SUMMARYEnzymes 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. Supplementary material available online at

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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.

Barnacles adhere permanently to surfaces by secreting and curing a thin interfacial adhesive underwater. Here, we show that the acorn barnacle Balanus amphitrite adheres by a two-step fluid secretion process, both contributing to adhesion. We found that, as barnacles grow, the first barnacle cement secretion (BCS1) is released at the periphery of the expanding base plate. Subsequently, a second, autofluorescent fluid (BCS2) is released. We show that secretion of BCS2 into the interface results, on average, in a 2-fold increase in adhesive strength over adhesion by BCS1 alone. The two secretions are distinguishable both spatially and temporally, and differ in morphology, protein conformation, and chemical functionality. The short time window for BCS2 secretion relative to the overall area increase demonstrates that it has a disproportionate, surprisingly powerful, impact on adhesion. The dramatic change in adhesion occurs without measurable changes in interface thickness and total protein content. A fracture mechanics analysis suggests the interfacial material's modulus or work of adhesion, or both, were substantially increased after BCS2 secretion. Addition of BCS2 into the interface generates highly networked amyloid-like fibrils and enhanced phenolic content. Both intertwined fibers and phenolic chemistries may contribute to mechanical stability of the interface through physically or chemically anchoring interface proteins to the substrate and intermolecular interactions. Our experiments point to the need to reexamine the role of phenolic components in barnacle adhesion, long discounted despite their prevalence in structural membranes of arthropods and crustaceans, as they may contribute to chemical processes that strengthen adhesion through intermolecular cross-linking.

The phase evolution during annealing of AlJNi multilayer samples prepared by ion-beam sputtering with composition modulation wavelengths A between 10 and 400 nm was determined using x-ray diffraction and differential scanning calorimeter measurements. Samples with average compositions of &n,N10,6,, and Al,,,N&,~ were investigated. For the Al,,40Ni0.60 samples the following results were obtained. A measure of the degree of periodic@ and the sharpness of the interfaces in a sample with A=80 mn was the large number (over 20) of peaks observed in small-angle x-ray scattering measurements. A sample with A=10 nm was transformed by heat treatment directly to the AlNi phase. In the A=80 nm sample, the first phase formed after annealing was the metastable Y,I phase. The r] phase was identified as Al,Ni,. In the 400 nm wavelength sample, both the metastable 17 phase and the stable Al,Ni formed after the first exothermic reaction. For the Alr,75Ni0,25 samples two results were obtained. A A=11.4 nm sample transformed directly on annealing into Al,Ni. The 77 phase was the first phase formed on annealing a A=100 nm sample. The difference in the component diffusivities and the concentration gradient play an important role in controlling phase formation and evolution.