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.