Influence of temperature and pearl rotation on biomineralization in the pearl oyster, Pinctada margaritifera.

The objective of this study was to observe the impact of temperature on pearl formation using an integrative approach describing the rotation of the pearls, the rate of nacre deposition, the thickness of the aragonite tablets and the biomineralizing potential of the pearl sac tissue though the expression level of some key genes. Fifty pearl oysters were grafted with magnetized nuclei to allow the rotation of the pearls to be described. Four months later, 32 of these pearl oysters were exposed to four temperatures (22, 26, 30 and 34°C) for 2 weeks. Results showed that the rotation speed differed according to the movement direction: pearls with axial movement had a significantly higher rotation speed than those with random movement. Pearl growth rate was influenced by temperature, with a maximum between 26 and 30°C but almost no growth at 34°C. Lastly, among the nine genes implicated in the biomineralization process, only Pmarg-Pif177 expression was significantly modified by temperature. These results showed that the rotation speed of the pearls was not linked to pearl growth or to the expression profiles of biomineralizing genes targeted in this study. On the basis of our results, we consider that pearl rotation is a more complex process than formerly thought. Mechanisms involved could include a strong environmental forcing in immediate proximity to the pearl. Another implication of our findings is that, in the context of ocean warming, pearl growth and quality can be expected to decrease in pearl oysters exposed to temperatures above 30°C.

Damage-tolerance strategies for nacre tablets.

Nacre, a natural armor, exhibits prominent penetration resistance against predatory attacks. Unraveling its hierarchical toughening mechanisms and damage-tolerance design strategies may provide significant inspiration for the pursuit of high-performance artificial armors. In this work, relationships between the structure and mechanical performance of nacre were investigated. The results show that other than their brick-and-mortar structure, individual nacre tablets significantly contribute to the damage localization of nacre. Affected by intracrystalline organics, the tablets exhibit a unique fracture behavior. The synergistic action of the nanoscale deformation mechanisms increases the energy dissipation efficiency of the tablets and contributes to the preservation of the structural and functional integrity of the shell.

Tissue inhibitor of metalloproteinase gene from pearl oyster Pinctada martensii participates in nacre formation.

Tissue inhibitors of metalloproteinases (TIMPs) are nature inhibitors of matrix metalloproteinases and play a vital role in the regulation of extracellular matrix turnover, tissue remodeling and bone formation. In this study, the molecular characterization of TIMP and its potential function in nacre formation was described in pearl oyster Pinctada martensii. The cDNA of TIMP gene in P. martensii (Pm-TIMP) was 901 bp long, containing a 5' untranslated region (UTR) of 51 bp, a 3' UTR of 169 bp, and an open reading fragment (ORF) of 681 bp encoding 226 amino acids with an estimated molecular mass of 23.37 kDa and a theoretical isoelectric point of 5.42; The predicted amino acid sequence had a signal peptide, 13 cysteine residues, a N-terminal domain and a C-terminal domain, similar to that from other species. Amino acid multiple alignment showed Pm-TIMP had the highest (41%) identity to that from Crassostrea gigas. Tissue expression analysis indicated Pm-TIMP was highly expressed in nacre formation related-tissues, including mantle and pearl sac. After decreasing Pm-TIMP gene expression by RNA interference (RNAi) technology in the mantle pallium, the inner nacreous layer of the shells showed a disordered growth. These results indicated that the obtained Pm-TIMP in this study participated in nacre formation.

The organic interlamellar layer in abalone nacre: Formation and mechanical response.

