PDMS networks meet barnacles: a complex and often toxic relationship.

The biological impact of chemical formulations used in various coating applications is essential in guiding the development of new materials that directly contact living organisms. To illustrate this point, an investigation addressing the impact of chemical compositions of polydimethylsiloxane networks on a common platform for foul-release biofouling management coatings was conducted. The acute toxicity of network components to barnacle larvae, the impacts of aqueous extracts of crosslinker, silicones and organometallic catalyst on trypsin enzymatic activity, and the impact of assembled networks on barnacle adhesion was evaluated. The outcomes of the study indicate that all components used in the formulation of the silicone network alter trypsin enzymatic activity and have a range of acute toxicity to barnacle larvae. Also, the adhesion strength of barnacles attached to PDMS networks correlates to the network formulation protocol. This information can be used to assess action mechanisms and risk-benefit analysis of PDMS networks.

Comparison of seven methods for DNA extraction from prosomata of the acorn barnacle, Amphibalanus amphitrite.

Next generation sequencing (NGS) technologies can provide an understanding of the molecular processes involved in marine fouling by Amphibalanus spp. barnacles. Here, seven methods for extracting DNA from A. amphitrite prosomata were assessed with respect to recovery, purity and size distribution. Methods incorporating organic extractions generally resulted in low recovery of fragmented DNA. The most promising method was the commercial E.Z.N.A. Blood DNA Mini kit, which provided tens of micrograms of DNA of sufficient molecular weight for use in long-read NGS library preparation. Other kits resulted in DNA preps suitable for short read length NGS platforms.

Resistance of Zwitterionic Peptide Monolayers to Biofouling.

Self-assembled monolayers (SAMs) are widely used in science and engineering, and recent progress has demonstrated the utility of zwitterionic peptides with alternating lysine (K) and glutamic acid (E) residues for antifouling purposes. Aiming at developing a peptide-based fouling-resistant SAM suitable for presentation of surface-attached pheromones for barnacle larvae, we have investigated five different peptide SAMs, where four are based on the EK motif, and the fifth was designed based on general principles for fouling resistance. The SAMs were formed by self-assembly onto gold substrates via cysteine residues on the peptides, and formation of SAMs was verified via ellipsometry, wettability, infrared reflection-absorption spectroscopy and cyclic voltammetry. Settlement of cypris larvae of the barnacle Balanus (=Amphibalanus) amphitrite, the target of pheromone studies, was tested. SAMs were also subjected to fouling assays using protein solutions, blood serum, and the bacterium Mycobacterium marinum. The results confirm the favorable antifouling properties of EK-containing peptides in most of the assays, although this did not apply to the barnacle larvae settlement test, where settlement was low on only one of the peptide SAMs. The one peptide that had antifouling properties for barnacles did not contain a pheromone motif, and would not be susceptible to degredation by common serine proteases. We conclude that the otherwise broadly effective antifouling properties of EK-containing peptide SAMs is not directly applicable to barnacles, and that great care must be exercised in the design of peptide-based SAMs for presentation of barnacle-specific ligands.

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

Mobile barnacle cypris larvae settle and metamorphose, transitioning to sessile juveniles with morphology and growth similar to that of adults. Because biofilms exist on immersed surfaces on which they attach, barnacles must interact with bacteria during initial attachment and subsequent growth. The objective of this study was to characterize the developing interface of the barnacle and substratum during this key developmental transition to inform potential mechanisms that promote attachment. The interface was characterized using confocal microscopy and fluorescent dyes to identify morphological and chemical changes to the interface and the status of bacteria present as a function of barnacle developmental stage. Staining revealed patchy material containing proteins and nucleic acids, reactive oxygen species amidst developing cuticle, and changes in bacteria viability at the developing interface. We found that as barnacles metamorphose from the cyprid to juvenile stage, proteinaceous materials with the appearance of coagulated liquid were released into and remained at the interface. It stained positive for proteins, including phosphoprotein, as well as nucleic acids. Regions of the developing cuticle and the patchy material itself stained for reactive oxygen species. Bacteria were absent until the cyprid was firmly attached, but populations died as barnacle development progressed. The oxidative environment may contribute to the cytotoxicity observed for bacteria and has the potential for oxidative crosslinking of cuticle and proteinaceous materials at the interface.

Imaging Active Surface Processes in Barnacle Adhesive Interfaces.

