Meta-analysis suggests negative, but pCO2-specific, effects of ocean acidification on the structural and functional properties of crustacean biomaterials.

Crustaceans comprise an ecologically and morphologically diverse taxonomic group. They are typically considered resilient to many environmental perturbations found in marine and coastal environments, due to effective physiological regulation of ions and hemolymph pH, and a robust exoskeleton. Ocean acidification can affect the ability of marine calcifying organisms to build and maintain mineralized tissue and poses a threat for all marine calcifying taxa. Currently, there is no consensus on how ocean acidification will alter the ecologically relevant exoskeletal properties of crustaceans. Here, we present a systematic review and meta-analysis on the effects of ocean acidification on the crustacean exoskeleton, assessing both exoskeletal ion content (calcium and magnesium) and functional properties (biomechanical resistance and cuticle thickness). Our results suggest that the effect of ocean acidification on crustacean exoskeletal properties varies based upon seawater pCO2 and species identity, with significant levels of heterogeneity for all analyses. Calcium and magnesium content was significantly lower in animals held at pCO2 levels of 1500-1999 µatm as compared with those under ambient pCO2. At lower pCO2 levels, however, statistically significant relationships between changes in calcium and magnesium content within the same experiment were observed as follows: a negative relationship between calcium and magnesium content at pCO2 of 500-999 µatm and a positive relationship at 1000-1499 µatm. Exoskeleton biomechanics, such as resistance to deformation (microhardness) and shell strength, also significantly decreased under pCO2 regimes of 500-999 µatm and 1500-1999 µatm, indicating functional exoskeletal change coincident with decreases in calcification. Overall, these results suggest that the crustacean exoskeleton can be susceptible to ocean acidification at the biomechanical level, potentially predicated by changes in ion content, when exposed to high influxes of CO2. Future studies need to accommodate the high variability of crustacean responses to ocean acidification, and ecologically relevant ranges of pCO2 conditions, when designing experiments with conservation-level endpoints.

Amphibalanus amphitrite begins exoskeleton mineralization within 48 hours of metamorphosis.

Barnacles are ancient arthropods that, as adults, are surrounded by a hard, mineralized, outer shell that the organism produces for protection. While extensive research has been conducted on the glue-like cement that barnacles use to adhere to surfaces, less is known about the barnacle exoskeleton, especially the process by which the barnacle exoskeleton is formed. Here, we present data exploring the changes that occur as the barnacle cyprid undergoes metamorphosis to become a sessile juvenile with a mineralized exoskeleton. Scanning electron microscope data show dramatic morphological changes in the barnacle exoskeleton following metamorphosis. Energy-dispersive X-ray spectroscopy indicates a small amount of calcium (8%) 1 h post-metamorphosis that steadily increases to 28% by 2 days following metamorphosis. Raman spectroscopy indicates calcite in the exoskeleton of a barnacle 2 days following metamorphosis and no detectable calcium carbonate in exoskeletons up to 3 h post-metamorphosis. Confocal microscopy indicates during this 2 day period, barnacle base plate area and height increases rapidly (0.001 mm2 h-1 and 0.30 µm h-1, respectively). These results provide critical information into the early life stages of the barnacle, which will be important for developing an understanding of how ocean acidification might impact the calcification process of the barnacle exoskeleton.

Laser imaging polarimetry of nacre.

Nacre is a complex biomaterial made of aragonite-tablet bricks and organic mortar that is considerably resilient against breakage. Nacre has been studied with a wide range of laboratory techniques, leading to understanding key fundamentals and informing the creation of bio-inspired materials. In this article, we present an optical polarimetric technique to investigate nacre, taking advantage of the translucence and birefringence of its microcomponents. We focus our study on 3 classes of mollusks that have nacreous shells: bivalve (Pinctada fucata), gastropod (Haliotis asinina and Haliotis rufescens) and cephalopod (Nautilus pompilius). We sent polarized light from a laser through thin samples of nacre and did imaging polarimetry of the transmitted light. We observed clear distinctions between the structures of bivalve and gastropod, due to the spatial variation of their birefringence. The patterns for cephalopod were more similar to bivalve than gastropod. Bleaching of the samples disrupted the transmitted light. Subsequent refilling of the bivalve and gastropod nacre samples with oil produced optical patterns similar to those of unbleached samples. In cephalopod samples, we found that bleaching produced irreversible changes in the optical pattern.

