The Geochemistry of Pāua as a Potential Proxy for Past and Present Environmental Change

<p>Nearshore New Zealand mollusca (shellfish) have the potential to be important archives of environmental conditions and change. Ambient ocean chemistry can be incorporated into the calcium carbonate (CaCO3) shell during the life span of the mollusc providing a high resolution temporal record of the chemical and physical changes of the environments the mollusc lived in. Previous studies on foraminifera and coral have shown that the substitution of magnesium or strontium for calcium (Mg, Sr/Ca) during the formation of the CaCO3 shell is directly correlated with ocean temperatures. Other divalent cations (e.g., Sr2+, Ba2+, Pb2+) can also provide information on ambient salinity, primary productivity or nutrient levels, and local anthropogenic pollution. This study uses new geochemical techniques that have been developed to measure the trace element chemistry of CaCO3 mollusc shells at high temporal resolution, using laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) in order to calibrate shell chemistry with environmental conditions. This study is the first to explore the use of the geochemistry of Haliotis iris as a potential proxy for (paleo-) environmental conditions. Pāua (Haliotis iris) were collected from six different localities around New Zealand and the Chatham Islands as well as a cultured environment (OceaNZ Blue Ltd). The shells were sectioned following the axis of maximum growth exposing both CaCO3 layers; the prismatic (predominantly calcite) and nacreous (aragonite) layers. The shells were analysed by LA-ICP-MS at 25 μm spot sizes through a high temporal transect of both layers. Observed differences in the element/Ca ratios between the prismatic and nacreous layer reflect the differing crystallinity of each layer. High temporal resolution Mg/Ca ratio data of the prismatic layer of the samples which grew in a cultured environment were compared with temperature and growth data supplied by OceaNZ Blue Ltd. The results showed that temperature was not the primary control on the uptake of Mg within the shells and that influences from biological factors including increased growth rate were also evident. Sr/Ca ratios show a weak inverse relationship with increased growth rate assumed. These results, however, are not reproducible within samples collected from the wild, showing that external factors (high wave energy, diet, predation, lack of food) place metabolic stress on the pāua. The monitoring of other element/Ca including Ba/Ca, Al/Ca, Pb/Ca and Zn/Ca ratios have the potential to provide information into the past frequency of storm events that deliver sediment into the oceans and remobilise other sediments and changing levels of environmental pollution. This is reflected through increased Al/Ca, Pb/Ca and Zn/Ca ratios during the winter season in a number of samples (n = 3) gained from the high resolution analysis of the prismatic layers. Overall, element/Ca ratios are difficult to correlate environmental conditions in samples from the wild as there are many different parameters influencing the uptake of element/Ca ratios with the shells of pāua. Uncertainties lie with a lack of understanding of the biological controls influencing pāua during biomineralisation including the transportation of the elements within organism to the extrapallial fluid to be biomineralised, ontogeny, and the rate and regularity of biomineralisation of shell material.</p>

The Geochemistry of Pāua as a Potential Proxy for Past and Present Environmental Change

