Abalone Shell Research Articles

The interstitial crystal-nucleating sheet in molluscan Haliotis rufescens shell: A bio-polymeric composite

The interstitial green sheets in abalone shell nacre are shown to be bifacially differentiated trilaminate polymeric complexes, with glycoprotein layers sandwiching a central core containing chitin. They share some common feature with the organic matrix layers between the aragonite tablets in the nacre and the periostracum, and show similarities to the myostracum. Thus, although the green sheet is reported to be unique to the abalone shell, it represents an interesting model for the study of molluscan shell biomineralization processes. Indeed, during shell formation, prismatic and spherulitic aragonite precedes and follows the deposition of the interstitial green polymeric composite sheets, and there is evidence to suggest that these sheets demark the interruption of nacre synthesis and serve to nucleate the resumption of calcium carbonate crystal growth. The green polymeric interstitial sheet purified from the abalone shell was investigated by spectroscopic and imaging techniques: FTIR, confocal microscopy, scanning and transmission electron microscopy, and by pyrolysis combined with GC-MS. Structural and compositional differences are observed between the surfaces of the two sides of the interstitial polymeric composite sheets. Moreover, comparative crystallization experiments on the green sheet sides also reveal asymmetry with respect to the nucleation of calcium carbonate. These findings suggest that these bifacially differentiated interstitial composites may play an active role in the mineral assembly processes, with one of the surfaces acting as a crystal nucleator.

Bio-inspired passive actuator simulating an abalone shell mechanism for structuralcontrol

An energy dispersion mechanism called ‘sacrificial bonds and hidden length’, which is foundin some biological systems, such as abalone shells and bones, is the inspiration fornew strategies for structural control. Sacrificial bonds and hidden length cansubstantially increase the stiffness and enhance energy dissipation in the constituentmolecules of abalone shells and bone. Having been inspired by the usefulness andeffectiveness of such a mechanism, which has evolved over millions of years andcountless cycles of evolutions, the authors employ the conceptual underpinnings ofthis mechanism to develop a bio-inspired passive actuator. This paper presents afundamental method for optimally designing such bio-inspired passive actuators forstructural control. To optimize the bio-inspired passive actuator, a simple methodutilizing the force–displacement–velocity (FDV) plots based on LQR control isproposed. A linear regression approach is adopted in this research to find theinitial values of the desired parameters for the bio-inspired passive actuator. Theillustrative examples, conducted by numerical simulation with experimental validation,suggest that the bio-inspired passive actuator based on sacrificial bonds and hiddenlength may be comparable in performance to state-of-the-art semi-active actuators.

ELNES Investigations of Interfaces in Abalone Shell

Extended abstract of a paper presented at Microscopy and Microanalysis 2010 in Portland, Oregon, USA, August 1 – August 5, 2010.

Proteomic analysis of the organic matrix of the abalone Haliotis asinina calcified shell

BackgroundThe formation of the molluscan shell is regulated to a large extent by a matrix of extracellular macromolecules that are secreted by the shell forming tissue, the mantle. This so called "calcifying matrix" is a complex mixture of proteins and glycoproteins that is assembled and occluded within the mineral phase during the calcification process. While the importance of the calcifying matrix to shell formation has long been appreciated, most of its protein components remain uncharacterised.ResultsRecent expressed sequence tag (EST) investigations of the mantle tissue from the tropical abalone (Haliotis asinina) provide an opportunity to further characterise the proteins in the shell by a proteomic approach. In this study, we have identified a total of 14 proteins from distinct calcified layers of the shell. Only two of these proteins have been previously characterised from abalone shells. Among the novel proteins are several glutamine- and methionine-rich motifs and hydrophobic glycine-, alanine- and acidic aspartate-rich domains. In addition, two of the new proteins contained Kunitz-like and WAP (whey acidic protein) protease inhibitor domains.ConclusionThis is one of the first comprehensive proteomic study of a molluscan shell, and should provide a platform for further characterization of matrix protein functions and interactions.

Mechanical properties and structure of Haliotis discus hannai Ino and Hemifusus tuba conch shells: a comparative study

Haliotis discus hannai Ino (abalone shell) and Hemifusus tuba conch shell have been studied for the purpose to comparatively investigate the mechanisms by which nature designs composites. It is shown that both shells are composed of aragonite and a small amount of proteins while the conch shell shows finer microstructure but lower strength than abalone shell. It is also shown that the fresh shells exhibits better property than those after heat-treatments. It is therefore supposed that the size of inorganic substance is not a dominant factor to improve strength, while both proteins in shells and the microstructure of inorganic matter also play important roles.

