Foamy oysters: vesicular microstructure production in the Gryphaeidae via emulsification

The vesicular microstructure is a very distinctive arrangement of calcite, consisting of hollow cavities (vesicles) of diverse sizes and shapes, usually elongated in the direction of shell thickening. It is uniquely found among living bivalves in a single oyster family, Gryphaeidae. The vesicles are distributed in lenses interleaved with compact foliated layers. We have studied the morphology and distribution of vesicles within the lenses using optical and electron microscopy, and micro-computed tomography. At a small scale, vesicles do not follow a classical von Neumann–Mullins route typical of ideal foams. At a larger scale, the initiation and evolution of a vesicular layer statistically proceed like a foam, with vesicles becoming more numerous, larger and more even in size. In summary, the vesicular material follows a foam-like coarsening to reduce the number of energetically costly interfaces. However, a steady state is never reached because the animal permanently introduces energy in the system by creating new vesicles. The fabrication of the vesicular material is mediated by the production of an emulsion between the extrapallial fluid and the precursor PILP of the calcitic walls within the thin extrapallial space. For this mechanism to proceed, the mantle cells must perform highly sophisticated behaviours of contact recognition and secretion. Accordingly, the vesicular material is under mixed physical–biological control.

Ocean acidification induces subtle shifts in gene expression and DNA methylation in mantle tissue of the Eastern oyster (Crassostrea virginica)

Early evidence suggests that DNA methylation can mediate phenotypic responses of marine calcifying species to ocean acidification (OA). Few studies, however, have explicitly studied DNA methylation in calcifying tissues through time. Here, we examined the phenotypic and molecular responses in the extrapallial fluid and mantle (fluid and tissue at the calcification site) in the Eastern oyster (Crassostrea virginica) exposed to experimental OA over 80 days. Oysters were reared under three experimental pCO2 treatments (‘control’, 580 μatm; ‘moderate OA’, 1000 uatm; ‘high OA’, 2800 μatm) and sampled at 6 time points (24 hours - 80 days). We found that high OA initially induced changes in the pH of the extrapallial fluid (pHEPF) relative to the external seawater, but the magnitude of this difference was highest at 9 days and diminished over time. Calcification rates were significantly lower in the high OA treatment compared to the other treatments. To explore how oysters regulate their extrapallial fluid, gene expression and DNA methylation were examined in the mantle-edge tissue of oysters from day 9 and 80 in the control and high OA treatments. Mantle tissue mounted a significant global molecular response (both in the transcriptome and methylome) to OA that shifted through time. Although we did not find individual genes that were significantly differentially expressed to OA, the pHEPF was correlated with the eigengene expression of several co-expressed gene clusters. A small number of OA-induced differentially methylated loci were discovered, which corresponded with a weak association between OA-induced changes in genome-wide gene body DNA methylation and gene expression. Gene body methylation, however, was not significantly correlated with the eigengene expression of pHEPF correlated gene clusters. These results suggest that in C. virginica, OA induces a subtle response in a large number of genes, but also indicates that plasticity at the molecular level may be limited. Our study highlights the need to re-assess the plasticity of tissue-specific molecular responses in marine calcifiers, as well as the role of DNA methylation and gene expression in mediating physiological and biomineralization responses to OA.

Architecture, Growth Dynamics and Biomineralization of Pulsed Sr-Labelled <i>Katelysia rhytiphora</i> (Mollusca, Bivalvia)

We use pulsed Sr-labelling experiments to visualize growth of aragonitic Katelysia rhytiphora (Mollusca, Bivalvia) shells. The outer compound composite prismatic structure is organized into three orders of prisms, and the inner crossed acicular structure consists of intersecting lamellae. Electron Backscatter Diffraction (EBSD) reveals substantial twinning of the aragonite crystals (> 46 %) and an overall reduced and strategically oriented anisotropy of the Young’s modulus in the whole shell compared to that of monolithic aragonite. All structural orders in both layers are enveloped by an organic sheath and the smallest mineralized units are nanogranules. Total organic contents are 2.2 (outer) and 1.4 wt. % (inner layer) and are, thus, intermediate between those of nacreous and crossed-lamellar shells. Prisms in the outer structure can be correlated to yearly, daily and sub-daily growth rates. Average daily growth rates at the ventral margin for the outer structure are 17 % higher than for the inner crossed acicular structure. The calcification front runs evenly across all structures and architectural orders independently of the current growth rate. Sharply defined transitions from labelled to unlabelled areas in the shell indicate that physiological processes driving calcification have no lag. This suggests that the extrapallial fluid cannot be very voluminous. Narrow increments of varying Sr content within labelled shell, despite constant Sr concentrations in seawater, suggest cyclic metabolic activity during calcification. Micro-Raman spectroscopy maps validate a low impact of high Sr-conditions on the aragonite crystal structure. Identical Sr-enrichment factors for labelled and ambient conditions support models of ion transport via a passive selective pathway to the mantle epithelium followed by calcification via amorphous calcium carbonate.

Transformation of amorphous calcium carbonate nanoparticles into aragonite controlled by ACCBP

Polymorph switching of calcium carbonate controlled by amorphous calcium carbonate-binding protein, an extrapallial fluid (EPF) protein from the pearl oyster, is investigated. The polymorph selection in nacre or pearl growth may be controlled not only by the nucleating template on the matrix but also by the physicochemical effects of EPF proteins.

Mussel shells of <i>Mytilus edulis</i> as bioarchives of the rare earth elements and yttrium distribution in seawater and the potential impact of pH and temperature on the partitioning behaviour

Mussel shells are potential bioarchives of proxies for changes of the physico-chemical conditions in the bivalve's habitat. One such proxy is the distribution of the Rare Earths and Yttrium (REY) in seawater, as REY speciation in seawater is sensitive to pH and temperature variations, due to the impact of these parameters on the activity of CO32− in seawater. We present a new protocol for sample preparation and determination of REY concentrations in bivalve shells, that includes sample treatment with NaOCl followed by REY separation and preconcentration. The data obtained was further used to calculate REY partition coefficients between shells of M. edulis and ambient seawater, and acquired results were then used in the investigation of the potential effects of pH and temperature on REY partitioning. Shells of M. edulis mussels from the North Sea show consistent shale-normalized ("SN") REY patterns that increase from the light REY to the middle REY and decrease from the middle REY to the heavy REY. Despite being different to the general seawater REYSN pattern, the shells still display distinct REY features of seawater such as a negative CeSN anomaly and small positive YSN and GdSN anomalies. Apparent partition coefficients for the REY between the shell and seawater (appDREYshell/seawater) are low and decrease strongly from the light REY (4.04 for La) to the heavy REY (0.34 for Lu). However, assuming that only the free REY3+ are incorporated into the shell, appDREY3&amp;plus;shell/seawater values are higher and rather similar for all REY (102.46 for La; 113.44 for Lu), but show a slight maximum at Tb (199.18). Although the impact of vital effects i.e. REY speciation in a mussel's extrapallial fluid from which the carbonate minerals precipitate, cannot be quantified yet, it appears that M. edulis shells are bioarchives of some REY features of seawater. We modelled the REYSN patterns of a hypothetical mussel shell at pH 8.2 and 7.6 and at temperatures of 25 and 5 °C assuming that only REY3+ are incorporated into the carbonate's crystal lattice. The results suggest that with lower pH, REY concentrations in a shells increase, but with little effect on the shape of the REYSN patterns, while a temperature change has an impact on the REYSN pattern, but only minor effects on REY concentrations.