cd. At different time of the embryonic development (50 days), some eggs were placed in non-contaminating conditions. During the experiment, the uptake and depuration kinetics, and distribution of these metals among the egg compartments (i.e. eggshell, vitellus, peri-vitelline fluid and embryo) were assessed. In parallel, experiments were conducted with sub-lethal concentrations of stable Ag and Cd (2 and 1 microgl(-1), respectively) to compare the metal behaviour at higher concentrations. From the spawning date up to 1 month of development, both metals were taken up efficiently by the eggs, reaching load/concentration ratio (LCR) of 1059+/-75 and 239+/-22 for (110m)Ag and (109)Cd, respectively. From this time onwards, (110m)Ag activity continued to increase in eggs, whereas (109)Cd kinetics displayed a significant decrease. Whatever the developmental stage, Cd was mainly associated with the eggshell all along the exposure experiment. In addition, both stable Cd concentrations and (109)Cd LCR remained low in the embryo all along the embryonic development, indicating that the eggshell acted as an efficient shield against the penetration of this metal. In contrast, (110m)Ag passed through the eggshell from day 30 onwards and was then accumulated in the embryo, which contained more than 40% of the whole egg metal burden at the end of the exposure period. In depuration conditions, it is noteworthy that Ag continued to accumulate in the embryo indicating translocation processes from the eggshell and a high affinity of the metal for the embryo tissues. Overall our results showed that at day 30 of the embryonic development the cuttlefish eggshell becomes permeable to Ag but not to Cd. Exposure to stable metals confirmed the saturation capacities of the eggshell for Cd and the Ag penetration properties.

Phylogenetic consistencies among chondrichthyan and teleost fishes in their bioaccumulation of multiple trace elements from seawater.

Multi-tracer experiments determined the accumulation from seawater of selected radioactive trace elements (Mn-54, Co-60, Zn-65, Cs-134, Am-241, Cd-109, Ag-110m, Se-75 and Cr-51) by three teleost and three chondrichthyan fish species to test the hypothesis that these phylogenetic groups have different bioaccumulation characteristics, based on previously established contrasts between the carcharhiniform chondrichthyan Scyliorhinus canicula (dogfish) and the pleuronectiform teleost Psetta maxima (turbot). Discriminant function analysis on whole body: water concentration factors (CFs) separated dogfish and turbot in two independent experiments. Classification functions grouped the perciform teleosts, seabream (Sparus aurata) and seabass (Dicentrarchus labrax), with turbot and grouped the chondrichthyans, undulate ray (Raja undulata; Rajiformes) and spotted torpedo (Torpedo marmorata; Torpediniformes), with dogfish, thus supporting our hypothesis. Hierarchical classificatory, multi-dimensional scaling and similarity analyses based on the CFs for the nine radiotracers, also separated all three teleosts (that aggregated lower in the hierarchy) from the three chondrichthyan species. The three chondrichthyans were also more diverse amongst themselves compared to the three teleosts. Particular trace elements that were more important in separating teleosts and chondrichthyans were Cs-134 that was elevated in teleosts and Zn-65 that was elevated in chondrichthyans, these differences being due to their differential rates of uptake rather than loss. Chondrichthyans were also higher in Cr-51, Co-60, Ag-110m and Am-241, whereas teleosts were higher only in Mn-54. These contrasts in bioaccumulation patterns between teleosts and chondrichthyans are interpreted in the context of both proximate causes of underlying differences in physiology and anatomy, as well as the ultimate cause of their evolutionary divergence over more than 500million years before present (MyBP). Our results and interpretation point to the possibility that radiation exposure regimes may be influenced by phylogeny, with implications for the adequacy of the marine reference organism approach in marine environmental protection.

New Insight into Marine Alkaloid Metabolic Pathways: Revisiting Oroidin Biosynthesis.

Sponge natural product biosynthesis: A highly sensitive in vivo protocol based on (14)C radiolabeled precursors and beta-imager autoradiography allowed the unraveling of the origin of the pyrrole 2-aminoimidazole-containing key biosynthetic intermediate oroidin. Proline and lysine are now proposed as the early precursors of the pyrrole and the 2-aminoimidazole moieties of oroidin respectively.

Bioaccumulation and detoxification processes of Hg in the king scallop Pecten maximus: field and laboratory investigations

Hg bioaccumulation was investigated in the king scallop Pecten maximus in the laboratory and in the field. In controlled conditions, scallops were exposed to (203)Hg through seawater, sediment and food in order to determine its uptake and depuration kinetics. In the field, Hg and metallothionein (MT) concentrations and the metal subcellular distribution were determined in scallops from two sites of the Bay of Seine (France) differently subjected to the Seine river inputs. While Hg concentrations in the whole soft parts and kidneys (viz. the highest accumulator organ) did not differ between scallops from both sites (74-156 ng g(-1)dry wt), they did for the digestive gland and the gills. According to the experimental results, a higher exposure to dissolved Hg might occur in the site close to the estuary whereas Hg would be mainly incorporated via the dietary pathway in the site away from the estuary. Within the cells of wild scallops, Hg was mainly associated to the cytosolic fraction in the digestive gland and gills (60-100%). However, the lack of relationship between Hg and MT levels suggests that Hg detoxification in P. maximus involves other, non-MT, soluble compounds. In kidneys, insoluble compounds played an important role in Hg sequestration. No effect of scallop age was observed neither on Hg and MT concentrations nor on the subcellular distribution of the metal. Finally, according to FAO/WHO recommendations (maximum weekly Hg intake), our results clearly indicate that the low Hg contents in the edible part of the king scallops from the Bay of Seine prevent any risk for human consumers.

