Shell Mineralization Research Articles

Coastal acidification impacts on shell mineral structure of bivalve mollusks.

Ocean acidification is occurring globally through increasing CO 2 absorption into the oceans creating particular concern for calcifying species. In addition to ocean acidification, near shore marine habitats are exposed to the deleterious effects of runoff from acid sulfate soils which also decreases environmental pH. This coastal acidification is being exacerbated by climate change‐driven sea‐level rise and catchment‐driven flooding. In response to reduction in habitat pH by ocean and coastal acidification, mollusks are predicted to produce thinner shells of lower structural integrity and reduced mechanical properties threatening mollusk aquaculture. Here, we present the first study to examine oyster biomineralization under acid sulfate soil acidification in a region where growth of commercial bivalve species has declined in recent decades. Examination of the crystallography of the shells of the Sydney rock oyster, Saccostrea glomerata, by electron back scatter diffraction analyses revealed that the signal of environmental acidification is evident in the structure of the biomineral. Saccostrea glomerata, shows phenotypic plasticity, as evident in the disruption of crystallographic control over biomineralization in populations living in coastal acidification sites. Our results indicate that reduced sizes of these oysters for commercial sale may be due to the limited capacity of oysters to biomineralize under acidification conditions. As the impact of this catchment source acidification will continue to be exacerbated by climate change with likely effects on coastal aquaculture in many places across the globe, management strategies will be required to maintain the sustainable culture of these key resources.

Diclofenac affects early embryo development in the marine bivalve Mytilus galloprovincialis

Diclofenac-DCF, one of the most widely prescribed non-steroidal anti-inflammatory drug, is globally detected in environmental compartments. Due to its occurrence in freshwater and potential impact on aquatic organisms, it has been added to the watch list of chemicals in the EU Water Directive; consequently, research on the impact of DCF in model aquatic organisms has great regulatory implications towards ecosystem health. DCF is also detected in coastal waters at concentrations from ng/L to 1 μg/L, as well as in marine organisms, such as the mussel Mytilus. Increasing evidence indicates that environmental concentrations of DCF have multiple impacts in adult mussels. Moreover, in M. galloprovincialis, DCF has been shown to affect early embryo development.The developmental effects of DCF in mussels were further investigated. DFC (1 and 10 μg/L) was added at different times post-fertilization (30 min and 24 hpf) and the effects were compared in the 48 hpf embryotoxicity assay. Shell mineralization and morphology were investigated by polarized light microscopy, X-Ray Spectrometry-XRD and Scanning Electron Microscopy-SEM. Transcriptional profiles of 12 selected genes physiologically regulated across early embryo development were assessed at 24 and 48 hpf.DCF induced shell malformations, irrespectively of concentration and time of exposure. DCF phenotypes were characterized by convex hinges, undulated edges, fractured shells. However, no changes in biomineralization were observed. DCF affected gene transcription at both times pf, in particular at 1 μg/L. The most affected genes were those involved in early shell formation (CS, CA, EP) and biotransformation (ABCB, GST). The results confirm that Mytilus early development represents a significant target for environmental concentrations of DCF. These data underline how the standard embryotoxicity assay, in combination with a structural and transcriptomic approach, represents a powerful tool for evaluating the early impact of pharmaceuticals on mussel embryos, and identification of the possible underlying mechanisms of action.

Hemocytes in the extrapallial space of Pinctada fucata are involved in immunity and biomineralization

In bivalves, the mantle tissue secretes organic matrix and inorganic ions into the extrapallial space (EPS) to form the shells. In addition, more and more evidences indicate the participation of hemocytes in shell mineralization, but no direct evidence has been reported that verifies the presence of hemocytes in the EPS, and their exact roles in biomineralization remain uncertain. Here, we identified hemocytes from the EPS of Pinctada fucata. Numerous components involved in cellular and humoral immunity were identified by proteome analysis, together with several proteins involved in calcium metabolism. The hemocytes exerted active phagocytosis and significantly upregulated the expression of immune genes after immune stimulation. A group of granulocytes were found to contain numerous calcium-rich vesicles and crystals, which serve as a calcium pool. During shell regeneration, some genes involved in calcium metabolism are upregulated. Strikingly, most of the shell matrix proteins were absent in the hemocytes, suggesting that they might not be solely responsible for directing the growth of the shell. Taken together, our results provided comprehensive information about the function of hemocytes in immunity and shell formation.

