Oxidase Activity of the Barnacle Adhesive Interface Involves Peroxide-Dependent Catechol Oxidase and Lysyl Oxidase Enzymes.

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.

Sequence basis of Barnacle Cement Nanostructure is Defined by Proteins with Silk Homology.

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.

Localization of Phosphoproteins within the Barnacle Adhesive Interface.

Barnacles permanently adhere to nearly any inert substrate using proteinaceous glue. The glue consists of at least ten major proteins, some of which have been isolated and sequenced. Questions still remain about the chemical mechanisms involved in adhesion and the potential of the glue to serve as a platform for mineralization of the calcified base plate. We tested the hypothesis that barnacle glue contains phosphoproteins, which have the potential to play a role in both adhesion and mineralization. Using a combination of phosphoprotein-specific gel staining and Western blotting with anti-phosphoserine antibody, we identified multiple phosphorylated proteins in uncured glue secretions from the barnacle Amphibalanus amphitrite The protein composition of the glue and the quantity and abundance of phosphoproteins varied distinctly among individual barnacles, possibly due to cyclical changes in the glue secretion over time. We assessed the location of the phosphoproteins within the barnacle glue layer using decalcified barnacle base plates and residual glue deposited by reattached barnacles. Phosphoproteins were found throughout the organic matrix of the base plate and within the residual glue. Staining within the residual glue appeared most intensely in regions where capillary glue ducts, which are involved in cyclical release of glue, had been laid down. Lastly, mineralization studies of glue proteins separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) indicated that proteins identified as phosphorylated possibly induce mineralization of calcium carbonate (CaCO3). These results contribute to our understanding of the protein composition of barnacle glue, and provide new insights into the potential roles of phosphoproteins in underwater bioadhesives.

Synergistic roles for lipids and proteins in the permanent adhesive of barnacle larvae.

Thoracian barnacles rely heavily upon their ability to adhere to surfaces and are environmentally and economically important as biofouling pests. Their adhesives have unique attributes that define them as targets for bio-inspired adhesive development. With the aid of multi-photon and broadband coherent anti-Stokes Raman scattering microscopies, we report that the larval adhesive of barnacle cyprids is a bi-phasic system containing lipids and phosphoproteins, working synergistically to maximize adhesion to diverse surfaces under hostile conditions. Lipids, secreted first, possibly displace water from the surface interface creating a conducive environment for introduction of phosphoproteins while simultaneously modulating the spreading of the protein phase and protecting the nascent adhesive plaque from bacterial biodegradation. The two distinct phases are contained within two different granules in the cyprid cement glands, implying far greater complexity than previously recognized. Knowledge of the lipidic contribution will hopefully inspire development of novel synthetic bioadhesives and environmentally benign antifouling coatings.

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.

The geography of mercury and PCBs in North Carolina's local seafood.

Mercury and PCBs are used by non-governmental organizations and federal agencies to inform seafood safety recommendations. Pollution dynamics suggest recommendations on the national scale may be too large to be accurate. We tested softshell and hardshell blue crab, white and pink shrimp, oysters, clams, spot, and mullet from fishers in each of the three North Carolina fishery districts. We measured mercury using EPA method 7473 and PCBs using a commercially available ELISA kit. Over 97% of samples were below the Environmental Protection Agency levels of concern for both mercury and PCBs. Mercury and PCBs have different spatial dynamics, but both differ significantly by water body, suggesting that seafood safety recommendations should occur by water body instead of at the national scale. This finding supports previous research suggesting that differences in water chemistry, terrestrial influence, and flushing time in a particular water body control the contaminant load in locally resident species.

Noradrenaline-functionalized hyperbranched fluoropolymer-poly(ethylene glycol) cross-linked networks as dual-mode, anti-biofouling coatings.