Biokinetics of Hg and Pb accumulation in the encapsulated egg of the common cuttlefish Sepia officinalis: Radiotracer experiments

Uptake and depuration kinetics of dissolved (203)Hg and (210)Pb were determined during the entire embryonic development of the eggs of the cuttlefish, Sepia officinalis (50d at 17 degrees C). (203)Hg and (210)Pb were accumulated continuously by the eggs all along the development time reaching load/concentration ratio (LCR) of 467+/-43 and 1301+/-126g, respectively. During the first month, most of the (203)Hg and (210)Pb remained associated with the eggshell indicating that the latter acted as an efficient shield against metal penetration. From this time onwards, (203)Hg accumulated in the embryo, indicating that it passed through the eggshell, whereas (210)Pb did not cross the chorion during the whole exposure time. It also demonstrated that translocation of Hg associated with the inner layers of the eggshell is a significant source of exposure for the embryo. This study highlighted that the maturing embryo could be subjected to the toxic effects of Hg in the coastal waters where the embryonic development is taking place.

Delineation of Pb contamination pathways in two Pectinidae: The variegated scallop Chlamys varia and the king scallop Pecten maximus

Bioaccumulation of Pb was determined in Chlamys varia and Pecten maximus exposed to (210)Pb via seawater, food and sediment. Both scallops readily concentrated dissolved Pb with whole-body 7-d concentration factors of 250+/-40 and 170+/-70, respectively. In both species, more than 70% of Pb taken up from seawater was strongly retained within tissues (biological half-life>1.5 month) whereas Pb ingested with phytoplankton was poorly assimilated (<20%). As P. maximus lives buried in the sediment, this exposure pathway was assessed and showed low bioaccumulation efficiency for sediment-bound Pb (transfer factor <0.015). Despite the poor transfer efficiency of Pb from food and sediment, the use of a global bioaccumulation model indicated that the particulate pathway (food and/or sediment) constituted the major bioaccumulation route of Pb in both scallops. Whatever the exposure pathway, the digestive gland and kidneys always played a major role in Pb accumulation. In scallop tissues, Pb was predominantly associated with the insoluble subcellular fraction, suggesting a low bioavailability of Pb for scallop consumers.

Comparative bioaccumulation kinetics of trace elements in Mediterranean marine sponges

While marine organisms such as bivalves, seagrasses and macroalgae are commonly used as biomonitors for the environment pollution assessment, widely distributed sponges received little attention as potential helpful species for monitoring programmes. In this study, the trace element and radionuclide bioaccumulation and retention capacities of some marine sponges were estimated in a species-comparative study using radiotracers technique. Six Mediterranean species were exposed to background dissolved concentrations of (110m)Ag, (241)Am, (109)Cd, (60)Co, (134)Cs, (54)Mn, (75)Se and (65)Zn allowing the assessment of the uptake and depuration kinetics for selected elements. Globally, massive demosponges Agelas oroides, Chondrosia reniformis and Ircinia variabilis displayed higher concentration factor (CF) than the erectile ones (Acanthella acuta, Cymbaxinella damicornis, Cymbaxinella verrucosa) at the end of exposure, suggesting that the morphology is a key factor in the metal bioaccumulation efficiency. Considering this observation, two exceptions were noted: (1) A. acuta reached the highest CF for (110m)Ag and strongly retained the accumulated metal without significant Ag loss when placed in depuration conditions and (2) C. reniformis did not accumulate Se as much as A. oroides and I. variabilis. These results suggest that peculiar metal uptake properties in sponges could be driven by specific metabolites or contrasting biosilification processes between species, respectively. This study demonstrated that sponges could be considered as valuable candidate for biomonitoring metal contamination but also that there is a need to experimentally highlight metal-dependant characteristic among species.

Differential bioaccumulation of 134Cs in tropical marine organisms and the relative importance of exposure pathways

Bioaccumulation of (134)Cs was determined in 5 tropical marine species: three bivalves (the oysters Isognomon isognomum and Malleus regula, and the clam Gafrarium pectinatum), one decapod (shrimp Penaeus stylirostris) and one alga (Lobophora variegata). Marine organisms were exposed to the radionuclides via different pathways: seawater (all of them), food (shrimp and bivalves) and sediment (bivalves). Our results indicate that the studied tropical species accumulate Cs similarly than species from temperate regions whereas retention capacities seems to be greater in the tropical species. Bioaccumulation capacities of the two oysters were similar for all the exposure pathways. The alga, and to a lesser extent the shrimp, concentrated dissolved Cs more efficiently than the bivalves (approx. 14 and 7 times higher, respectively). Assimilation efficiencies of Cs in bivalves and shrimp after a single feeding with radiolabelled food were comprised between 7.0 ± 0.4 and 40.7 ± 4.3%, with a variable retention time (half-life -Tb1/2- ranging from 16 ± 3 to 89 ± 55 d). Although the clam lives buried in the sediment, this exposure pathway resulted in low bioaccumulation efficiency for sediment-bound Cs (mean transfer factor: 0.020 ± 0.001) that was lower than the two oyster species, which are not used to live in this media (0.084 ± 0.003 and 0.080 ± 0.005). Nonetheless, Cs accumulated from sediment was similarly absorbed (61.6 ± 9.7 to 79.2 ± 2.3%) and retained (Tb1/2: 37 ± 2 to 58 ± 25 d) for the three bivalves species. Despite the poor transfer efficiency of Cs from food, the use of a global bioaccumulation model indicated that the trophic pathways was the main uptake route of Cs in the bivalves and shrimp. In shelled organisms, shells played a non-negligible role in Cs uptake, and their composition and structure might play a major role in this process. Indeed, most of the Cs taken up from seawater and sediment was principally located on the hard parts of the bivalves and shrimp, with the exception of G. pectinatum, where Cs was mainly distributed in the soft-parts.