Geochemical and Crystallographic Study of Turbo Torquatus (Mollusca: Gastropoda) From Southwestern Australia

AbstractShells of the marine gastropod Turbo torquatus were sampled from three different locations along the Western Australian coastline, namely Marmion Lagoon (31°S), Rottnest Island (32°S), and Hamelin Bay (34°S). Marmion Lagoon and Rottnest Island have similar sea surface temperature ranges that are ∼1°C warmer than Hamelin Bay, with all sites influenced by the warm southward flowing Leeuwin Current. The shells were characterized using crystallographic, spectroscopic, and geochemical analyses. Shell mineral composition varies between the three sites suggesting the influence of sea surface temperature, oxygen consumption, and/or bedrock composition on shell mineralogy and preferential incorporation and/or elemental discrimination of Mg, P, and S. Furthermore, T. torquatus was found to exert control over the incorporation of most, if not all, the elements measured here, suggesting strong biological regulation. At all levels of testing, the concentrations of Li varied significantly, which indicates that this trace element may not be a suitable environmental proxy. Variation in Sr concentration between sites and between specimens reflects combined environmental and biological controls suggesting that Sr/Ca ratios in T. torquatus cannot be used to estimate sea surface temperature without experimentally accounting for metabolic and growth effects. The mineral composition and microstructure of T. torquatus shells may help identify sea surface temperature variations on geological time scales. These findings support the previously hypothesized involvement of an active selective pathway across the calcifying mantle of T. torquatus for most, if not all, the elements measured here.

MSP22.8 is a protease inhibitor-like protein involved in shell mineralization in the edible mussel Mytilus galloprovincialis.

The mussel shell protein 22.8 (MSP22.8) is recognized by a monoclonal antibody (M22.8) directed against larvae of the mussel Mytilus galloprovincialis. After being secreted by cells of the mantle‐edge epithelium into the extrapallial (EP) space (the gap between the mantle and the shell), the protein is detected in the extrapallial fluid (EPF) and EP hemocytes and finally becomes part of the shell matrix framework in adult specimens of M. galloprovincialis. In the work described here, we show how MSP22.8 is detected in EPF samples from different species of mussels (M. galloprovincialis, Mytilus edulis, and Xenostrobus securis), and also as a shell matrix protein in M. galloprovincialis, Mytilus chilensis, and Perna canaliculus. A multistep purification strategy was employed to isolate the protein from the EPF, which was then analyzed by mass spectrometry in order to identify it. The results indicate that MSP22.8 is a serpin‐like protein that has great similarity with the protease inhibitor‐like protein‐B1, reported previously for Mytilus coruscus. We suggest that MSP22.8 is part of a system offering protection from proteolysis during biomineralization and is also part of the innate immune system in mussels.

Characterization of pinctada fucata mantle gene 2 (pfmg2) in pearl oyster

ABSTRACTBiomineralization is a complicated process which involves multiple gene products in Pinctada fucata. Here, Pinctada fucata mantle gene 2 (pfmg2) was characterized in P. fucata. The putative PFMG2 protein has a Calponin Homology (CH) domain and an EF hand that can bind calcium. PFMG2 shares homology with transgelin in other species. Multi-tissue reverse transcriptase polymerase chain reaction (RT-PCR) indicates that pfmg2 mRNA is expressed ubiquitously, but its expression is high in the mantle tissue of P. fucata. Furthermore, in situ hybridization shows that pfmg2 localized at the edge of the mantle. Our results suggest that PFMG2 plays an important role in the shell mineralization process in pearl oyster. The analysis and the functional characterization of PFMG2 in P. fucata will provide new insight into the mineralization process.

On Mineral Retrosynthesis of a Complex Biogenic Scaffold

Synergistic relations between organic molecules and mineral precursors regulate biogenic mineralization. Given the remarkable material properties of the egg shell as a biogenic ceramic, it serves as an important model to elucidate biomineral growth. With established roles of complex anionic biopolymers and a heterogeneous organic scaffold in egg shell mineralization, the present study explores the regulation over mineralization attained by applying synthetic polymeric counterparts (polyethylene glycol, poly(acrylic acid), poly(aspartic acid) and poly(4-styrenesulfonic acid-co-maleic acid)) as additives during remineralization of decalcified eggshell membranes. By applying Mg2+ ions as a co-additive species, mineral retrosynthesis is achieved in a manner that modulates the polymorph and structure of mineral products. Notable features of the mineralization process include distinct local wettability of the biogenic organic scaffold by mineral precursors and mineralization-induced membrane actuation. Overall, the form, structure and polymorph of the mineralization products are synergistically affected by the additive and the content of Mg2+ ions. We also revisit the physicochemical nature of the biomineral scaffold and demonstrate the distinct spatial distribution of anionic biomolecules associated with the scaffold-mineral interface, as well as highlight the hydrogel-like properties of mammillae-associated macromolecules.