Surface plasmon resonance imaging (SPRI) and voltammetry were used simultaneously to monitor Amphibalanus (=Balanus) amphitrite barnacles reattached and grown on gold-coated glass slides in artificial seawater. Upon reattachment, SPRI revealed rapid surface adsorption of material with a higher refractive index than seawater at the barnacle/gold interface. Over longer time periods, SPRI also revealed secretory activity around the perimeter of the barnacle along the seawater/gold interface extending many millimeters beyond the barnacle and varying in shape and region with time. Ex situ experiments using attenuated total reflectance infrared (ATR-IR) spectroscopy confirmed that reattachment of barnacles was accompanied by adsorption of protein to surfaces on similar time scales as those in the SPRI experiments. Barnacles were grown through multiple molting cycles. While the initial reattachment region remained largely unchanged, SPRI revealed the formation of sets of paired concentric rings having alternately darker/lighter appearance (corresponding to lower and higher refractive indices, respectively) at the barnacle/gold interface beneath the region of new growth. Ex situ experiments coupling the SPRI imaging with optical and FTIR microscopy revealed that the paired rings coincide with molt cycles, with the brighter rings associated with regions enriched in amide moieties. The brighter rings were located just beyond orifices of cement ducts, consistent with delivery of amide-rich chemistry from the ducts. The darker rings were associated with newly expanded cuticle. In situ voltammetry using the SPRI gold substrate as the working electrode revealed presence of redox active compounds (oxidation potential approx 0.2 V vs Ag/AgCl) after barnacles were reattached on surfaces. Redox activity persisted during the reattachment period. The results reveal surface adsorption processes coupled to the complex secretory and chemical activity under barnacles as they construct their adhesive interfaces.

Effects of Toxic Leachate from Commercial Plastics on Larval Survival and Settlement of the Barnacle Amphibalanus amphitrite.

Plastic pollution represents a major and growing global problem. It is well-known that plastics are a source of chemical contaminants to the aquatic environment and provide novel habitats for marine organisms. The present study quantified the impacts of plastic leachates from the seven categories of recyclable plastics on larval survival and settlement of barnacle Amphibalanus (=Balanus) amphitrite. Leachates from plastics significantly increased barnacle nauplii mortality at the highest tested concentrations (0.10 and 0.50 m(2)/L). Hydrophobicity (measured as surface energy) was positively correlated with mortality indicating that plastic surface chemistry may be an important factor in the effects of plastics on sessile organisms. Plastic leachates significantly inhibited barnacle cyprids settlement on glass at all tested concentrations. Settlement on plastic surfaces was significantly inhibited after 24 and 48 h, but settlement was not significantly inhibited compared to the controls for some plastics after 72-96 h. In 24 h exposure to seawater, we found larval toxicity and inhibition of settlement with all seven categories of recyclable commercial plastics. Chemical analysis revealed a complex mixture of substances released in plastic leachates. Leaching of toxic compounds from all plastics should be considered when assessing the risks of plastic pollution.

Incorporation of silicone oil into elastomers enhances barnacle detachment by active surface strain.

Silicone-oil additives are often used in fouling-release silicone coatings to reduce the adhesion strength of barnacles and other biofouling organisms. This study follows on from a recently reported active approach to detach barnacles, which was based on the surface strain of elastomeric materials, by investigating a new, dual-action approach to barnacle detachment using Ecoflex®-based elastomers incorporated with poly(dimethylsiloxane)-based oil additives. The experimental results support the hypothesis that silicone-oil additives reduce the amount of substratum strain required to detach barnacles. The study also de-coupled the two effects of silicone oils (ie surface-activity and alteration of the bulk modulus) and examined their contributions in reducing barnacle adhesion strength. Further, a finite element model based on fracture mechanics was employed to qualitatively understand the effects of surface strain and substratum modulus on barnacle adhesion strength. The study demonstrates that dynamic substratum deformation of elastomers with silicone-oil additives provides a bifunctional approach towards management of biofouling by barnacles.

Dynamic surface deformation of silicone elastomers for management of marine biofouling: laboratory and field studies using pneumatic actuation.

Many strategies have been developed to improve the fouling release (FR) performance of silicone coatings. However, biofilms inevitably build on these surfaces over time. Previous studies have shown that intentional deformation of silicone elastomers can be employed to detach biofouling species. In this study, inspired by the methods used in soft-robotic systems, controlled deformation of silicone elastomers via pneumatic actuation was employed to detach adherent biofilms. Using programmed surface deformation, it was possible to release > 90% of biofilm from surfaces in both laboratory and field environments. A higher substratum strain was required to remove biofilms accumulated in the field environment as compared with laboratory-grown biofilms. Further, the study indicated that substratum modulus influences the strain needed to de-bond biofilms. Surface deformation-based approaches have potential for use in the management of biofouling in a number of technological areas, including in niche applications where pneumatic actuation of surface deformation is feasible.

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.

Confocal microscopy-based goniometry of barnacle cyprid permanent adhesive.