Chitinase of Bacillus licheniformis from oyster shell as a probe to detect chitin in marine shells

Bacillus licheniformis CBFOS-03 is a chitinase producing bacteria isolated from oyster (Crassostrea gigas) shell waste. We have cloned and expressed the chi18B gene of B. licheniformis CBFOS-03, which encodes a glycohydrolase family 18 chitinase (GH18). Chi18B is a predicted 598 amino acid protein that consists of a catalytic domain (GH18), a fibronectin type III domain (Fn3), and a chitin binding domain (CBD). Purified Chi18B showed optimum chitinase activity at pH 9 and 55 degrees C, and activity was stimulated with 25 mM Mn2+. In kinetic analysis, Chi18B showed Km values of 9.07 +/- 0.65 microM and 129.27 +/- 0.38 microM with the substrates 4-methylumbelliferyl-N-N'-diacetylchitobiose and alpha-chitin, respectively. Studies of C-terminal deletion constructs revealed that the GH18 domain with one amino acid in C-terminal region was sufficient for chitinase activity; however, fusions of full length and CBD-deleted constructs to green florescent protein (GFP) and yellow florescent protein (YFP) suggest that the C-terminus is supposedly important in binding to shell powder. Full length Chi18B with GFP showed green fluorescence with oyster shell powder, but GH18+Fn3 with GFP did not. Similarly, full length Chi18B with YFP showed yellow fluorescence with clam (Chamelea gallina) shell and disk abalone (Haliotis discus) shell powder, but GH18+Fn3 with YFP construct did not. So, the CBD domain of Chi18B appears to play an important role in binding of oyster and other marine shells. It is likely to be used as a probe to identify the presence of chitin in marine shells like oyster shell, clam shell, and disk abalone shell using fusions of Chi18B with fluorescent proteins.

SAWTOOTH BEHAVIOUR OF NATURAL BIO-MACROMOLECULES AND SYNTHETIC POLYMERS

In this paper, we present molecular dynamic simulation of natural and synthetic polymers, including tropocollagen (TC), Lustrin A and poly-acrylic acid (PAA), located inside the bone, abalone shells and bio-mimetic materials, respectively. The objective of these simulations is to investigate the well-known `sawtooth' behaviour observed in atomic force microscopy (AFM) experiments of bio-minerals. We have observed the sawtooth behaviour in the force-extension curves of Lustrin A and PAA only when their interactions with calcareous minerals are considered. On the other hand, the TC does not show the sawtooth behaviour even in the presence of calcareous minerals. We have also simulated a NC1 α-helix and a β-sheet domain to investigate the structural effect on the sawtooth behaviour. Based on these simulation results, we propose a new toughening mechanism that the electrostatic force in the organic–inorganic interface plays a critical role in the sawtooth behaviour. These simulated phenomena can explain some of the AFM experiment observation and provide insight into the molecular mechanism of the super toughness of bio-minerals.

The role of organic intertile layer in abalone nacre

Characterization of the growth surfaces removed from red and green abalone ( Haliotis) shells shows a terraced cone mode of mineralization in which the organic layer is deposited periodically and regulates the formation of tiles with ~ 500 nm thickness. The details of the mineral and organic layer surface are revealed by atomic force microscopy; the surface roughness and the thickness of the tiles in the terraces and organic intertile layer were measurement. Nanoindentation experiments at the top of the terraced cones confirm a hardness of the same order as that of completely mineralized surfaces. Indentation of the organic layer provides a force-deflection curve that can be expressed as tension on a centrally-loaded membrane. The results show that the dry organic layer is very stiff and deforms inelastically or cracks under the indenter, whereas in the fully hydrated state it shows a low modulus and strength and great extensibility. This strongly suggests that this organic interlayer acquires considerable strength and stiffness as a result of the drying process, which is consistent with a T g of approximately 200 °C for chitin. The chitin network that forms the structural component of the intertile layer is revealed and the orientation and spacing are measured. Terraced cones broken under the force of a flexing and shrinking organic layer enable the estimation of the tensile strength of the abalone when loaded through the fracture of the mineral bridges. Calculations show consistency with earlier tensile strength measurements of < 10 MPa.

Fishing from past to present: continuity and resilience of red abalone fisheries on the Channel Islands, California

Archaeological data from coastal shell middens provide a window into the structure of ancient marine ecosystems and the nature of human impacts on fisheries that often span millennia. For decades Channel Island archaeologists have studied Middle Holocene shell middens visually dominated by large and often whole shells of the red abalone (Haliotis rufescens). Here we use modern ecological data, historical accounts, commercial red abalone catch records, and zooarchaeological data to examine long-term spatial and temporal variation in the productivity of red abalone fisheries on the Northern Channel Islands, California (USA). Historical patterns of abundance, in which red abalone densities increase from east to west through the islands, extend deep into the Holocene. The correlation of historical and archaeological data argue for long-term spatial continuity in productive red abalone fisheries and a resilience of abalone populations despite dramatic ecological changes and intensive human predation spanning more than 8000 years. Archaeological, historical, and ecological data suggest that California kelp forests and red abalone populations are structured by a complex combination of top-down and bottom-up controls.

