Barnacle Balanus amphitrite Adheres by a Stepwise Cementing Process

Abstract

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.

Acorn Barnacles Secrete Phase‐Separating Fluid to Clear Surfaces Ahead of Cement Deposition

“…However, the gelatinous phase lacks autofluorescence associated with quinone tanning in cuticular tissues, suggesting these chemistries may help to retain the lipidaceous phase as the secretion spreads. We note that Burden et al detected an increase in phenolic compounds at the adhesive interface after ecdysis 21. While Burden et al attributed the phenolic compounds to BCS2, the surface‐cleaning fluid is also secreted after ecdysis and could have given rise to the spectral changes they observed.…”

Barnacle biology before, during and after settlement and metamorphosis: a study of the interface

“…It is possible that the developing cuticle undergoes tearing, especially in later stages of metamorphosis, concurrent with substantial changes to the barnacle shape. Consistent with the suggestion of oxidative crosslinking at the developing interface, recent studies of adult barnacle interfaces using in situ cyclic voltammetry have revealed electrochemical behavior consistent with the presence of redox active compounds (Golden et al, 2016), as well as spectroscopic evidence for the presence of phenolic compounds in interfacial barnacle fluids (Burden et al, 2012). Quinone tanning can be driven by free radicals or enzymes and mediates crosslinking in diverse systems and contexts, including sclerotized insect cuticle and mussel byssal threads (Korytowski et al, 1987;Waite, 1995;Suderman et al, 2006;Yang et al, 2014).…”

“…The generation of ROS, or free radicals, can explain the observed mortality of bacterial populations (Bandyopadhyay et al, 1999;Cabiscol et al, 2000;Fang, 2004) under the barnacle. The data show that oxidative chemistries were present in several interface features including the cuticle, the patchy material and within a capillary, a feature that delivers one type of barnacle cement secretion to the interface (Burden et al, 2012). The bacterial staining showed that the ROS was largely distinct from the bacteria and from features of the barnacle.…”

“…Juvenile attachment mechanisms have been less well studied, but it was found that a proteinaceous juvenile cement appears to increase adhesion within 2 weeks of settlement and metamorphosis (Yule and Walker, 1984), earlier than the adult cement system is known to develop (40 days later) (Walker, 1973). Much later, adult barnacles use a combination of mostly proteinaceous secretions (Walker, 1971;Burden et al, 2012Burden et al, , 2014, which become insoluble over time (Kamino et al, 1996;Naldrett and Kaplan, 1997;Kamino, 2001;Dickinson et al, 2009).…”

“…Recent insight into the interfacial fluids of barnacles revealed two distinct secretions differing spatiotemporally as the adult barnacle grows, one released at the periphery and the other delivered periodically via the network of capillaries and ducts (Burden et al, 2012) connected to the cement gland (Yule and Walker, 1987). In this study, our driving question was: how does the interface between the barnacle and the substrate change during the morphological transition of cyprid to juvenile?…”