Biomineralisation by earthworms: an investigation into the stability and distribution of amorphous calcium carbonate

Background Many biominerals form from amorphous calcium carbonate (ACC), but this phase is highly unstable when synthesised in its pure form inorganically. Several species of earthworm secrete calcium carbonate granules which contain highly stable ACC. We analysed the milky fluid from which granules form and solid granules for amino acid (by liquid chromatography) and functional group (by Fourier transform infrared (FTIR) spectroscopy) compositions. Granule elemental composition was determined using inductively coupled plasma-optical emission spectroscopy (ICP-OES) and electron microprobe analysis (EMPA). Mass of ACC present in solid granules was quantified using FTIR and compared to granule elemental and amino acid compositions. Bulk analysis of granules was of powdered bulk material. Spatially resolved analysis was of thin sections of granules using synchrotron-based μ-FTIR and EMPA electron microprobe analysis. Results The milky fluid from which granules form is amino acid-rich (≤ 136 ± 3 nmol mg−1 (n = 3; ± std dev) per individual amino acid); the CaCO3 phase present is ACC. Even four years after production, granules contain ACC. No correlation exists between mass of ACC present and granule elemental composition. Granule amino acid concentrations correlate well with ACC content (r ≥ 0.7, p ≤ 0.05) consistent with a role for amino acids (or the proteins they make up) in ACC stabilisation. Intra-granule variation in ACC (RSD = 16%) and amino acid concentration (RSD = 22–35%) was high for granules produced by the same earthworm. Maps of ACC distribution produced using synchrotron-based μ-FTIR mapping of granule thin sections and the relative intensity of the ν2: ν4 peak ratio, cluster analysis and component regression using ACC and calcite standards showed similar spatial distributions of likely ACC-rich and calcite-rich areas. We could not identify organic peaks in the μ-FTIR spectra and thus could not determine whether ACC-rich domains also had relatively high amino acid concentrations. No correlation exists between ACC distribution and elemental concentrations determined by EMPA. Conclusions ACC present in earthworm CaCO3 granules is highly stable. Our results suggest a role for amino acids (or proteins) in this stability. We see no evidence for stabilisation of ACC by incorporation of inorganic components.

Ocean acidification impacts mussel control on biomineralisation

Ocean acidification is altering the oceanic carbonate saturation state and threatening the survival of marine calcifying organisms. Production of their calcium carbonate exoskeletons is dependent not only on the environmental seawater carbonate chemistry but also the ability to produce biominerals through proteins. We present shell growth and structural responses by the economically important marine calcifier Mytilus edulis to ocean acidification scenarios (380, 550, 750, 1000 µatm pCO2). After six months of incubation at 750 µatm pCO2, reduced carbonic anhydrase protein activity and shell growth occurs in M. edulis. Beyond that, at 1000 µatm pCO2, biomineralisation continued but with compensated metabolism of proteins and increased calcite growth. Mussel growth occurs at a cost to the structural integrity of the shell due to structural disorientation of calcite crystals. This loss of structural integrity could impact mussel shell strength and reduce protection from predators and changing environments.

The effect of unlocking RGD-motifs in collagen I on pre-osteoblast adhesion and differentiation

Denaturation of extracellular matrix proteins exposes cryptic binding sites. It is hypothesized that binding of cell adhesion receptors to these cryptic binding sites regulates cellular behaviour during tissue repair and regeneration. To test this hypothesis, we quantify the adhesion of pre-osteoblastic cells to native (Col) and partially-denatured (pdCol) collagen I using single-cell force spectroscopy. During early stages of cell attachment (≤180 s) pre-osteoblasts (MC3T3-E1) adhered significantly stronger to pdCol compared to Col. RGD (Arg-Gly-Asp)-containing peptides suppressed this elevated cell adhesion. We show that the RGD-binding α5β1- and αv-integrins mediated pre-osteoblast adhesion to pdCol, but not to Col. On pdCol pre-osteoblasts had a higher focal adhesion kinase tyrosine-phosphorylation level that correlated with enhanced spreading and motility. Moreover, pre-osteoblasts cultured on pdCol showed a pronounced matrix mineralization activity. Our data suggest that partially-denatured collagen exposes RGD-motifs that trigger binding of α5β1- and αv-integrins. These integrins initiate cellular processes that stimulate osteoblast adhesion, spreading, motility and differentiation. Taken together, these quantitative insights reveal an approach for the development of alternative collagen I- based surfaces for tissue engineering applications.