Polarimetry of Pinctada fucata nacre indicates myostracal layer interrupts nacre structure.

The inner layer of many bivalve and gastropod molluscs consists of iridescent nacre, a material that is structured like a brick wall with bricks consisting of crystalline aragonite and mortar of organic molecules. Myostracal layers formed during shell growth at the point of muscle attachment to the shell can be found interspersed within the nacre structure. Little has been done to examine the effect the myostracal layer has on subsequent nacre structure. Here we present data on the structure of the myostracal and nacre layers from a bivalve mollusc, Pinctada fucata. Scanning electron microscope imaging shows the myostracal layer consists of regular crystalline blocks. The nacre before the layer consists of tablets approximately 400 nm thick, while after the myostracal layer the tablets are approximately 500 nm thick. A new technique, imaging polarimetry, indicates that the aragonite crystals within the nacre following the myostracal layer have greater orientation uniformity than before the myostracal layer. The results presented here suggest a possible interaction between the myostracal layer and subsequent shell growth.

Oxygen spectroscopy and polarization-dependent imaging contrast (PIC)-mapping of calcium carbonate minerals and biominerals.

X-ray absorption near-edge structure (XANES) spectroscopy and spectromicroscopy have been extensively used to characterize biominerals. Using either Ca or C spectra, unique information has been obtained regarding amorphous biominerals and nanocrystal orientations. Building on these results, we demonstrate that recording XANES spectra of calcium carbonate at the oxygen K-edge enables polarization-dependent imaging contrast (PIC) mapping with unprecedented contrast, signal-to-noise ratio, and magnification. O and Ca spectra are presented for six calcium carbonate minerals: aragonite, calcite, vaterite, monohydrocalcite, and both hydrated and anhydrous amorphous calcium carbonate. The crystalline minerals reveal excellent agreement of the extent and direction of polarization dependences in simulated and experimental XANES spectra due to X-ray linear dichroism. This effect is particularly strong for aragonite, calcite, and vaterite. In natural biominerals, oxygen PIC-mapping generated high-magnification maps of unprecedented clarity from nacre and prismatic structures and their interface in Mytilus californianus shells. These maps revealed blocky aragonite crystals at the nacre-prismatic boundary and the narrowest calcite needle-prisms. In the tunic spicules of Herdmania momus, O PIC-mapping revealed the size and arrangement of some of the largest vaterite single crystals known. O spectroscopy therefore enables the simultaneous measurement of chemical and orientational information in CaCO3 biominerals and is thus a powerful means for analyzing these and other complex materials. As described here, PIC-mapping and spectroscopy at the O K-edge are methods for gathering valuable data that can be carried out using spectromicroscopy beamlines at most synchrotrons without the expense of additional equipment.

Polarization dependence of aragonite calcium L-edge XANES spectrum indicates c and b axes orientation.

Calcium carbonate minerals are frequently found in biomineral structures and are the predominant mineral in invertebrates. While there are several calcium carbonate polymorphs, aragonite and calcite are the two most commonly found in biogenic systems. Currently, calcium L-edge X-ray absorption near-edge structure (XANES) spectra are used to distinguish between different calcium carbonate polymorphs, including calcite and aragonite, while oxygen and carbon K-edge XANES spectra are often used to determine the c axis orientation of a given calcium carbonate crystal. By doing a full analysis of the calcite and aragonite calcium L-edge XANES spectrum for both geologic and biogenic systems, we were able to show that aragonite has a polarization-dependent peak while calcite does not. Analysis based on both multiplet models and density functional calculations show how the polarization dependence arises from directional bonds between the calcium and oxygen atoms within aragonite. These data not only enable an interpretation of the aragonite calcium L-edge XANES spectrum but also the ability to determine the orientation of the c and b axes of aragonite crystals within a biomineral sample.

Crystal lattice tilting in prismatic calcite.