The strategy of decorating antibiofouling hyperbranched fluoropolymer-poly(ethylene glycol) (HBFP-PEG) networks with a settlement sensory deterrent, noradrenaline (NA), and the results of biofouling assays are presented. This example of a dual-mode surface, which combines both passive and active modes of antibiofouling, works in synergy to improve the overall antibiofouling efficiency against barnacle cyprids. The HBFP-PEG polymer surface, prior to modification with NA, was analyzed by atomic force microscopy, and a significant distribution of topographical features was observed, with a nanoscopic roughness measurement of 110 ± 8 nm. NA attachment to the surface was probed by secondary ion mass spectrometry to quantify the extent of polymer chain-end substitution with NA, where a 3- to 4-fold increase in intensity for a fragment ion associated with NA was observed and 39% of the available sites for attachment were substituted. Cytoskeletal assays confirmed the activity of tethered NA on adhering oyster hemocytes. Settlement assays showed deterrence toward barnacle cyprid settlement, while not compromising the passive biofouling resistance of the surface. This robust strategy demonstrates a methodology for the incorporation of actively antibiofouling moieties onto a passively antibiofouling network.

Involvement of reactive oxygen species in the electrochemical inhibition of barnacle (Amphibalanus amphitrite) settlement.

The role of reactive oxygen species (ROS) in electrochemical biofouling inhibition was investigated using a series of abiotic tests and settlement experiments with larvae of the barnacle Amphibalanus amphitrite, a cosmopolitan fouler. Larval settlement, a measure of biofouling potential, was reduced from 43% +/- 14% to 5% +/- 6% upon the application of pulsed electric signals. The application of ROS scavengers such as glutathione and catalase counteracted the inhibitory effects of the electric signals, allowing settlement, and thus indicating that ROS are antifouling agents. Based on the experimental evidence, the proposed mechanism for ROS-based fouling prevention with interdigitated electrodes involved the electrochemical generation of hydrogen peroxide by oxygen reduction, and its likely reduction to hydroxyl radicals. Either hydroxyl radicals or products of hydroxyl radical reactions appeared to be the main deterrents of larval settlement.

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.

Oxidative iron species and ocean challenges: a perspective.

The idea of using oxidative iron (Fe(6+)) to manage fouling and potentially invasive and pathogenic species in ballast water has merit and is attractive when viewed in the broadest context. Ferrate (Fe(6+)) has potential in ballast water management because it reduces a complex global problem to a single issue, viz.how to dispose of the waste which is predominantly Fe(3+). Waste iron disposal must be considered carefully because iron limits photosynthesis in oligotrophic oceans, alters physiological processes in bacteria and animals, produces reactive oxygen species, causes nitrosative stress and increased availability enhances the virulence of pathogenic bacteria. The case is made that the oxidative iron waste should be recycled rather than discharged into the ocean.

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).

Molluscs as multidisciplinary models in environment toxicology.

The primary goal of this paper is to stimulate discussion and promote the use of mollusc models and multidisciplinary research approaches in the field of environmental toxicology. Molluscs are effective models because they are ubiquitous, have highly conserved control and regulatory pathways that are often homologous to vertebrate systems, and are extremely sensitive to anthropogenic inputs. We have attempted to provide initial references as an avenue into the literature and as a means for researchers to expand their thoughts to regions outside of their own area of expertise. The authors are particularly interested in developing multidisciplinary international collaborative efforts.

Crustacean peptide and peptide-like pheromones and kairomones.

Crustacean peptide pheromones, kairomones, and substituted amino sugar kairomones are reviewed from a historical perspective. These crustacean information molecules are secondary functions of structural polymers. They are partial hydrolysis products, generated usually by the action of trypsin-like enzymes on proteins, and glycosidase enzymes on glycoproteins and proteoglycans. Structure-function studies based upon synthetic mimics of peptide information molecules show neutral amino acids with a basic carboxyl terminal are active in modifying physiological and or behavioral responses. Behaviorally active substituted amino sugar mimics are disaccharide hydrolysis products of heparin and chondroitin sulfate. Similar molecules are also used as information molecules by a variety of other marine organisms indicating they are a common biological theme.

Responses of mud snails from low and high imposex sites to sex pheromones.