Insights from sodium into the impacts of elevated pCO2 and temperature on bivalve shell formation

Ocean acidification and warming are predicted to affect the ability of marine bivalves to build their shells, but little is known about the underlying mechanisms. Shell formation is an extremely complex process requiring a detailed understanding of biomineralization processes. Sodium incorporation into the shells would increase if bivalves rely on the exchange of Na+/H+ to maintain homeostasis for shell formation, thereby shedding new light on the acid-base and ionic regulation at the calcifying front. Here, we investigated the combined effects of seawater pH (8.1, 7.7 and 7.4) and temperature (16 and 22°C) on the growth and sodium composition of the shells of the blue mussel, Mytilus edulis, and the Yesso scallop, Patinopecten yessoensis. Exposure of M. edulis to low pH (7.7 and 7.4) caused a significant decrease of shell formation, whereas a 6°C warming significantly depressed the rate of shell growth in P. yessoensis. On the other hand, while the amount of Na incorporated into the shells of P. yessoensis did not increase in acidified seawater, an increase of Na/Cashell with decreasing pH was observed in M. edulis, the latter agreeing well with the aforementioned hypothesis. Moreover, a combined analysis of the shell growth and sodium content provides a more detailed understanding of shell formation processes. Under acidified conditions, mussels may maintain more alkaline conditions favorable for calcification, but a significant decrease of shell formation indicates that the mineralization processes are impaired. The opposite occurs in scallops; virtually unaffected shell growth implies that shell mineralization functions well. Finding of the present study may pave the way for deciphering the mechanisms underlying the impacts of ocean acidification and warming on bivalve shell formation.

Morphometrics and mineral composition of shell whorls in three species of giant African snails from Abeokuta, Nigeria

Archachatina marginata (Swainson), Achatina achatina (Linnaeus) and Achatina fulica Bowdich were examined for their shell whorl morphometrics (20 snails per species) and the calcium, iron and phosphorus content in the shell whorls (10 snails per species). The species differed in the degree of shell mineralization (the highest in the medium-sized A. achatina, intermediate in the smallest-sized A. fulica and the smallest in the largest-sized A. marginata) which was not ageor environment-dependent, since all the experimental snails were 12 months old adults of 6 whorls, originating from the same snail farm. In all three species the degree of shell mineralization decreased from the apical (oldest) to the body (youngest) whorl, thus depending entirely on the whorl’s age. The inter-specific differences in calcium content should be considered when using shells for medical or agricultural purposes.

Dietary Probiotics Affect Gastrointestinal Microbiota, Histological Structure and Shell Mineralization in Turtles.

Probiotics are widely used in nutrition, and their mode of action is intensively studied in mammals and birds; however, it is almost unknown in reptiles. In the present study, Trachemys scripta scripta and Sternotherus odoratus were used to assess the effects of dietary probiotics on chelonian gastrointestinal tract microecology. In the first, 20-week experiment, 40 young T. s. scripta were randomly distributed to four experimental groups: 1st, (CON)–with no additives; 2nd, (SSPA) with Bacillus subtilis PB6; 3rd, (MSP)–with multiple strain probiotic; and 4th, (SSPB) with Bacillus subtilis C-3102. The first study has shown that SSPA and MSP decreased the numbers of total bacteria, Enterobacteriace, Staphylococcus sp. and Streptococcus sp. excreted to water and increased the villous height and mucosa thickness in duodenum. SSPB improved the duodenal microstructure; however, it also increased numbers of kanamycin and vancomycin resistant bacteria, Staphylococcus sp. and Streptococcus sp., in water. In the second, 52-week experiment, 30 S. odoratus were randomly assigned to three dietary treatments. CON, SSPA and MSP groups. The MSP preparation increased the body weight gain, crude ash, Ca and P share in the turtles’ shells. Both probiotics affected duodenal histomorphology. SSPA decreased the villous height, while MSP increased the villous height and mucosa thickness, and decreased the crypt depth. SSPA decreased the concentrations of bacteria excreted to water. In the case of intestinal microbiota, bacteria suppressing effects were observed in the case of both probiotics. MSP increased the number of Bifidobacterium sp. and Lactobacillus sp./Enteroccoccus sp., and decreased the number of Clostridium perfringens and Campylobacter sp. in the small intestine. In the large intestine it lowered, amongst others, Bacteroides–Pervotella cluster, Clostridium leptum subgroup and Clostridium perfringens numbers. The above-mentioned results suggest that probiotics are useful in turtle nutrition due to their positive effects on growth performance, shell mineralization, duodenal histomorphology and microbiota.