Biological adhesives are materials of particular interest in the fields of bio-inspired technology and antifouling research. The adhesive of adult barnacles has received much attention over the years; however, the permanent adhesive of the cyprid - the colonisation stage of barnacles - is a material about which very little is presently known. We applied confocal laser-scanning microscopy to the measurement of contact angles between the permanent adhesive of barnacle cyprid larvae and self-assembled monolayers of OH- and CH3-terminated thiols. Measurement of contact angles between actual bioadhesives and surfaces has never previously been achieved and the data may provide insight into the physicochemical properties and mechanism of action of these functional materials. The adhesive is a dual-phase system post-secretion, with the behaviour of the components governed separately by the surface chemistry. The findings imply that the cyprid permanent adhesion process is more complex than previously thought, necessitating broad re-evaluation of the system. Improved understanding will have significant implications for the production of barnacle-resistant coatings as well as development of bio-inspired glues for niche applications.

Barnacle Balanus amphitrite adheres by a stepwise cementing process.

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.

Characterization of the adhesive plaque of the barnacle Balanus amphitrite: amyloid-like nanofibrils are a major component.

The nanoscale morphology and protein secondary structure of barnacle adhesive plaques were characterized using atomic force microscopy (AFM), far-UV circular dichroism (CD) spectroscopy, transmission Fourier transform infrared (FTIR) spectroscopy, and Thioflavin T (ThT) staining. Both primary cement (original cement laid down by the barnacle) and secondary cement (cement used for reattachment) from the barnacle Balanus amphitrite (= Amphibalanus amphitrite) were analyzed. Results showed that both cements consisted largely of nanofibrillar matrices having similar composition. Of particular significance, the combined results indicate that the nanofibrillar structures are consistent with amyloid, with globular protein components also identified in the cement. Potential properties, functions, and formation mechanisms of the amyloid-like nanofibrils within the adhesive interface are discussed. Our results highlight an emerging trend in structural biology showing that amyloid, historically associated with disease, also has functional roles.

Amphibalanus amphitrite begins exoskeleton mineralization within 48 hours of metamorphosis.

Barnacles are ancient arthropods that, as adults, are surrounded by a hard, mineralized, outer shell that the organism produces for protection. While extensive research has been conducted on the glue-like cement that barnacles use to adhere to surfaces, less is known about the barnacle exoskeleton, especially the process by which the barnacle exoskeleton is formed. Here, we present data exploring the changes that occur as the barnacle cyprid undergoes metamorphosis to become a sessile juvenile with a mineralized exoskeleton. Scanning electron microscope data show dramatic morphological changes in the barnacle exoskeleton following metamorphosis. Energy-dispersive X-ray spectroscopy indicates a small amount of calcium (8%) 1 h post-metamorphosis that steadily increases to 28% by 2 days following metamorphosis. Raman spectroscopy indicates calcite in the exoskeleton of a barnacle 2 days following metamorphosis and no detectable calcium carbonate in exoskeletons up to 3 h post-metamorphosis. Confocal microscopy indicates during this 2 day period, barnacle base plate area and height increases rapidly (0.001 mm2 h-1 and 0.30 µm h-1, respectively). These results provide critical information into the early life stages of the barnacle, which will be important for developing an understanding of how ocean acidification might impact the calcification process of the barnacle exoskeleton.

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.

Nanoscale structures and mechanics of barnacle cement.

Polymerized barnacle glue was studied by atomic force microscopy (AFM), scanning electron microscopy (SEM), Fourier transform infrared (FTIR) spectroscopy and chemical staining. Nanoscale structures exhibiting rod-shaped, globular and irregularly-shaped morphologies were observed in the bulk cement of the barnacle Amphibalanus amphitrite (=Balanus amphitrite) by AFM. SEM coupled with energy dispersive X-ray (EDX) provided chemical composition information, making evident the organic nature of the rod-shaped nanoscale structures. FTIR spectroscopy gave signatures of beta-sheet and random coil conformations. The mechanical properties of these nanoscale structures were also probed using force spectroscopy and indentation with AFM. Indentation data yielded higher elastic moduli for the rod-shaped structures when compared with the other structures in the bulk cement. Single molecule AFM force-extension curves on the matrix of the bulk cement often exhibited a periodic sawtooth-like profile, observed in both the extend and retract portions of the force curve. Rod-shaped structures stained with amyloid protein-selective dyes (Congo red and thioflavin-T) revealed that about 5% of the bulk cement were amyloids. A dominant 100 kDa cement protein was found to be mechanically agile, using repeating hydrophobic structures that apparently associate within the same protein or with neighbors, creating toughness on the 1-100 nm length scale.

