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.

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.

Molt-dependent transcriptomic analysis of cement proteins in the barnacle Amphibalanus amphitrite.

BACKGROUND: A complete understanding of barnacle adhesion remains elusive as the process occurs within and beneath the confines of a rigid calcified shell. Barnacle cement is mainly proteinaceous and several individual proteins have been identified in the hardened cement at the barnacle-substrate interface. Little is known about the molt- and tissue-specific expression of cement protein genes but could offer valuable insight into the complex multi-step processes of barnacle growth and adhesion. METHODS: The main body and sub-mantle tissue of the barnacle Amphibalanus amphitrite (basionym Balanus amphitrite) were collected in pre- and post-molt stages. RNA-seq technology was used to analyze the transcriptome for differential gene expression at these two stages and liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) was used to analyze the protein content of barnacle secretions. RESULTS: We report on the transcriptomic analysis of barnacle cement gland tissue in pre- and post-molt growth stages and proteomic investigation of barnacle secretions. While no significant difference was found in the expression of cement proteins genes at pre- and post-molting stages, expression levels were highly elevated in the sub-mantle tissue (where the cement glands are located) compared to the main barnacle body. We report the discovery of a novel 114kD cement protein, which is identified in material secreted onto various surfaces by adult barnacles and with the encoding gene highly expressed in the sub-mantle tissue. Further differential gene expression analysis of the sub-mantle tissue samples reveals a limited number of genes highly expressed in pre-molt samples with a range of functions including cuticular development, biominerialization, and proteolytic activity. CONCLUSIONS: The expression of cement protein genes appears to remain constant through the molt cycle and is largely confined to the sub-mantle tissue. Our results reveal a novel and potentially prominent protein to the mix of cement-related components in A. amphitrite. Despite the lack of a complete genome, sample collection allowed for extended transcriptomic analysis of pre- and post-molt barnacle samples and identified a number of highly-expressed genes. Our results highlight the complexities of this sessile marine organism as it grows via molt cycles and increases the area over which it exhibits robust adhesion to its substrate.

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.

Divalent-anion salt effects in polyelectrolyte multilayer depositions.

We systematically investigate the effects of divalent anions on the assembly of polyelectrolyte multilayers by fabricating polystyrene sulfonate (PSS)/polyallylamine hydrochloride (PAH) multilayer films from aqueous solutions containing SO(4)(2-), HPO(4)(2-), or organic dicarboxylate dianions. The chosen concentrations of these anions (i.e., ≤0.05 M) allow us to isolate their effects on the assembly process from those of the polyelectrolyte solubility or solution ionic strength (maintained constant at µ = 1.00 M by added NaCl). Compared to a control film prepared from solutions containing only Cl(-) anions, stratified multilayers deposited in the presence of dianions exhibit increased UV absorbance, thickness, and roughness. From the dependence of film properties on the solution concentration of SO(4)(2-) and number of polyelectrolyte layers deposited, we derive a generic model for the PSS/PAH multilayer formation that involves adsorption of PAH aggregates formed in solution via electrostatic interactions of PAH with bridging dianions. Experiments using HPO(4)(2-) and organic dicarboxylate species of varying structure indicate that the separation, rigidity, and angle between the discrete negatively charged sites in the dianion govern the formation of the PAH aggregates, and therefore also the properties of the multilayer film. A universal linear relationship between film UV absorbance and thickness is observed among all dianion types or concentrations, consistent with the model.

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.

Comparative analysis of stalked and acorn barnacle adhesive proteomes.

Barnacles interest the scientific community for multiple reasons: their unique evolutionary trajectory, vast diversity and economic impact-as a harvested food source and also as one of the most prolific macroscopic hard biofouling organisms. A common, yet novel, trait among barnacles is adhesion, which has enabled a sessile adult existence and global colonization of the oceans. Barnacle adhesive is primarily composed of proteins, but knowledge of how the adhesive proteome varies across the tree of life is unknown due to a lack of genomic information. Here, we supplement previous mass spectrometry analyses of barnacle adhesive with recently sequenced genomes to compare the adhesive proteomes of Pollicipes pollicipes (Pedunculata) and Amphibalanus amphitrite (Sessilia). Although both species contain the same broad protein categories, we detail differences that exist between these species. The barnacle-unique cement proteins show the greatest difference between species, although these differences are diminished when amino acid composition and glycosylation potential are considered. By performing an in-depth comparison of the adhesive proteomes of these distantly related barnacle species, we show their similarities and provide a roadmap for future studies examining sequence-specific differences to identify the proteins responsible for functional differences across the barnacle tree of life.

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.

Direct Observation of Corrosive Wear by In Situ Scanning Probe Microscopy.

Tribocorrosion involves mechanical wear in a corrosive environment, damaging the protective oxide layer of passivating alloys and increasing material loss rates. Here, we develop a nanoscale, in situ technique using scanning probe microscopy in an electrochemical cell to explore the phase-by-phase tribocorrosion behavior of a heat-treated duplex stainless-steel alloy with secondary phases. We found that under anodic potentials well within the passive oxide region, sliding mechanical contact initiated pitting corrosion and increased electrochemical cell current localized to regions undergoing pitting. Secondary phases were most vulnerable to pitting corrosion during sliding, particularly secondary austenite which is chromium-depleted relative to the matrix steel phases. Under certain conditions, even sigma phases of high nobility were damaged from pits that originate from chromium nitrides. Initiation sites coincide with nanoscale surface voids created at chromium nitride inclusions under a threshold contact stress. Below the initiation stress, no pitting or corrosive wear was observed on sensitized phases. Material loss ceased to propagate when sliding stresses were removed but accelerated when sliding contact stresses were increased. Wear rates and current in the cell were both linearly correlated with material loss. Electrochemical current data were used to monitor oxide penetration spatially but could not be used to quantify material loss. In situ tribocorrosion using a scan probe tip is a viable platform to resolve mechanisms of failure that originate at the nanoscale on actively passivated metal surfaces.