<p>Nearshore New Zealand mollusca (shellfish) have the potential to be important archives of environmental conditions and change. Ambient ocean chemistry can be incorporated into the calcium carbonate (CaCO3) shell during the life span of the mollusc providing a high resolution temporal record of the chemical and physical changes of the environments the mollusc lived in. Previous studies on foraminifera and coral have shown that the substitution of magnesium or strontium for calcium (Mg, Sr/Ca) during the formation of the CaCO3 shell is directly correlated with ocean temperatures. Other divalent cations (e.g., Sr2+, Ba2+, Pb2+) can also provide information on ambient salinity, primary productivity or nutrient levels, and local anthropogenic pollution. This study uses new geochemical techniques that have been developed to measure the trace element chemistry of CaCO3 mollusc shells at high temporal resolution, using laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) in order to calibrate shell chemistry with environmental conditions. This study is the first to explore the use of the geochemistry of Haliotis iris as a potential proxy for (paleo-) environmental conditions. Pāua (Haliotis iris) were collected from six different localities around New Zealand and the Chatham Islands as well as a cultured environment (OceaNZ Blue Ltd). The shells were sectioned following the axis of maximum growth exposing both CaCO3 layers; the prismatic (predominantly calcite) and nacreous (aragonite) layers. The shells were analysed by LA-ICP-MS at 25 μm spot sizes through a high temporal transect of both layers. Observed differences in the element/Ca ratios between the prismatic and nacreous layer reflect the differing crystallinity of each layer. High temporal resolution Mg/Ca ratio data of the prismatic layer of the samples which grew in a cultured environment were compared with temperature and growth data supplied by OceaNZ Blue Ltd. The results showed that temperature was not the primary control on the uptake of Mg within the shells and that influences from biological factors including increased growth rate were also evident. Sr/Ca ratios show a weak inverse relationship with increased growth rate assumed. These results, however, are not reproducible within samples collected from the wild, showing that external factors (high wave energy, diet, predation, lack of food) place metabolic stress on the pāua. The monitoring of other element/Ca including Ba/Ca, Al/Ca, Pb/Ca and Zn/Ca ratios have the potential to provide information into the past frequency of storm events that deliver sediment into the oceans and remobilise other sediments and changing levels of environmental pollution. This is reflected through increased Al/Ca, Pb/Ca and Zn/Ca ratios during the winter season in a number of samples (n = 3) gained from the high resolution analysis of the prismatic layers. Overall, element/Ca ratios are difficult to correlate environmental conditions in samples from the wild as there are many different parameters influencing the uptake of element/Ca ratios with the shells of pāua. Uncertainties lie with a lack of understanding of the biological controls influencing pāua during biomineralisation including the transportation of the elements within organism to the extrapallial fluid to be biomineralised, ontogeny, and the rate and regularity of biomineralisation of shell material.</p>

Molecular Characterization of Carbonic Anhydrase II (CA II) and Its Potential Involvement in Regulating Shell Formation in the Pacific Abalone, Haliotis discus hannai

Carbonic anhydrases (CAs) are a family of metalloenzymes that can catalyze the reversible interconversion of CO2/HCO3–, ubiquitously present in both prokaryotes and eukaryotes. In the present study, a CA II (designated as HdhCA II) was sequenced and characterized from the mantle tissue of the Pacific abalone. The complete sequence of HdhCA II was 1,169 bp, encoding a polypeptide of 349 amino acids with a NH2-terminal signal peptide and a CA architectural domain. The predicted protein shared 98.57% and 68.59% sequence identities with CA II of Haliotis gigantea and Haliotis tuberculata, respectively. Two putative N-linked glycosylation motifs and two cysteine residues could potentially form intramolecular disulfide bond present in HdhCA II. The phylogenetic analysis indicated that HdhCA II was placed in a gastropod clade and robustly clustered with CA II of H. gigantea and H. tuberculata. The highest level of HdhCA II mRNA expression was detected in the shell forming mantle tissue. During ontogenesis, the mRNA of HdhCA II was detected in all stages, with larval shell formation stage showing the highest expression level. The in situ hybridization results detected the HdhCA II mRNA expression in the epithelial cells of the dorsal mantle pallial, an area known to express genes involved in the formation of a nacreous layer in the shell. This is the first report of HdhCA II in the Pacific abalone, and the results of this study indicate that this gene might play a role in the shell formation of abalone.

Carbonic Anhydrase in Pacific Abalone Haliotis discus hannai: Characterization, Expression, and Role in Biomineralization