We analyzed the calcitic prismatic layers in Atrina rigida (Ar), Haliotis iris (Hi), Haliotis laevigata (HL), Haliotis rufescens (Hrf), Mytilus californianus (Mc), Pinctada fucata (Pf), Pinctada margaritifera (Pm) shells, and the aragonitic prismatic layer in the Nautilus pompilius (Np) shell. Dramatic structural differences were observed across species, with 100-µm wide single-crystalline prisms in Hi, HL and Hrf, 1-µm wide needle-shaped calcite prisms in Mc, 1-µm wide spherulitic aragonite prisms in Np, 20-µm wide single-crystalline calcite prisms in Ar, and 20-µm wide polycrystalline calcite prisms in Pf and Pm. The calcite prisms in Pf and Pm are subdivided into sub-prismatic domains of orientations, and within each of these domains the calcite crystal lattice tilts gradually over long distances, on the order of 100 µm, with an angle spread of crystal orientation of 10-20°. Furthermore, prisms in Pf and Pm are harder than in any other calcite prisms analyzed, their nanoparticles are smaller, and the angle spread is strongly correlated with hardness in all shells that form calcitic prismatic layers. One can hypothesize a causal relationship of these correlated parameters: greater angle spread may confer greater hardness and resistance to wear, thus providing Pf and Pm with a structural advantage in their environment. This is the first structure-property relationship thus far hypothesized in mollusk shell prisms.

Anisotropy of chemical bonds in collagen molecules studied by X-ray absorption near-edge structure (XANES) spectroscopy.

Collagen type I fibrils are the major building blocks of connective tissues. Collagen fibrils are anisotropic supramolecular structures, and their orientation can be revealed by polarized light microscopy and vibrational microspectroscopy. We hypothesized that the anisotropy of chemical bonds in the collagen molecules, and hence their orientation, might also be detected by X-ray photoemission electron spectromicroscopy (X-PEEM) and X-ray absorption near-edge structure (XANES) spectroscopy, which use linearly polarized synchrotron light. To test this hypothesis, we analyzed sections of rat-tail tendon, composed of parallel arrays of collagen fibrils. The results clearly indicate that XANES-PEEM is sensitive to collagen fibril orientation and, more specifically, to the orientations of carbonyl and amide bonds in collagen molecules. These data suggest that XANES-PEEM is a promising technique for characterizing the chemical composition and structural organization at the nanoscale of collagen-based connective tissues, including tendons, cartilage, and bone.

Asprich peptides are occluded in calcite and permanently disorder biomineral crystals.

Macromolecules are a minority but important component of the minerals formed by living organisms, or biominerals. The role these macromolecules play at the early stages of biomineral formation, as well as their long-term and long-range effects on the mature biomineral, is poorly understood. A 42-amino acid peptide, asp2, was derived from the Asprich family of proteins. In this study we present X-ray absorption near-edge structure spectroscopy and X-ray photoelectron emission microscopy data from the asp2 peptide, the calcite (CaCO(3)) crystals, and the peptide + crystal composites. The results clearly show that asp2 is occluded in fully formed biomineral crystals and slightly but permanently disorders the crystal structure at short- and long-range distances.

Nacre protein fragment templates lamellar aragonite growth.

Proteins play a major role in the formation of all biominerals. In mollusk shell nacre, complex mixtures and assemblies of proteins and polysaccharides were shown to induce aragonite formation, rather than the thermodynamically favored calcite (both aragonite and calcite are CaCO(3) polymorphs). Here we used N16N, a single 30 amino acid-protein fragment originally inspired by the mineral binding site of N16, a protein in the nacre layer of the Japanese pearl oysters (Pinctada fucata). In a calcite growth solution this short peptide induces in vitro biomineralization. This model biomineral was analyzed using X-ray PhotoElectron Emission spectroMicroscopy (X-PEEM) and found to be strikingly similar to natural nacre: lamellar aragonite with interspersed N16N layers. This and other findings combined suggest a hypothetical scenario in which in vivo three proteins (N16, Pif80, and Pif97) and a polysaccharide (chitin) work in concert to form lamellar nacre.

Mechanism of calcite co-orientation in the sea urchin tooth.