Temporal tracking of mineralization and transcriptional developments of shell formation during the early life history of pearl oyster Pinctada maxima

Molluscan larval ontogeny is a highly conserved process comprising three principal developmental stages. A characteristic unique to each of these stages is shell design, termed prodissoconch I, prodissoconch II and dissoconch. 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. Scanning electron microscopy and X-ray diffraction analysis of P. maxima larvae and juveniles collected throughout post-embryonic ontogenesis, document the 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 on the microarray platform PmaxArray 1.0. A number of transcripts are reported as differentially expressed in correlation to the mineralization events of P. maxima larval ontogeny. Some of those isolated are known shell matrix genes while others are novel; these are discussed in relation to potential shell formation roles. This interdisciplinary investigation has linked the shell developments of P. maxima larval ontogeny with corresponding gene expression profiles, furthering the elucidation of shell biomineralization.

Fusion or non fusion of coral fragments in Acropora

Corals inhabit high energy environments where frequent disturbances result in physical damage to coralla, including fragmentation, as well as generating and mobilizing large sediment clasts. The branching growth form common in the Acropora genus makes it particularly susceptible to such disturbances and therefore useful for study of the fate of large sediment clasts. Living Acropora samples with natural, extraneous, broken coral branches incorporated on their living surface and dead Acropora skeletons containing embedded clasts of isolated branch sections of Acropora were observed and/or collected from the reef flat of Heron Reef, southern Great Barrier Reef and Bargara, Australia respectively. Here we report three different outcomes when pebble-sized coral branches became lodged on living coral colonies during sedimentation events in natural settings in Acropora: 1) Where live coral branches produced during a disturbance event come to rest on probable genetic clone-mate colonies they become rapidly stabilised leading to complete soft tissue and skeletal fusion; 2) Where the branch and underlying colony are not clone-mates, but may still be the same or similar species, the branches still may be stabilised rapidly by soft tissue, but then one species will overgrow the other; and 3) Where branches represent dead skeletal debris, they are treated like any foreign clast and are surrounded by clypeotheca and incorporated into the corallum by overgrowth. The retention of branch fragments on colonies in high energy reef flat settings may suggest an active role of coral polyps to recognise and fuse with each other. Also, in all cases the healing of disturbed tissue and subsequent skeletal growth is an adaptation important for protecting colonies from invasion by parasites and other benthos following disturbance events and may also serve to increase corallum strength. Knowledge of such adaptations is important in studies of coral behaviour during periods of environmental stress.

Geochemistry-based coral palaeoclimate studies and the potential of ‘non-traditional’ (non-massive Porites) corals : recent developments and future progression