Preparation of Calcite and Aragonite Complex Layer Materials Inspired from Biomineralization

Abalone shell is natural inorganic/organic hybrid material, which is biologically constructed by using two calcium carbonates, aragonite and calcite. Aragonite can provide an enhanced mechanical support for the shell, and the stable phase, calcite, acts as the outer layer. In the current study, large-area aragonite film is deposited onto a calcite film in the presence of magnesium ion to biomimetically construct an aragonite−calcite complex structure. A calcite film is fabricated on a silicon wafer by a controlled phase transformation of amorphous calcium carbonate film at 120 °C, which is used as a substrate to induce the deposition of the aragonite layer with the addition of magnesium ion. We demonstrated the importance of the transition process from calcite to aragonite during the formation of the aragonite−calcite complex layer. This study could be used to mimic a sharp transition process of calcite to aragonite in vitro without any organic macromolecules.

Crystal orientations in nacreous layers of organic–inorganic biocomposites

Abalone shell comprises a bio-composite material, combining the properties of inorganic calcite intergrown with organic nacre. This paper reports about the microstructure of this composite. By examining the Kikuchi patterns obtained for nacre ( Haliotis discus hannai) using transmission electron microscopy, we have shown that the tiles within nacre have specific orientations. The stereographic projection spheres for the tiles of nacre can be divided into two main types, namely a right oriented region and a left oriented region with respect to the c axis as a reference plane (001). The cluster character of nacre can be explained in terms of the growth mechanism of the ‘Christmas tree’ pattern. The orientation of the c-axis in the nacreous layer is elucidated for the first time. We demonstrate the use of the soluble protein obtained from the tiles of nacre in in vitro calcium carbonate crystallization.

Bioinspired Inorganic/polymer Thin Films

AbstractStudies of hard biological materials such as marine shells, animal teeth, horns and bones have produced fascinating ideas for mimicking their micro/nanostructure in the lab. The nacre in the abalone shell has a well-defined organic/inorganic structure that has a fracture resistance that is much higher than the individual constituents. By using biocompatible materials we have fabricated zirconium nitride/ polymethylmethacrylate alternating layers that are based on the structure of nacre. A combination of DC-magnetron sputtering and pulsed laser deposition on (100) silicon substrates was used to fabricate multilayers in a single chamber without breaking the vacuum. The ZrN films showed nanocrystalline columnar growth on the silicon substrates or on the PMMA nanolayer. High resolution SEM analysis at the inorganic/organic interface revealed well formed, uniform thickness inorganic films which are separated by the polymeric layer (30-90 nm). The ratio of the ceramic/polymer is the same as in nacre. Nanoindentation hardness values of ˜ 20GPa were measured on both the ZrN single film, similar to published values, and the ZrN/PMMA composite layers and the elastic modulus remained constant, independent of the number of layers.

Effect of eight benthic diatoms as feed on the growth of red abalone (Haliotis rufescens) postlarvae

The growth rate of abalone post larvae of Haliotis rufescens fed ad libitum with a benthic monoalgal diatom culture maintained as monocultures on a semi-commercial scale, was evaluated and correlated with the biochemical composition of the diatoms. The cell size (7.0 × 4.0 µm to 21.0 × 7.5 µm), protein percentage (7.42% to 13.66%), and ash content (49.03% to 59.61%) were different among diatom strains; lipid percentage, nitrogen free extract, and energy content (Kcal g−1) were similar among diatom strains. The values of essential and non-essential amino and fatty acids composition differed among diatom strains. Differences in the abalone shell length and orthogonal analyses revealed postlarval growth was dependent on the quality of the food source. Postlarvae abalone displaying the longest shell lengths were fed Nitzschia thermalis var. minor and Amphiprora paludosa var. hyalina (1,712.0 ± 61 µm and 1,709 ± 67 µm, respectively), followed by Navicula incerta (1,413.3 ± 43 µm). The fatty acid content of benthic diatoms and abalone growth rate were not correlated.