Temporal Tracking of Mineralization and Transcriptional Events Associated with Shell Formation During the Early Life History of Pearl Oyster Pinctada maxima

Background: Molluscan larval ontogeny is a highly conserved process comprising three principal developmental stages; trochophore, veliger and metamorphosis into the juvenile. A characteristic that is unique to each of these stages is shell design, termed prodissoconch I, prodissoconch II and dissoconch in bivalves. These shells vary in morphology, mineralogy and microstructure. The discrete temporal transitions in shell biomineralization between these larval stages are utilized in this study to investigate transcriptional involvement in several distinct biomineralization events. Methods: Scanning electron microscopy, X-ray diffraction and microarray differential gene expression analysis were used to document temporal transitions in morphology, mineralogy and microstructure of larval shells and the genes directing their biomineralization. Results: P. maxima larvae and juveniles collected throughout post-embryonic ontogenesis are described in terms of mineralogy and microstructure of each shelled stage as well as establishing a timeline for transitions in biomineralization. P. maxima larval samples most representative of these biomineralization distinctions and transitions were analyzed for differential gene expression with the microarray platform PmaxArray 1.0. A number of known shell matrix genes and novel transcripts are reported as differentially expressed in correlation to the mineralization events of P. maxima larval ontogeny. However, only a single transcript, PM066, was noted as being expressed before and after the transition to the adult shell design. No other known/putative adult shell matrix genes from P. maxima were detected in association with the larval shells prodissococh I and II, suggesting that their expression is either below detection limits or an almost entirely different set of genes is potentially responsible for larval and adult shell mineralization. Conclusion: This interdisciplinary investigation has linked the shell developments of P. maxima larval ontogeny with corresponding gene expression profiles, furthering the elucidation of bivalve development and shell biomineralization. Keywords: Biomineralization, gene expression, microarray, mollusc, ontogeny, pearl oyster.

Biomaterials. Crystalline eyes of chitons inspire materials scientists.

Mollusk makes hundreds of eyes from shell mineral.

Data set for the proteomic inventory and quantitative analysis of chicken eggshell matrix proteins during the primary events of eggshell mineralization and the active growth phase of calcification.

Chicken eggshell is a biomineral composed of 95% calcite calcium carbonate mineral and of 3.5% organic matrix proteins. The assembly of mineral and its structural organization is controlled by its organic matrix. In a recent study [1], we have used quantitative proteomic, bioinformatic and functional analyses to explore the distribution of 216 eggshell matrix proteins at four key stages of shell mineralization defined as: (1) widespread deposition of amorphous calcium carbonate (ACC), (2) ACC transformation into crystalline calcite aggregates, (3) formation of larger calcite crystal units and (4) rapid growth of calcite as columnar structure with preferential crystal orientation. The current article detailed the quantitative analysis performed at the four stages of shell mineralization to determine the proteins which are the most abundant. Additionally, we reported the enriched GO terms and described the presence of 35 antimicrobial proteins equally distributed at all stages to keep the egg free of bacteria and of 81 proteins, the function of which could not be ascribed.

Quantitative proteomics provides new insights into chicken eggshell matrix protein functions during the primary events of mineralisation and the active calcification phase

Eggshell is a bioceramic composed of 95% calcium carbonate mineral and 3.5% organic matrix. Its structural organisation is controlled by its organic matrix. We have used quantitative proteomics to study four key stages of shell mineralisation: 1) widespread deposition of amorphous calcium carbonate (ACC), 2) ACC transformation into crystalline calcite aggregates, 3) formation of larger calcite crystal units and 4) development of a columnar structure with preferential calcite crystal orientation. This approach explored the distribution of 216 shell matrix proteins found at the four stages. Variations in abundance according to these calcification events were observed for 175 proteins. A putative function related to the mineralisation process was predicted by bioinformatics for 77 of them and was further characterised.We confirmed the important role of lysozyme, ovotransferrin, ovocleidin-17 and ovocleidin-116 for shell calcification process, characterised major calcium binding proteins (EDIL3, ALB, MFGE8, NUCB2), and described novel proteoglycans core proteins (GPC4, HAPLN3). We suggest that OVAL and OC-17 play a role in the stabilisation of ACC. Finally, we report proteins involved in the regulation of proteins driving the mineralisation. They correspond to numerous molecular chaperones including CLU, PPIB and OCX21, protease and protease inhibitors including OVM and CST3, and regulators of phosphorylation.