Understanding the natural variability of the Earth's climate system and accurately identifying potential anthropogenic influences requires long term, geographically distributed records of key climate indicators, such as temperature and precipitation that extend prior to the last 400. years of the Holocene. Reef corals provide an excellent source of high resolution climate records, and importantly represent the tropical marine environment where palaeoclimate data are urgently required. Recent decades have seen significant improvement in our understanding of coral biomineralisation, the associated uptake of geochemical proxies and methods of identifying and understanding the effects of both early and late, post depositional diagenetic alteration. These processes all have significant implications for interpreting geochemical proxies relevant to palaeoclimatic reconstructions. This paper reviews the current 'state of the art' in terms of coral based palaeoclimate reconstructions and highlights a key remaining problem. The majority of coral based palaeoclimate research has been derived from massive colonies of Porites. However, massive Porites are not globally abundant and may not provide material of a particular age of interest in those regions where they are present. Therefore, there is great potential for alternate coral genera to act as complimentary climate archives. While it remains critical to consider five key factors - vital effects, differential growth morphologies, geochemical heterogeneity in the skeletal ultrastructure, transfer equation selection and diagenetic screening of skeletal material - in order to allow the highest level of accuracy in coral palaeoclimate reconstructions, it is also important to develop alternate taxa for palaeoclimate studies in regions where Porites colonies are absent or rare. Currently as many as nine genera other than Porites have proven at least limited utility in palaeothermometry, most of which are found in the Atlantic/Caribbean region where massive Porites do not exist. Even branching taxa such as Acropora have significant potential to preserve environmental archives. Increasing this capability will greatly expand the number of potential geochemical archives available for longer term temporal records of palaeoclimate.

Major Cellular and Physiological Impacts of Ocean Acidification on a Reef Building Coral

As atmospheric levels of CO2 increase, reef-building corals are under greater stress from both increased sea surface temperatures and declining sea water pH. To date, most studies have focused on either coral bleaching due to warming oceans or declining calcification due to decreasing oceanic carbonate ion concentrations. Here, through the use of physiology measurements and cDNA microarrays, we show that changes in pH and ocean chemistry consistent with two scenarios put forward by the Intergovernmental Panel on Climate Change (IPCC) drive major changes in gene expression, respiration, photosynthesis and symbiosis of the coral, Acropora millepora, before affects on biomineralisation are apparent at the phenotype level. Under high CO2 conditions corals at the phenotype level lost over half their Symbiodinium populations, and had a decrease in both photosynthesis and respiration. Changes in gene expression were consistent with metabolic suppression, an increase in oxidative stress, apoptosis and symbiont loss. Other expression patterns demonstrate upregulation of membrane transporters, as well as the regulation of genes involved in membrane cytoskeletal interactions and cytoskeletal remodeling. These widespread changes in gene expression emphasize the need to expand future studies of ocean acidification to include a wider spectrum of cellular processes, many of which may occur before impacts on calcification.

Morphological differentiation observed in manganese oxidizing bacterial colonies

Bacteria play a vital role in bringing about Mn(II) oxidation in the natural environment. A study was conducted to identify the potential threat offered by these bacteria in bringing about biomineralisation of manganese dioxide on titanium surfaces exposed to seawater. During the study it was observed that the bacteria such as Pseudomonas and Bacillus formed brown colonies on agar plates amended with Mn2+ indicating their ability to oxidize Mn(II). These colonies showed distinct morphologies when grown on plates containing Mn(II) while they formed normal colonies in the absence of Mn.(II).Hence it is possible that these morphologically distinct structures produced by the bacterial colonies assist these bacteria to perform this function of Mn-oxidation.

Crystallization of SrCO3 on a self-assembled monolayer substrate: an in-situ synchrotron X-ray study

Self-assembled monolayers (SAMs) of alkanethiols on gold surfaces show great promise in controlling the nucleation and growth of inorganic minerals from solution. In doing so, they mimic the role of some biogenic macromolecules in natural biomineralisation processes. Crystallization on SAM surfaces is usually monitored ex-situ; by allowing the process to commence and to evolve for some time, removing the substrate from the mother solution, and then examining it using microscopy, diffraction etc. We present here for the first time, the use of high energy monochromatic synchrotron X-radiation in conjunction with a two dimensional detector to monitor in situ, in a time resolved fashion, the growth of SrCO3 (strontianite) crystals on a SAM substrate.