Carbonic anhydrases (CAs) are universal zinc ion containing metalloenzymes that play a pivotal role in various physiological processes. In this study, a CA I (designated as Hdh CA I) was isolated and characterized from the mantle tissue of Pacific abalone, Haliotis discus hannai. The full-length cDNA sequence of Hdh CA I was 1,417-bp in length, encoding a protein of 337 amino acids with molecular weight of 37.58 kDa. Hdh CA I sequence possessed a putative signal peptide of 22 amino acids and a CA catalytic function domain. The predicted protein shared 94 and 78% sequence identities with Haliotis gigantea and Haliotis tuberculata CA I, respectively. Results of phylogenetic analysis indicated that Hdh CA I was evolutionarily close to CA I of H. gigantea and H. tuberculata with high bootstrap values. Significantly higher levels of Hdh CA I mRNA transcript were found in mantle than other examined tissues. In situ hybridization results showed strong hybridization signals in epithelial cells of the dorsal mantle pallial, an area known to synthesize and secrete proteins responsible for the nacreous layer formation of shell. This is the first study on Hdh CA I in H. discus hannai and the results may contribute to further study its physiological functions in shell biomineralization of abalone.

Analysis of Finnish Blue Mussel (Mytilus edulis L.) Shell: Biomineral Ultrastructure, Organic-Rich Interfacial Matrix and Mechanical Behavior

Studying various marine biomineralized ultrastructures reveals the appearance of common architectural designs and building blocks in materials with fascinating mechanical properties that match perfectly to their biological tasks. Advanced mechanical properties of biological materials are attributed to evolutionary optimized molecular architectures and structural hierarchy. One example which has not yet been structurally investigated in great detail is the shell of Mytilus edulis L. (Finnish blue mussel) found in the archipelago of SW-Finland. Through a combination of various state-of-the-art techniques such as high-resolution electron microscopy imaging, Fourier-transformed infrared spectroscopy, powder X-ray diffraction, synchrotron wide-angle X-ray diffraction, nanoindentation and protein analysis, both the inorganic mineralized components as well as the organic-rich matrix were extensively characterized. We found very similar ultra-architecture across the shell of M. edulis L. as compared to the widely studied and closely related M. edulis. However, we also found interesting differences, for instance in the thickness and degree of orientation of the mineralized layers indicating dissimilar properties and related alterations in the biomineralization processes. Our results show that the shell of M. edulis L. has a gradient of mechanical properties, with the increase in the stiffness and the hardness from anterior to the posterior region of the shell. The shell is made from distinct and recognizable mineralized layers each varying in thickness and microstructural features. At posterior regions of the shell, moving from dorsal to ventral side, these layers are an oblique prismatic layer, a prismatic layer and a nacreous layer, in which the oblique prismatic layer is found to be the main and thickest mineralized layer of the shell. Probing the calcified rods in the oblique prismatic layer using high resolution SEM imaging revealed opening of channels with a diameters of 40-50 nm and lengths up to a micrometer extending through the rods. The chitin and protein have been found to be the main component of the organic-rich interfacial matrix as expected. Protein analysis showed two abundant proteins with sizes around 100 kD and 45 kD which likely not only regulates biomineralization and adhesion of the crystals but also governing the intrinsic-extrinsic toughening in the shell. Overall, this detailed analysis provides new structural insights into biomineralization of marine shells in general.

The analysis of freshwater pearl mussel shells using µ-XRF (micro-x-ray fluorescence) and the applicability for environmental reconstruction

Freshwater pearl mussel is a highly threatened species, and many populations are currently on the brink of local extinction. For example, in south Finland, only two populations are currently viable. Even though the reasons for the mussels’ demise are relatively well known, the long-term impacts of water quality are not completely resolved. Here, µ-XRF analysis and historical records were used to evaluate whether the differences in water chemistry or past environmental changes in three rivers in southern Finland are visible in mussel shell chemistry. The results show that the cracks inside mussel shells, invisible to the naked eye, may greatly affect the elemental composition results. Further, anomalies which could be related to inclusion of detrital matter inside the shells were detected. Manganese (Mn) seems to be related to mussel growth dynamics, especially in the nacreous layer, while high values of iron (Fe) and Mn are also present at the top sections of the prismatic layer. Line scan analysis revealed high variation between replicates. The µ-XRF method could be used as prescreening method in mussel sclerochemistry studies, but more studies are needed to clarify the ability of FPM shells to reliably record the environmental conditions.