The interlamellar organic layer plays a key role in establishing the tensile mechanical response of nacre, while changing the compressive response in only a marginal manner. We conduct observations on the epithelial layer of the abalone foot in direct contact with the extrapallial layer where the deposition process takes place and identify cilia, microvilli, and secretory cells which determine the deposition of chitin to form the interlamellar organic layer. On the basis of these observations we propose a mechanism for the deposition of interlamellar organic layers. We show that the fraction of pores, as well as their diameter, grow rapidly as the interlamellar layer is extended uniaxially or biaxially, and compare these calculations with the observed values. In the calculations we assume a Poisson's ratio equal to zero in the plane of the lamellae. This assumption is justified by the thickness of the organic layer (20-50 nm) being equal to twice the diameter of the chitin fibrils; consequently the expansion of the membrane occurs at a constant thickness. As an illustration of this effect, an externally applied strain of 0.5 increases the initial pore diameter (typically equal to 20-50 nm) to a value of ten times (0.2-0.5 µm). These calculations explain the observations of large pores reported in the literature and interpret them as the result of externally applied loads.

Strain rate hardening: a hidden but critical mechanism for biological composites?

Natural materials such as nacre, bone, collagen and spider silk boast unusual combinations of stiffness, strength and toughness. Behind this performance is a staggered microstructure, which consists of stiff and elongated inclusions embedded in a softer and more deformable matrix. The micromechanics of deformation and failure associated with this microstructure are now well understood at the "unit cell" level, the smallest representative volume for this type of material. However, these mechanisms only translate to high performance if they propagate throughout large volumes, an important condition which is often overlooked. Here we present, for the first time, a model which captures the conditions for either spreading of deformations or localization, which determines whether a staggered composite is brittle or deformable at the macroscale. The macroscopic failure strain for the material was calculated as function of the viscoplastic properties of the interfaces and the severity of the defect. As expected, larger strains at failure can be achieved when smaller defects are present within the material, or with more strain hardening at the interface. The model also shows that strain rate hardening is a powerful source of large deformations for the material as well, a result we confirmed and validated with tensile experiments on glass-polydimethylsiloxane (PDMS) nacre-like staggered composites. An important implication is that natural materials, largely made of rate-dependent materials, could rely on strain rate hardening to tolerate initial defects and damage to maintain their functionality. Strain rate hardening could also be harnessed and optimized in bio-inspired composites in order to maximize their overall performance.

Organic interlamellar layers, mesolayers and mineral nanobridges: contribution to strength in abalone (Haliotis rufescence) nacre.

The contributions of mesolayers, organic interlamellar layers and nanoasperities/mineral bridges to the strength of nacre from red abalone (Haliotis rufescens) shell nacre are investigated. Samples were demineralized and deproteinized to separate the organic and mineral components, respectively. Tensile tests were performed on both the isolated organic constituent and the isolated mineral. The strength of the isolated organic component suggests that growth bands play an important role in the mechanical behavior as they are thick regions of protein that are a significant fraction (∼0.4) of the total organic content. The thickness variation of the nacre tablets was measured and found to be a small fraction of the mean tablet thickness (0.568µm); the standard deviation is 26nm, indicating that the wedge mechanism of toughening does not operate in the nacre investigated. Results obtained from the isolated mineral validate the importance of the organic constituent as the mechanical properties decline greatly when the organic component is removed. The results presented herein add to the understanding of the mechanical response of the organic interlayers and growth bands and their effect on the toughness of the abalone nacre.

Temperature and food influence shell growth and mantle gene expression of shell matrix proteins in the pearl oyster Pinctada margaritifera.

In this study, we analyzed the combined effect of microalgal concentration and temperature on the shell growth of the bivalve Pinctada margaritifera and the molecular mechanisms underlying this biomineralization process. Shell growth was measured after two months of rearing in experimental conditions, using calcein staining of the calcified structures. Molecular mechanisms were studied though the expression of 11 genes encoding proteins implicated in the biomineralization process, which was assessed in the mantle. We showed that shell growth is influenced by both microalgal concentration and temperature, and that these environmental factors also regulate the expression of most of the genes studied. Gene expression measurement of shell matrix protein thereby appears to be an appropriate indicator for the evaluation of the biomineralization activity in the pearl oyster P. margaritifera under varying environmental conditions. This study provides valuable information on the molecular mechanisms of mollusk shell growth and its environmental control.