Dentin phosphoprotein frameshift mutations in hereditary dentin disorders and their variation patterns in normal human population

Background: Dentin phosphoprotein ( DPP) is the most abundant non-collagenous protein in dentin, which is highly phosphorylated and plays key roles in dentin biomineralisation. The aetiology of isolated hereditary dentin disorders in most affected familie

Grazing under experimental hypercapnia and elevated temperature does not affect the radula of a chiton (Mollusca, Polyplacophora, Lepidopleurida)

Chitons (class Polyplacophora) are benthic grazing molluscs with an eight-part aragonitic shell armature. The radula, a serial tooth ribbon that extends internally more than half the length of the body, is mineralised on the active feeding teeth with iron magnetite apparently as an adaptation to constant grazing on rocky substrates. As the anterior feeding teeth are eroded they are shed and replaced with a new row. The efficient mineralisation and function of the radula could hypothetically be affected by changing oceans in two ways: changes in seawater chemistry (pH and pCO(2)) may impact the biomineralisation pathway, potentially leading to a weaker or altered density of the feeding teeth; rising temperatures could increase activity levels in these ectothermic animals, and higher feeding rates could increase wear on the feeding teeth beyond the animals' ability to synthesise, mineralise, and replace radular rows. We therefore examined the effects of pH and temperature on growth and integrity in the radula of the chiton Leptochiton asellus. Our experiment implemented three temperature (similar to 10, 15, 20 degrees C) and two pCO(2) treatments (similar to 400 mu atm, pH 8.0; similar to 2000 mu atm, pH 7.5) for six treatment groups. Animals (n = 50) were acclimated to the treatment conditions for a period of 4 weeks. This is sufficient time for growth of ca. 7-9 new tooth rows or 20% turnover of the mineralised portion. There was no significant difference in the number of new (non-mineralised) teeth or total tooth row count in any treatment. Examination of the radulae via SEM revealed no differences in microwear or breakage on the feeding cusps correlating to treatment groups. The shell valves also showed no signs of dissolution. As a lineage, chitons have survived repeated shifts in Earth's climate through geological time, and at least their radulae may be robust to future perturbations.

Comment on: Fruit and seed biomineralization and its effect on preservation by E. Messager et al.; in: Archaeological and Anthropological Sciences (2010) 2:25–34. DOI 10.1007/s12520-010-0024-1

This article provides a brief critique of a recent article on biomineralisation and preservation. It gives a summary of the difference between biomineralisation and mineral replacement, and addresses problems with the interpretation of FT-IR data. The lack of contextual information for the samples studied is another problem which is highlighted.

Ferritin in iron containing granules from the fat body of the honeybees Apis mellifera and Scaptotrigona postica

It is already known that the behaviour of the honeybee Apis mellifera is influenced by the Earth's magnetic field. Recently it has been proposed that iron-rich granules found inside the fat body cells of this honeybee had small magnetite crystals that were responsible for this behaviour. In the present work, we studied the iron containing granules from queens of two species of honeybees (A. mellifera and Scaptotrigona postica) by electron microscopy methods in order to clarify this point. The granules were found inside rough endoplasmic reticulum cisternae. Energy dispersive X-ray analysis of granules from A. mellifera showed the presence of iron, phosphorus and calcium. The same analysis performed on the granules of S. postica also indicated the presence of these elements along with the additional element magnesium. The granules of A. mellifera were composed of apoferritin-like particles in the periphery while in the core, clusters of organised particles resembling holoferritin were seen. The larger and more mineralised granules of S. postica presented structures resembling ferritin cores in the periphery, and smaller electron dense particles inside the bulk. Electron spectroscopic images of the granules from A. mellifera showed that iron, oxygen and phosphorus were co-localised in the ferritin-like deposits. These results indicate that the iron-rich granules of these honeybees are formed by accumulation of ferritin and its degraded forms together with elements present inside the rough endoplasmic reticulum, such as phosphorus, calcium and magnesium. It is suggested that the high level of phosphate in the milieu would prevent the crystallisation of iron oxides in these structures, making very unlikely their participation in magnetoreception mechanisms. They are most probably involved in iron homeostasis. (C) 2001 Elsevier B.V. Ltd. All rights reserved.