Publications by authors named "Beatriz Orihuela"

31 Publications

begins exoskeleton mineralization within 48 hours of metamorphosis.

R Soc Open Sci 2020 Sep 30;7(9):200725. Epub 2020 Sep 30.

Department of Biology, The College of New Jersey, 2000 Pennington Road, Ewing, NJ 08628, USA.

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 mm h and 0.30 µm h, 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.
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http://dx.doi.org/10.1098/rsos.200725DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7540746PMC
September 2020

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

Anal Biochem 2019 12 17;586:113441. Epub 2019 Sep 17.

Center for Bio/Molecular Science and Engineering, Naval Research Laboratory, Washington, DC, 20375, USA. Electronic address:

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.
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http://dx.doi.org/10.1016/j.ab.2019.113441DOI Listing
December 2019

Adhesion of acorn barnacles on surface-active borate glasses.

Philos Trans R Soc Lond B Biol Sci 2019 10 9;374(1784):20190203. Epub 2019 Sep 9.

Chemistry Division, US Naval Research Laboratory, 4555 Overlook Avenue SW, Washington, DC 20375, USA.

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 and BO) 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'.
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http://dx.doi.org/10.1098/rstb.2019.0203DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6745471PMC
October 2019

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

PLoS One 2018 10;13(12):e0208352. Epub 2018 Dec 10.

Center for Bio/Molecular Science and Engineering, Naval Research Laboratory, Washington, D.C., United States of America.

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.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0208352PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6287898PMC
May 2019

Resistance of Zwitterionic Peptide Monolayers to Biofouling.

Langmuir 2019 02 28;35(5):1818-1827. Epub 2018 Aug 28.

Duke University Marine Laboratory, Nicholas School of the Environment, Duke University , Beaufort , North Carolina 28516-9721 , United States.

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.
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http://dx.doi.org/10.1021/acs.langmuir.8b01625DOI Listing
February 2019

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

Adv Sci (Weinh) 2018 Jun 14;5(6):1700762. Epub 2018 Mar 14.

Chemistry Division Naval Research Laboratory 4555 Overlook Ave. SW Washington DC 20375 USA.

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 ( (=) ) 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 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.
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http://dx.doi.org/10.1002/advs.201700762DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6010908PMC
June 2018

Multivariate analysis of attachment of biofouling organisms in response to material surface characteristics.

Biointerphases 2017 12 29;12(5):051003. Epub 2017 Dec 29.

Department of Chemistry, University at Buffalo, The State University of New York, Buffalo, New York 14260.

Multivariate analyses were used to investigate the influence of selected surface properties (Owens-Wendt surface energy and its dispersive and polar components, static water contact angle, conceptual sign of the surface charge, zeta potentials) on the attachment patterns of five biofouling organisms (Amphibalanus amphitrite, Amphibalanus improvisus, Bugula neritina, Ulva linza, and Navicula incerta) to better understand what surface properties drive attachment across multiple fouling organisms. A library of ten xerogel coatings and a glass standard provided a range of values for the selected surface properties to compare to biofouling attachment patterns. Results from the surface characterization and biological assays were analyzed separately and in combination using multivariate statistical methods. Principal coordinate analysis of the surface property characterization and the biological assays resulted in different groupings of the xerogel coatings. In particular, the biofouling organisms were able to distinguish four coatings that were not distinguishable by the surface properties of this study. The authors used canonical analysis of principal coordinates (CAP) to identify surface properties governing attachment across all five biofouling species. The CAP pointed to surface energy and surface charge as important drivers of patterns in biological attachment, but also suggested that differentiation of the surfaces was influenced to a comparable or greater extent by the dispersive component of surface energy.
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http://dx.doi.org/10.1116/1.5008988DOI Listing
December 2017

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

ACS Appl Mater Interfaces 2017 Apr 22;9(13):11493-11505. Epub 2017 Mar 22.

Nicholas School of the Environment and Earth Sciences, Duke University Marine Laboratory , Beaufort, North Carolina 28516, United States.

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.
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http://dx.doi.org/10.1021/acsami.7b01185DOI Listing
April 2017

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

Sci Rep 2016 11 8;6:36219. Epub 2016 Nov 8.

Chemistry Division, Code 6176, US Naval Research Laboratory, 4555 Overlook Ave, SW, Washington, DC, USA.

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.
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http://dx.doi.org/10.1038/srep36219DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5099703PMC
November 2016

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

J Exp Biol 2017 01 3;220(Pt 2):194-207. Epub 2016 Nov 3.

Duke University Marine Laboratory, Marine Science and Conservation, 135 Duke Marine Lab Road, Beaufort, NC 28516, USA.

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.
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http://dx.doi.org/10.1242/jeb.145094DOI Listing
January 2017

Recyclable plastics as substrata for settlement and growth of bryozoans Bugula neritina and barnacles Amphibalanus amphitrite.

Environ Pollut 2016 Nov 25;218:973-980. Epub 2016 Aug 25.

Marine Laboratory, Nicholas School of the Environment, Duke University, