Shell Structure and Growth in the Base Plate of the Barnacle Amphibalanus amphitrite.

The base plate of the acorn barnacle Amphibalanus amphitrite (equivalent to Balanus amphitrite) is composed of hierarchically scaled, mutually aligned calcite grains, adhered to the substratum via layered cuticular tissue and protein. Acorn barnacles grow by expanding and lengthening their side plates, under which the cuticle is stretched, and adhesive proteins are secreted. In barnacles with mineralized base plates, such as A. amphitrite, a mineralization front follows behind, radially expanding the base plate at the periphery. In this study, we show that the new mineralization develops above the adhesion layers in a unique trilayered structure. Calcite crystallites in each of the layers have distinct sizes, varying from coarse-grained (>1 µm across) in the upper layer, to fine-grained (∼1 µm) in the middle layer, to nanoparticulate (∼40 nm) in the basal layer. The fine-grained crystallites dominate the growth front, comprising the bulk of the shell at the periphery, with later coarse grain development on the top of the base plate (toward the barnacle interior) and nanocrystalline calcite templating underneath in contact with the cuticle/protein layer. While the coarse-grained calcite on the upper surface contains a range of crystal orientations, the underlying fine-grained and nanocrystalline calcite are mutually oriented to within a few degrees of each other. Electron diffraction and X-ray absorption spectroscopy confirm that all of the crystallites are calcite, and metastable aragonite or amorphous calcium carbonate (ACC) phases are not observed. The complex morphology of the leading edge of the base plate suggests that crystallization initiates with the emplacement of mutually aligned fine-grained calcite, followed by the accumulation of coarser grains above and nucleation of highly oriented nanocrystalline grains below.

Growth and development of the barnacle Amphibalanus amphitrite: time and spatially resolved structure and chemistry of the base plate.

The radial growth and advancement of the adhesive interface to the substratum of many species of acorn barnacles occurs underwater and beneath an opaque, calcified shell. Here, the time-dependent growth processes involving various autofluorescent materials within the interface of live barnacles are imaged for the first time using 3D time-lapse confocal microscopy. Key features of the interface development in the striped barnacle, Amphibalanus (= Balanus) amphitrite were resolved in situ and include advancement of the barnacle/substratum interface, epicuticle membrane development, protein secretion, and calcification. Microscopic and spectroscopic techniques provide ex situ material identification of regions imaged by confocal microscopy. In situ and ex situ analysis of the interface support the hypothesis that barnacle interface development is a complex process coupling sequential, timed secretory events and morphological changes. This results in a multi-layered interface that concomitantly fulfills the roles of strongly adhering to a substratum while permitting continuous molting and radial growth at the periphery.

Identifying the dynamic compressive stiffness of a prospective biomimetic elastomer by an inverse method.

Soft elastomeric materials that mimic real soft human tissues are sought to provide realistic experimental devices to simulate the human body's response to blast loading to aid the development of more effective protective equipment. The dynamic mechanical behavior of these materials is often measured using a Kolsky bar because it can achieve both the high strain rates (>100s(-1)) and the large strains (>20%) that prevail in blast scenarios. Obtaining valid results is challenging, however, due to poor dynamic equilibrium, friction, and inertial effects. To avoid these difficulties, an inverse method was employed to determine the dynamic response of a soft, prospective biomimetic elastomer using Kolsky bar tests coupled with high-speed 3D digital image correlation. Individual tests were modeled using finite elements, and the dynamic stiffness of the elastomer was identified by matching the simulation results with test data using numerical optimization. Using this method, the average dynamic response was found to be nearly equivalent to the quasi-static response measured with stress-strain curves at compressive strains up to 60%, with an uncertainty of ±18%. Moreover, the behavior was consistent with the results in stress relaxation experiments and oscillatory tests although the latter were performed at lower strain levels.

Barnacles resist removal by crack trapping.

We study the mechanics of pull-off of a barnacle adhering to a thin elastic layer which is bonded to a rigid substrate. We address the case of barnacles having acorn shell geometry and hard, calcarious base plates. Pull-off is initiated by the propagation of an interface edge crack between the base plate and the layer. We compute the energy release rate of this crack as it grows along the interface using a finite element method. We also develop an approximate analytical model to interpret our numerical results and to give a closed-form expression for the energy release rate. Our result shows that the resistance of barnacles to interfacial failure arises from a crack-trapping mechanism.

Barnacle cement: a polymerization model based on evolutionary concepts.

Enzymes and biochemical mechanisms essential to survival are under extreme selective pressure and are highly conserved through evolutionary time. We applied this evolutionary concept to barnacle cement polymerization, a process critical to barnacle fitness that involves aggregation and cross-linking of proteins. The biochemical mechanisms of cement polymerization remain largely unknown. We hypothesized that this process is biochemically similar to blood clotting, a critical physiological response that is also based on aggregation and cross-linking of proteins. Like key elements of vertebrate and invertebrate blood clotting, barnacle cement polymerization was shown to involve proteolytic activation of enzymes and structural precursors, transglutaminase cross-linking and assembly of fibrous proteins. Proteolytic activation of structural proteins maximizes the potential for bonding interactions with other proteins and with the surface. Transglutaminase cross-linking reinforces cement integrity. Remarkably, epitopes and sequences homologous to bovine trypsin and human transglutaminase were identified in barnacle cement with tandem mass spectrometry and/or western blotting. Akin to blood clotting, the peptides generated during proteolytic activation functioned as signal molecules, linking a molecular level event (protein aggregation) to a behavioral response (barnacle larval settlement). Our results draw attention to a highly conserved protein polymerization mechanism and shed light on a long-standing biochemical puzzle. We suggest that barnacle cement polymerization is a specialized form of wound healing. The polymerization mechanism common between barnacle cement and blood may be a theme for many marine animal glues.