Sea urchin teeth are remarkable and complex calcite structures, continuously growing at the forming end and self-sharpening at the mature grinding tip. The calcite (CaCO(3)) crystals of tooth components, plates, fibers, and a high-Mg polycrystalline matrix, have highly co-oriented crystallographic axes. This ability to co-orient calcite in a mineralized structure is shared by all echinoderms. However, the physico-chemical mechanism by which calcite crystals become co-oriented in echinoderms remains enigmatic. Here, we show differences in calcite c-axis orientations in the tooth of the purple sea urchin ( Strongylocentrotus purpuratus ), using high-resolution X-ray photoelectron emission spectromicroscopy (X-PEEM) and microbeam X-ray diffraction (muXRD). All plates share one crystal orientation, propagated through pillar bridges, while fibers and polycrystalline matrix share another orientation. Furthermore, in the forming end of the tooth, we observe that CaCO(3) is present as amorphous calcium carbonate (ACC). We demonstrate that co-orientation of the nanoparticles in the polycrystalline matrix occurs via solid-state secondary nucleation, propagating out from the previously formed fibers and plates, into the amorphous precursor nanoparticles. Because amorphous precursors were observed in diverse biominerals, solid-state secondary nucleation is likely to be a general mechanism for the co-orientation of biomineral components in organisms from different phyla.

The grinding tip of the sea urchin tooth exhibits exquisite control over calcite crystal orientation and Mg distribution.

The sea urchin tooth is a remarkable grinding tool. Even though the tooth is composed almost entirely of calcite, it is used to grind holes into a rocky substrate itself often composed of calcite. Here, we use 3 complementary high-resolution tools to probe aspects of the structure of the grinding tip: X-ray photoelectron emission spectromicroscopy (X-PEEM), X-ray microdiffraction, and NanoSIMS. We confirm that the needles and plates are aligned and show here that even the high Mg polycrystalline matrix constituents are aligned with the other 2 structural elements when imaged at 20-nm resolution. Furthermore, we show that the entire tooth is composed of 2 cooriented polycrystalline blocks that differ in their orientations by only a few degrees. A unique feature of the grinding tip is that the structural elements from each coaligned block interdigitate. This interdigitation may influence the fracture process by creating a corrugated grinding surface. We also show that the overall Mg content of the tooth structural elements increases toward the grinding tip. This probably contributes to the increasing hardness of the tooth from the periphery to the tip. Clearly the formation of the tooth, and the tooth tip in particular, is amazingly well controlled. The improved understanding of these structural features could lead to the design of better mechanical grinding and cutting tools.

Transient amorphous calcium phosphate in forming enamel.

Enamel, the hardest tissue in the body, begins as a three-dimensional network of nanometer size mineral particles, suspended in a protein gel. This mineral network serves as a template for mature enamel formation. To further understand the mechanisms of enamel formation we characterized the forming enamel mineral at an early secretory stage using X-ray absorption near-edge structure (XANES) spectromicroscopy, transmission electron microscopy (TEM), FTIR microspectroscopy and polarized light microscopy. We show that the newly formed enamel mineral is amorphous calcium phosphate (ACP), which eventually transforms into apatitic crystals. Interestingly, the size, shape and spatial organization of these amorphous mineral particles and older crystals are essentially the same, indicating that the mineral morphology and organization in enamel is determined prior to its crystallization. Mineralization via transient amorphous phases has been previously reported in chiton teeth, mollusk shells, echinoderm spicules and spines, and recent reports strongly suggest the presence of transient amorphous mineral in forming vertebrate bones. The present finding of transient ACP in murine tooth enamel suggests that this strategy might be universal.

Gradual ordering in red abalone nacre.

Red abalone (Haliotis rufescens) nacre is a layered composite biomineral that contains crystalline aragonite tablets confined by organic layers. Nacre is intensely studied because its biologically controlled microarchitecture gives rise to remarkable strength and toughness, but the mechanisms leading to its formation are not well understood. Here we present synchrotron spectromicroscopy experiments revealing that stacks of aragonite tablet crystals in nacre are misoriented with respect to each other. Quantitative measurements of crystal orientation, tablet size, and tablet stacking direction show that orientational ordering occurs not abruptly but gradually over a distance of 50 microm. Several lines of evidence indicate that different crystal orientations imply different tablet growth rates during nacre formation. A theoretical model based on kinetic and gradual selection of the fastest growth rates produces results in qualitative and quantitative agreement with the experimental data and therefore demonstrates that ordering in nacre is a result of crystal growth kinetics and competition either in addition or to the exclusion of templation by acidic proteins as previously assumed. As in other natural evolving kinetic systems, selection of the fastest-growing stacks of tablets occurs gradually in space and time. These results suggest that the self-ordering of the mineral phase, which may occur completely independently of biological or organic-molecule control, is fundamental in nacre formation.