Imposex, male secondary sexual characteristics in female snails, is a morphological indicator of sub-lethal exposure to organotin compounds. The relation between imposex and behavioral responses to sex pheromones was studied. Responses of snails to sex specific pheromones were determined in laboratory assays. Females and males from a low imposex site and females, imposex females and males from a high imposex site were tested. The snails from the low imposex site showed the expected strong behavioral dimorphism with females responding to male pheromone and males responding to female pheromone. In assays using snails from the high imposex site, female and imposex females showed no dimorphism in response to pheromones and males showed a relatively weak dimorphism. As there was no breeding or egg capsule deposition at the high imposex site, we conclude that even the morphologically normal snails were behaviorally and reproductively compromised.

Pharmaceuticals as antifoulants: concept and principles.

The hypothesis that pharmaceuticals, with their known syntheses, chemical properties and primary mechanism of action would be an efficient source of new antifouling agents compatible with existing antifouling coating technology was tested. Twenty-three compounds at concentrations from 5 micrograms ml-1 to 40 ng ml-1 were tested for toxicity and inhibition of settlement of barnacle larvae. The compounds had a wide range of solubility in water and covered nine primary mechanisms of action in vertebrates. The upper level of potency was chosen because compounds that are highly potent have greater practical potential. The goal was to find compounds with high inhibition of settlement and low toxicity. Of the 23 compounds tested, 22 had significant effects on barnacle larvae. The variety of chemical structures and their variation in water solubility support the hypothesis that pharmaceuticals that are compatible with existing coatings technology should be considered as antifouling agents. Moreover, factors such as coating compatibility and environmental fate should be addressed early in the development process.

Inhibition of barnacle larval settlement and crustacean toxicity of some hoplonemertine pyridyl alkaloids.

Hoplonemertines are carnivorous marine worms, which prey upon crustaceans and annelids. They paralyze their prey by injecting alkaloids with a stylet-bearing proboscis. The dermis of these animals also secretes alkaloids to repel predators. Besides affecting central and peripheral nervous system nicotinic receptors, some pyridyl alkaloids also activate certain chemoreceptor neurons in crustacean walking legs, which sense environmental chemicals. Anabaseine (2-[3-pyridyl]-3,4,5,6-tetrahydropyridyl) and 2,3'-bipyridyl (2,3'-BP) are two nemertine alkaloids, which potently paralyze crustaceans. Anabaseine is an agonist of vertebrate as well as invertebrate nicotinic receptors. While 2,3'-BP is non-toxic to mice, it is toxic to crustaceans. We tested a variety of nemertine pyridyl alkaloids for inhibition of barnacle (Balanus amphitrite) larval settlement and for crustacean toxicity in order to determine whether toxicity could be dissociated from inhibition of larval settlement. We prepared eight C-methylated 2,3'-BP isomers to determine where substitution is permitted without loss of activity. Anti-settlement and toxicity activities were not always related. For instance, 4'-methyl-2,3'-BP displayed only 3% of the crayfish paralytic activity of 2,3'-BP, but inhibited settlement almost 2-fold more effectively. Two other isomers displaying exceptional anti-settlement activity were the 4- and 5-methyl-2,3'-BPs; these also displayed high crustacean toxicity. Nemertelline inhibited barnacle settlement at concentrations similar to 2,3'-BP but was 136-fold less toxic when injected into crayfish. Thus, certain bipyridyls and tetrapyridyls may be useful anti-fouling additives.

Chemical mediation of egg capsule deposition by mud snails.

Mud snails (Ilyanassa obsoleta = Nassarius obsoletus = Nassa obsoleta) deposit eggs in protective capsules on hard substrata in soft bottom environments. We studied sites of egg capsule deposition and snail movement responses to odors to determine if chemoreception plays a role in deposition site selection. From results of field surveys, laboratory experiments, and field experiments, we conclude that mud snails use chemoreception for capsule deposition. Attractive odors originate from mud snail and whelk egg capsules and from living bivalves. Evidence for attractive odors from conspecifics is equivocal. Capsules are deposited on living odor sources and nearby hard substrates. We hypothesize that deposition of capsules on living substrates increases the likelihood that embryos will survive by decreasing the chance of smothering of embryos by sediments.