Amorphous calcium carbonate controls avian eggshell mineralization: A new paradigm for understanding rapid eggshell calcification.

Avian eggshell mineralization is the fastest biogenic calcification process known in nature. How this is achieved while producing a highly crystalline material composed of large calcite columnar single crystals remains largely unknown. Here we report that eggshell mineral originates from the accumulation of flat disk-shaped amorphous calcium carbonate (ACC) particles on specific organic sites on the eggshell membrane, which are rich in proteins and sulfated proteoglycans. These structures known as mammillary cores promote the nucleation and stabilization of a amorphous calcium carbonate with calcitic short range order which predetermine the calcite composition of the mature eggshell. The amorphous nature of the precursor phase was confirmed by the diffuse scattering of X-rays and electrons. The nascent calcitic short-range order of this transient mineral phase was revealed by infrared spectroscopy and HRTEM. The ACC mineral deposited around the mammillary core sites progressively transforms directly into calcite crystals without the occurrence of any intermediate phase. Ionic speciation data suggest that the uterine fluid is equilibrated with amorphous calcium carbonate, throughout the duration of eggshell mineralization process, supporting that this mineral phase is constantly forming at the shell mineralization front. On the other hand, the transient amorphous calcium carbonate mineral deposits, as well as the calcite crystals into which they are converted, form by the ordered aggregation of nanoparticles that support the rapid mineralization of the eggshell. The results of this study alter our current understanding of avian eggshell calcification and provide new insights into the genesis and formation of calcium carbonate biominerals in vertebrates.

Deposition of annual growth lines in the apex of the common limpet (Patella vulgata) from Shetland Islands, UK and Norway: Evidence from field marking and shell mineral content of annual line deposition

The abundance, accessibility and value of limpets as a source of food and bait for coastal peoples have resulted in their high frequency in shell middens worldwide. The limpet Patella vulgata is found in middens from the Mediterranean to Norway, and morphometric and sclerochronological analyses of its shell can provide insight into harvesting patterns and paleoenvironmental variables valuable in reconstructing climate. Previous work with P. vulgata has relied on lines on the exterior of the shell, or on lines exiting on the shell surface in shell cross-section, as annual or sub-annual markers. Shell damage may compromise these lines and limit the use of some shells, but growth lines are also found in the better-preserved shell apex. We investigated whether the growth lines in the apex of P. vulgata from two locations in Northern Europe are annual using calcein-marking and recapture. Investigations were performed at one site in the Shetland Islands (UK) and at one site at the northern limit of P. vulgata's range in Northern Norway. We also used laser ablation to measure the concentration of minerals in the shells of two individuals from Shetland to determine if patterns of minerals suggested as bioproxies for temperature and productivity varied annually. All individuals deposited one growth line in the apex during their year in the field, and the lowest ratios of Sr/Ca and Ba/Ca and to a lesser extent Mg/Ca were coincident with annual lines on the shells from Shetland. Growth at both sites was modelled using the von Bertalanffy growth function, and apex growth was nearly five times faster in Shetland than in Norway, probably a result of differences in temperature between the two locations.

Shell Mineral Composition of Artificially and Naturally Incubated Eggs of Ornate Box Turtles (Terrapene ornata ornata)

Ornate box turtles (Terrapene ornata ornata) are a threatened species in Illinois. In 2011, a head-start program initiated by the United States Fish and Wildlife Service Upper Mississippi River National Wildlife and Fish Refuge was conducted at two zoological facilities. Variable success in artificial incubation and hatching of eggs was noted between the institutions. In 2012, artificially incubated eggs from eight clutches (n = 28) and nine batched clutches of unknown total egg count incubated in situ had the hatched egg shells submitted for mineral content analysis. All minerals showed significant differences, except sulfur. Future analysis of fertile and infertile eggs or successful and unsuccessful hatches would provide additional interpretation for use of shell mineral analysis for captive propagation of this species.

