Barnacles adhere by producing a mixture of cement proteins (CPs) that organize into a permanently bonded layer displayed as nanoscale fibers. These cement proteins share no homology with any other marine adhesives, and a common sequence-basis that defines how nanostructures function as adhesives remains undiscovered. Here we demonstrate that a significant unidentified portion of acorn barnacle cement is comprised of low complexity proteins; they are organized into repetitive sequence blocks and found to maintain homology to silk motifs. Proteomic analysis of aggregate bands from PAGE gels reveal an abundance of Gly/Ala/Ser/Thr repeats exemplified by a prominent, previously unidentified, 43 kDa protein in the solubilized adhesive. Low complexity regions found throughout the cement proteome, as well as multiple lysyl oxidases and peroxidases, establish homology with silk-associated materials such as fibroin, silk gum sericin, and pyriform spidroins from spider silk. Distinct primary structures defined by homologous domains shed light on how barnacles use low complexity in nanofibers to enable adhesion, and serves as a starting point for unraveling the molecular architecture of a robust and unique class of adhesive nanostructures.

Oxidases are found to play a growing role in providing functional chemistry to marine adhesives for the permanent attachment of macrofouling organisms. Here, we demonstrate active peroxidase and lysyl oxidase enzymes in the adhesive layer of adult Amphibalanus amphitrite barnacles through live staining, proteomic analysis, and competitive enzyme assays on isolated cement. A novel full-length peroxinectin (AaPxt-1) secreted by barnacles is largely responsible for oxidizing phenolic chemistries; AaPxt-1 is driven by native hydrogen peroxide in the adhesive and oxidizes phenolic substrates typically preferred by phenoloxidases (POX) such as laccase and tyrosinase. A major cement protein component AaCP43 is found to contain ketone/aldehyde modifications via 2,4-dinitrophenylhydrazine (DNPH) derivatization, also called Brady's reagent, of cement proteins and immunoblotting with an anti-DNPH antibody. Our work outlines the landscape of molt-related oxidative pathways exposed to barnacle cement proteins, where ketone- and aldehyde-forming oxidases use peroxide intermediates to modify major cement components such as AaCP43.

Acorn barnacles are major marine fouling organisms. Their success is largely due to an ability to adhere to diverse substrates via a sub-micron thick proteinaceous adhesive layer that develops as the organism molts and expands its base. Recent work has expanded the set of proteins identified within the adhesive interface, but one outstanding question concerns their spatial distribution throughout the organism. Here, we employ immunological analysis of Amphibalanus amphitrite tissue sections and identify the presence of two cement proteins, AaCP19-1 and AaCP43-1, in areas far removed from the adhesive interface. Confocal imaging reveals specific staining along different tissue linings of the organism as well as other non-cementing regions. Additionally, we employ a modified, pressure cycling technology approach to recover protein from histological tissue sections to perform proteomics analysis. Mass spectrometry analysis of proteins recovered from transverse histological sections of the upper portion of barnacles indicates the presence of these same proteins, complementing the immunostaining observations. The proteomics analysis also revealed the presence of other proteins first identified in the adhesive layer. While some proteins are clearly enriched at the surface interface, our findings challenge the concept that cement proteins are exclusive to the substrate interface and suggest they may have an expanded physiological role beyond substrate adhesion-related processes of A. amphitrite.

Concerns about the bioaccumulation of toxic antifouling compounds have necessitated the search for alternative strategies to combat marine biofouling. Because many biologically essential minerals have deleterious effects on organisms at high concentration, one approach to preventing the settlement of marine foulers is increasing the local concentration of ions that are naturally present in seawater. Here, we used surface-active borate glasses as a platform to directly deliver ions (Na + , Mg 2+ and BO 4 3− ) to the adhesive interface under acorn barnacles ( Amphibalanus ( =Balanus ) amphitrite ). Additionally, surface-active glasses formed reaction layers at the glass–water interface, presenting another challenge to fouling organisms. Proteomics analysis showed that cement deposited on the gelatinous reaction layers is more soluble than cement deposited on insoluble glasses, indicating the reaction layer and/or released ions disrupted adhesion processes. Laboratory experiments showed that the majority (greater than 79%) of adult barnacles re-attached to silica-free borate glasses for 14 days could be released and, more importantly, barnacle larvae did not settle on the glasses. The formation of microbial biofilms in field tests diminished the performance of the materials. While periodic water jetting (120 psi) did not prevent the formation of biofilms, weekly cleaning did dramatically reduce macrofouling on magnesium aluminoborate glass to levels below a commercial foul-release coating. This article is part of the theme issue ‘Transdisciplinary approaches to the study of adhesion and adhesives in biological systems’.

Hydrolysable borate glasses form surface layers in aqueous environments that can resist barnacle adhesion by physical and/or chemical mechanisms. After 14 d re‐attachment periods, the adhesion strength of barnacles attached to these glasses are comparable to silicone antifouling coatings. Furthermore, the glasses remain optically transparent from 400 to 1700 nm, presenting a promising class of materials for underwater optics.