Organic–inorganic interfaces and spiral growth in nacre

Nacre, the crown jewel of natural materials, has been extensively studied owing to its remarkable physical properties for over 160 years. Yet, the precise structural features governing its extraordinary strength and its growth mechanism remain elusive. In this paper, we present a series of observations pertaining to the red abalone (Haliotis rufescens) shell's organic–inorganic interface, organic interlayer morphology and properties, large-area crystal domain orientations and nacre growth. In particular, we describe unique lateral nano-growths and paired screw dislocations in the aragonite layers, and demonstrate that the organic material sandwiched between aragonite platelets consists of multiple organic layers of varying nano-mechanical resilience. Based on these novel observations and analysis, we propose a spiral growth model that accounts for both [001] vertical propagation via helices that surround numerous screw dislocation cores and simultaneous 〈010〉 lateral growth of aragonite sheet structure. These new findings may aid in creating novel organic–inorganic micro/nano composites through synthetic or biomineralization pathways.

Characterization of the Organic-Inorganic Interface of Abalone Shell Nacre

Extended abstract of a paper presented at Microscopy and Microanalysis 2008 in Albuquerque, New Mexico, USA, August 3 – August 7, 2008

In situ transmission electron microscopy observation of reversible deformation in nacre organic matrix

The deformation behavior of the organic polymer matrix of the biocomposite nacre structure in abalone shell was investigated by in situ straining during transmission electron microscopy (TEM). We observed strong adhesion to mineral plates and high ductility of the organic matrix, confirming a crack-bridging toughening mechanism. In addition, direct observation of reversible mechanical behavior was made in the viscoelastic reformation of matrix ligaments after failure. Crystalline β-sheet structures identified through electron diffraction suggested the presence of protein structures similar to spider or cocoon silk, and the reversible mechanism was attributed to hydration-induced unfolding and refolding of domains in these silklike proteins. This work provides further insight into the molecular and nanoscale behavior of nacre organic matrix and its contribution to bulk mechanical performance.

Human impacts on ancient shellfish: a 10,000 year record from San Miguel Island, California

We use measurements of more than 11,000 marine shells from 41 archaeological components to construct a 10,000 year record of human impacts on ancient mussel and abalone stocks on San Miguel Island, California. General reductions in the mean size of mussel and abalone shells gathered through time are attributed to growing human population and predation pressure. Based on comparison with historically documented changes in shellfish communities caused by the local extinction of sea otters in the 19th century, changes in mean shell size and the abundance of other shellfish species may have been facilitated by Native American predation on sea otters as early as 7500 years ago. Despite having measurable impacts on local ecosystems, Native Americans on San Miguel harvested huge quantities of shellfish throughout the Holocene. Such long-term harvests appear to have been sustained by an early emphasis on fishing at lower trophic levels, by periodically shifting village locations, and by intensifying the use of finfish and sea mammals through time. This pattern of “fishing up the food web” contrasts with many modern fisheries, suggesting that the study of ancient fisheries can help us better manage our own endangered coastal ecosystems.

Structure and mechanical properties of selected biological materials

Mineralized biological tissues offer insight into how nature has evolved these components to optimize multifunctional purposes. These mineral constituents are weak by themselves, but interact with the organic matrix to produce materials with unexpected mechanical properties. The hierarchical structure of these materials is at the crux of this enhancement. Microstructural features such as organized, layered organic/inorganic structures and the presence of porous and fibrous elements are common in many biological components. The organic and inorganic portions interact at the molecular and micro-levels synergistically to enhance the mechanical function. In this paper, we report on recent progress on studies of the abalone and Araguaia river clam shells, arthropod exoskeletons, antlers, tusks, teeth and bird beaks.

Spectral confocal reflection microscopy using a white light source

We present a reflection confocal microscope incorporating a white light supercontinuum source and spectral detection. The microscope provides images resolved spatially in three-dimensions, in addition to spectral resolution covering the wavelength range 450-650 nm. Images and reflection spectra of artificial and natural specimens are presented, showing features that are not normally revealed in conventional microscopes or confocal microscopes using discrete-line lasers. The specimens include thin film structures on semiconductor chips, iridescent structures in Papilio blumei butterfly scales, nacre from abalone shells and opal gemstones. Quantitative size and refractive index measurements of transparent beads are derived from spectral interference bands.

An Historic Chinese Abalone Fishery on California’s Northern Channel Islands

Beginning in the 1850s, Chinese abalone fishermen developed an intensive commercial fishery focused on the abundant black abalone (Haliotis cracherodii) stocks of Alta and Baja California. They systematically harvested and dried tons of abalone meat and shells from intertidal waters and shipped them to markets in mainland China and America. Several legislative attempts were made to curtail Chinese involvement in the fishing industry, claiming they were harvesting abalone without regard to size. Recent research documents the abundance, distribution, and constituents of historic abalone sites and discusses the impact of “Chinese” abalone fishermen on San Miguel Island, California. Thousands of shell measurements show that the Chinese harvested larger abalones than those collected by Native American foragers for 10,000 years prior to European contact, providing important data on local ecology during the early historic period.