Quantitative proteomics and bioinformatic analysis provide new insight into protein function during avian eggshell biomineralization.

The calcitic avian eggshell protects the developing embryo and, moreover, ensures that the nutritious table egg remains free of pathogens. The eggshell is formed by nucleation upon a fibrous scaffold (the eggshell membranes) followed by an interaction between the growing mineral crystals and the shell organic matrix. This interaction leads to a highly ordered shell microstructure and texture which contribute to its exceptional mechanical properties. Shell mineralization occurs in three distinct phases of calcification (initiation, growth and termination), which are associated with distinct populations of matrix proteins that are secreted into the acellular uterine fluid as modulators of the process. The recent development of high-throughput methods has led to the identification of many proteins in the shell, but little is known concerning their role in shell formation. In order to determine precisely the importance of particular proteins relative to eggshell mineralization, this project used qualitative and quantitative proteomics of the uterine fluid constituents, coupled with bioinformatic analysis, to predict the functional role of proteins secreted at each of the three main stages of shell calcification. Besides its relevance to food production and to hen reproduction, eggshell calcification is furthermore a relevant model for studying calcium carbonate biomineralization on a two-dimensional membrane support. Better understanding of this process will provide insight into the fabrication of ceramics at ambient pressure and temperature.

Effect of hen age, environmental temperature, and oviposition time on egg shell quality and egg shell and serum mineral contents in laying and broiler breeder hens

The aim of the study was to evaluate egg shell quality characteristics, mineral content in the egg shell, and serum mineral concentration during the egg formation process as influenced by oviposition time and ambient temperature at the beginning and end of the laying cycle in laying hens and broiler breeders. Egg shell quality deteriorated significantly at the higher temperature and was lower in broiler breeders than in laying hens. The Ca (P = 0.047) and P (P = 0.018) contents of the egg shell were significantly higher at the higher temperature. The highest (P = 0.028) shell Ca content (352 g/kg) was in eggs laid in the morning, but the highest P (P = 0.030; 1.43 g/kg) and Mg (P = 0.001; 3.88 g/kg) contents were in eggs laid in the afternoon. Broiler breeder egg shells contained significantly more P (P = 0.004) and Mg (P = 0.001) than did those from laying hens. Serum Ca and P levels remained constant throughout the day whereas serum Mg and Zn levels decreased, the rate of decrease in Zn content being the same in all treatments, but with the amounts being greater in laying hens than broiler breeders. The results demonstrate that shell quality characteristics are more severely affected by different factors implemented in this trial compared to the shell mineral composition and especially compared to the serum mineral content.  

Non-equilibrium fractionation of stable carbon isotopes in chemosynthetic mussels

Chemosynthetic bivalves from deep-sea hydrothermal vents exploit the energy derived from chemical compounds, such as methane, sulfide or hydrogen, using symbiotic bacteria that are able to fix inorganic carbon. Available chemical resources in their habitat vary widely at various scales, from the vent field scale to the micro-habitat scale. Parallel to this environmental heterogeneity, Bathymodiolus species are considered to be flexible in their energy acquisition pathways.The goal of this study was to determine whether the isotopic compositions archived in the shells of hydrothermal vent mussels could trace chemical energy sources and their variability over spatial and temporal scales. Two different species (Bathymodiolus azoricus and Bathymodiolus thermophilus) inhabiting three vent fields with contrasted geochemical features on the Mid Atlantic Ridge (MAR; Rainbow and Menez Gwen) and the East Pacific Rise (EPR; 9°47′N), were considered for carbon isotopes and growth rate variation along the shell length. The study revealed that 13C fractionation between shells and seawater is higher than expected from calcite–bicarbonate equilibrium fractionation, suggesting a significant influence of the chemosynthetic pathway on the shell composition. Furthermore, significant differences in δ13Cshell fractionation with respect to seawater are observed between sites and habitats of the two MAR vent fields, suggesting that different chemosynthetic pathway (e.g. methanotrophic and thiotrophic) could lead to variable enrichments of the shell in 13C. Mussels supposed to rely more largely on methanotrophy (at Rainbow where free sulfide is unavailable) display a lower δ13Cshell values than mussels relying also on sulfide-oxidizing symbiosis (at Menez Gwen). Variability in δ13Cshell between habitats, or between individuals within the same assemblage, could thus reflect differences in the symbiosis activity at a micro-habitat scale. These isotopic signatures could provide useful information on the relationships between micro-habitat properties, symbiont activity and shell mineralization.