Identification of methionine -rich insoluble proteins in the shell of the pearl oyster, Pinctada fucata

The molluscan shell is a biomineral that comprises calcium carbonate and organic matrices controlling the crystal growth of calcium carbonate. The main components of organic matrices are insoluble chitin and proteins. Various kinds of proteins have been identified by solubilizing them with reagents, such as acid or detergent. However, insoluble proteins remained due to the formation of a solid complex with chitin. Herein, we identified these proteins from the nacreous layer, prismatic layer, and hinge ligament of Pinctada fucata using mercaptoethanol and trypsin. Most identified proteins contained a methionine-rich region in common. We focused on one of these proteins, NU-5, to examine the function in shell formation. Gene expression analysis of NU-5 showed that NU-5 was highly expressed in the mantle, and a knockdown of NU-5 prevented the formation of aragonite tablets in the nacre, which suggested that NU-5 was required for nacre formation. Dynamic light scattering and circular dichroism revealed that recombinant NU-5 had aggregation activity and changed its secondary structure in the presence of calcium ions. These findings suggest that insoluble proteins containing methionine-rich regions may be important for scaffold formation, which is an initial stage of biomineral formation.

Effects of the Sintering Process on Nacre-Derived Hydroxyapatite Scaffolds for Bone Engineering

A hydroxyapatite scaffold is a suitable biomaterial for bone tissue engineering due to its chemical component which mimics native bone. Electronic states which present on the surface of hydroxyapatite have the potential to be used to promote the adsorption or transduction of biomolecules such as protein or DNA. This study aimed to compare the morphology and bioactivity of sinter and nonsinter marine-based hydroxyapatite scaffolds. Field emission scanning electron microscopy (FESEM) and micro-computed tomography (microCT) were used to characterize the morphology of both scaffolds. Scaffolds were co-cultured with 5 × 104/cm2 of MC3T3-E1 preosteoblast cells for 7, 14, and 21 days. FESEM was used to observe the cell morphology, and MTT and alkaline phosphatase (ALP) assays were conducted to determine the cell viability and differentiation capacity of cells on both scaffolds. Real-time polymerase chain reaction (rtPCR) was used to identify the expression of osteoblast markers. The sinter scaffold had a porous microstructure with the presence of interconnected pores as compared with the nonsinter scaffold. This sinter scaffold also significantly supported viability and differentiation of the MC3T3-E1 preosteoblast cells (p < 0.05). The marked expression of Col1α1 and osteocalcin (OCN) osteoblast markers were also observed after 14 days of incubation (p < 0.05). The sinter scaffold supported attachment, viability, and differentiation of preosteoblast cells. Hence, sinter hydroxyapatite scaffold from nacreous layer is a promising biomaterial for bone tissue engineering.

Cloning, characterization, and functional analysis of chitinase-like protein 1 in the shell of Pinctada fucata

Biomineralization, especially shell formation, is a sophisticated process regulated by various matrix proteins. Pinctada fucata chitinase-like protein 1 (Pf-Clp1), which belongs to the GH18 family, was discovered by our group using in-depth proteomic analysis. However, its function is still unclear. In this study, we first obtained the full-length cDNA sequence of Pf-Clp1 by RACE. Real-time polymerase chain reaction results revealed that Pf-Clp1 was highly expressed in the important biomineralization tissues, the mantle edge and the mantle pallial. We expressed and purified recombinant protein rPf-Clp1 in vitro to investigate the function of Pf-Clp1 on CaCO3 crystallization. Scanning electron microscopy imaging and Raman spectroscopy revealed that rPf-Clp1 was able to affect the morphologies of calcite crystal in vitro. Shell notching experiments suggested that Pf-Clp1 might function as a negative regulator during shell formation in vivo. Knockdown of Pf-Clp1 by RNAi led to the overgrowth of aragonite tablets, further confirming its potential negative regulation on biomineralization, especially in the nacreous layer. Our work revealed the potential function of molluscan Clp in shell biomineralization for the first time and unveiled some new understandings toward the molecular mechanism of shell formation.