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.

Molecular Recognition of Structures Is Key in the Polymerization of Patterned Barnacle Adhesive Sequences.

The permanent adhesive produced by adult barnacles is held together by tightly folded proteins that form amyloid-like materials distinct among marine foulants. In this work, we link stretches of alternating charged and noncharged linear sequences from a family of adhesive proteins to their role in forming fibrillar nanomaterials. Using recombinant proteins and short barnacle cement derived peptides (BCPs), we find a central sequence with charged motifs of the pattern [Gly/Ser/Val/Thr/Ala-X], where X are charged amino acids, to exert specific control over timing, structure, and morphology of fibril formation. While most BCPs remain dormant, the core segment demonstrates rapid polymerization as well as an ability to template other peptides with no propensity for self-assembly. Patterned charge domains assemble dormant peptides through a specific antiparallel ß-sheet structure as measured by FTIR. While charged domains favor an antiparallel structure, BCPs without charged domains switch fibril assembly to favor simpler parallel ß-sheet aggregates. In addition to activation, charged domains direct nanofibers to grow into discrete microns long fibrils similar to the natural adhesive, while segments without such domains only form short branched aggregates. The assembly of adhesive sequences through recognition of structured templates outlines a strategy used by barnacles to control physical mechanisms of underwater adhesive delivery, activation, and curing based on molecular recognition between proteins.

Pressure cycling technology for challenging proteomic sample processing: application to barnacle adhesive.

Successful proteomic characterization of biological material depends on the development of robust sample processing methods. The acorn barnacle Amphibalanus amphitrite is a biofouling model for adhesive processes, but the identification of causative proteins involved has been hindered by their insoluble nature. Although effective, existing sample processing methods are labor and time intensive, slowing progress in this field. Here, a more efficient sample processing method is described which exploits pressure cycling technology (PCT) in combination with protein solvents. PCT aids in protein extraction and digestion for proteomics analysis. Barnacle adhesive proteins can be extracted and digested in the same tube using PCT, minimizing sample loss, increasing throughput to 16 concurrently processed samples, and decreasing sample processing time to under 8 hours. PCT methods produced similar proteomes in comparison to previous methods. Two solvents which were ineffective at extracting proteins from the adhesive at ambient pressure (urea and methanol) produced more protein identifications under pressure than highly polar hexafluoroisopropanol, leading to the identification and description of >40 novel proteins at the interface. Some of these have homology to proteins with elastomeric properties or domains involved with protein-protein interactions, while many have no sequence similarity to proteins in publicly available databases, highlighting the unique adherent processes evolved by barnacles. The methods described here can not only be used to further characterize barnacle adhesive to combat fouling, but may also be applied to other recalcitrant biological samples, including aggregative or fibrillar protein matrices produced during disease, where a lack of efficient sample processing methods has impeded advancement. Data are available via ProteomeXchange with identifier PXD012730.

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.

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.

High-performance nanomaterials formed by rigid yet extensible cyclic ß-peptide polymers.

Organisms have evolved biomaterials with an extraordinary convergence of high mechanical strength, toughness, and elasticity. In contrast, synthetic materials excel in stiffness or extensibility, and a combination of the two is necessary to exceed the performance of natural biomaterials. We bridge this materials property gap through the side-chain-to-side-chain polymerization of cyclic ß-peptide rings. Due to their strong dipole moments, the rings self-assemble into rigid nanorods, stabilized by hydrogen bonds. Displayed amines serve as functionalization sites, or, if protonated, force the polymer to adopt an unfolded conformation. This molecular design enhances the processability and extensibility of the biopolymer. Molecular dynamics simulations predict stick-slip deformations dissipate energy at large strains, thereby, yielding toughness values greater than natural silks. Moreover, the synthesis route can be adapted to alter the dimensions and displayed chemistries of nanomaterials with mechanical properties that rival nature.

Below the Hall-Petch Limit in Nanocrystalline Ceramics.

Reducing the grain size of metals and ceramics can significantly increase strength and hardness, a phenomenon described by the Hall-Petch relationship. The many studies on the Hall-Petch relationship in metals reveal that when the grain size is reduced to tens of nanometers, this relationship breaks down. However, experimental data for nanocrystalline ceramics are scarce, and the existence of a breakdown is controversial. Here we show the Hall-Petch breakdown in nanocrystalline ceramics by performing indentation studies on fully dense nanocrystalline ceramics fabricated with grain sizes ranging from 3.6 to 37.5 nm. A maximum hardness occurs at a grain size of 18.4 nm, and a negative (or inverse) Hall-Petch relationship reduces the hardness as the grain size is decreased to around 5 nm. At the smallest grain sizes, the hardness plateaus and becomes insensitive to grain size change. Strain rate studies show that the primary mechanism behind the breakdown, negative, and plateau behavior is not diffusion-based. We find that a decrease in density and an increase in dissipative energy below the breakdown correlate with increasing grain boundary volume fraction as the grain size is reduced. The behavior below the breakdown is consistent with structural changes, such as increasing triple-junction volume fraction. Grain- and indent-size-dependent fracture behavior further supports local structural changes that corroborate current theories of nanocrack formation at triple junctions. The synergistic grain size dependencies of hardness, elasticity, energy dissipation, and nanostructure of nanocrystalline ceramics point to an opportunity to use the grain size to tune the strength and dissipative properties.

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.