Transformation mechanism of amorphous calcium carbonate into calcite in the sea urchin larval spicule.

Sea urchin larval spicules transform amorphous calcium carbonate (ACC) into calcite single crystals. The mechanism of transformation is enigmatic: the transforming spicule displays both amorphous and crystalline properties, with no defined crystallization front. Here, we use X-ray photoelectron emission spectromicroscopy with probing size of 40-200 nm. We resolve 3 distinct mineral phases: An initial short-lived, presumably hydrated ACC phase, followed by an intermediate transient form of ACC, and finally the biogenic crystalline calcite phase. The amorphous and crystalline phases are juxtaposed, often appearing in adjacent sites at a scale of tens of nanometers. We propose that the amorphous-crystal transformation propagates in a tortuous path through preexisting 40- to 100-nm amorphous units, via a secondary nucleation mechanism.

Assignment of polarization-dependent peaks in carbon K-edge spectra from biogenic and geologic aragonite.

Many biominerals, including mollusk and echinoderm shells, avian eggshells, modern and fossil bacterial sediments, planktonic coccolithophores, and foraminifera, contain carbonates in the form of biogenic aragonite or calcite. Here we analyze biogenic and geologic aragonite using different kinds of surface- and bulk-sensitive X-ray absorption near-edge structure (XANES) spectroscopy at the carbon K-edge, as well as high-resolution scanning transmission X-ray microscopy (STXM). Besides the well-known main pi* and sigma* carbonate peaks, we observed and fully characterized four minor peaks, at energies between the main pi* and sigma* peaks. As expected, the main peaks are similar in geologic and biogenic aragonite, while the minor peaks differ in relative intensity. In this and previous work, the minor peaks appear to be the ones most affected in biomineralization processes, hence the interest in characterizing them. Peak assignment was achieved by correlation of polarization-dependent behavior of the minor peaks with that of the main pi* and sigma* peaks. The present characterization provides the background for future studies of aragonitic biominerals.

Probing the organic-mineral interface at the molecular level in model biominerals.

It is widely known that macromolecules, such as proteins, can control the nucleation and growth of inorganic solids in biomineralizing organisms. However, what is not known are the complementary molecular interactions, organization, and rearrangements that occur when proteins interact with inorganic solids during the formation of biominerals. The organic-mineral interface (OMI) is expected to be the site for these phenomena, and is therefore extraordinarily interesting to investigate. In this report, we employ X-ray absorption near edge (XANES) spectromicroscopy to investigate the electronic structure of both calcium carbonate mineral crystals and polypeptides, and detect changing bonds at the OMI during crystal growth in the presence of polypeptides. We acquired XANES spectra from calcium carbonate crystals grown in the presence of three mollusk nacre-associated polypeptides (AP7N, AP24N, n16N) and in the presence of a sea urchin spicule matrix protein, LSM34. All these model biominerals gave similar results, including the disruption of CO bonds in calcite and enhancement of the peaks associated with C-H bonds and C-O bonds in peptides, indicating ordering of the amino acid side chains in the mineral-associated polypeptides and carboxylate binding. This is the first evidence of the mutual effect of calcite on peptide chain and peptide chain on calcite during biomineralization. We also show that these changes do not occur when Asp and Glu are replaced in the n16N sequence with Asn and Gln, respectively, demonstrating that carboxyl groups in Asp and Glu do participate in polypeptide-mineral molecular associations.

Architecture of columnar nacre, and implications for its formation mechanism.

We analyze the structure of Haliotis rufescens nacre, or mother-of-pearl, using synchrotron spectromicroscopy and x-ray absorption near-edge structure spectroscopy. We observe imaging contrast between adjacent individual nacre tablets, arising because different tablets have different crystal orientations with respect to the radiation's polarization vector. Comparing previous data and our new data with models for columnar nacre growth, we find the data are most consistent with a model in which nacre tablets are nucleated by randomly distributed sites in the organic matrix layers.