Adhesion of acorn barnacles on surface-active borate glasses.

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+, Mg2+ and BO43-) 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'.

Base plate mechanics of the barnacle Balanus amphitrite (=Amphibalanus amphitrite).

The mechanical properties of barnacle base plates were measured using a punch test apparatus, with the purpose of examining the effect that the base plate flexural rigidity may have on adhesion mechanics. Base plate compliance was measured for 43 Balanus amphitrite (=Amphibalanus amphitrite) barnacles. Compliance measurements were used to determine flexural rigidity (assuming a fixed-edge circular plate approximation) and composite modulus of the base plates. The barnacles were categorized by age and cement type (hard or gummy) for statistical analyses. Barnacles that were 'hard' (> or =70% of the base plate thin, rigid cement) and 'gummy' (>30% of the base plate covered in compliant, tacky cement) showed statistically different composite moduli but did not show a difference in base plate flexural rigidity. The average flexural rigidity for all barnacles was 0.0020 Nm (SEM +/- 0.0003). Flexural rigidity and composite modulus did not differ significantly between 3-month and 14-month-old barnacles. The relatively low flexural rigidity measured for barnacles suggests that a rigid punch approximation is not sufficient to account for the contributions to adhesion mechanics due to flexing of real barnacles during release.

Inhibition of barnacle (Amphibalanus amphitrite) cyprid settlement by means of localized, pulsed electric fields.

The increasing needs for environmental friendly antifouling coatings have led to investigation of new alternatives for replacing copper and TBT-based paints. In this study, results are presented from larval settlement assays of the barnacle Amphibalanus (= Balanus) amphitrite on planar, interdigitated electrodes (IDE), having 8 or 25 mum of inter-electrode spacing, upon the application of pulsed electric fields (PEF). Using pulses of 100 ms in duration, 200 Hz in frequency and 10 V in pulse amplitude, barnacle settlement below 5% was observed, while similar IDE surfaces without pulse application had an average of 40% settlement. The spacing between the electrodes did not affect cyprid settlement. Assays with lower PEF amplitudes did not show significant settlement inhibition. On the basis of the settlement assays, the calculated minimum energy requirement to inhibit barnacle settlement is 2.8 W h m(-2).

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

Marine macrofoulers (e.g., barnacles, tubeworms, mussels) create underwater adhesives capable of attaching themselves to almost any material. The difficulty in removing these organisms frustrates maritime and oceanographic communities, and fascinates biomedical and industrial communities seeking synthetic adhesives that cure and hold steadfast in aqueous environments. Protein analysis can reveal the chemical composition of natural adhesives; however, developing synthetic analogs that mimic their performance remains a challenge due to an incomplete understanding of adhesion processes. Here, it is shown that acorn barnacles (Amphibalanus (=Balanus) amphitrite) secrete a phase-separating fluid ahead of growth and cement deposition. This mixture consists of a phenolic laden gelatinous phase that presents a phase rich in lipids and reactive oxygen species at the seawater interface. Nearby biofilms rapidly oxidize and lift off the surface as the secretion advances. While phenolic chemistries are ubiquitous to arthropod adhesives and cuticles, the findings demonstrate that A. amphitrite uses these chemistries in a complex surface-cleaning fluid, at a substantially higher relative abundance than in its adhesive. The discovery of this critical step in underwater adhesion represents a missing link between natural and synthetic adhesives, and provides new directions for the development of environmentally friendly biofouling solutions.

Characterization of longitudinal canal tissue in the acorn barnacle Amphibalanus amphitrite.

The morphology and composition of tissue located within parietal shell canals of the barnacle Amphibalanus amphitrite are described. Longitudinal canal tissue nearly spans the length of side shell plates, terminating near the leading edge of the specimen basis in proximity to female reproductive tissue located throughout the peripheral sub-mantle region, i.e. mantle parenchyma. Microscopic examination of stained longitudinal canal sections reveal the presence of cell nuclei as well as an abundance of micron-sized spheroids staining positive for basic residues and lipids. Spheroids with the same staining profile are present extensively in ovarioles, particularly within oocytes which are readily identifiable at various developmental stages. Mass spectrometry analysis of longitudinal canal tissue compared to tissue collected from the mantle parenchyma reveals a nearly 50% overlap of the protein profile with the greatest number of sequence matches to vitellogenin, a glycolipoprotein playing a key role in vitellogenesis-yolk formation in developing oocytes. The morphological similarity and proximity to female reproductive tissue, combined with mass spectrometry of the two tissues, provides compelling evidence that one of several possible functions of longitudinal canal tissue is supporting the female reproductive system of A. amphitrite, thus expanding the understanding of the growth and development of this sessile marine organism.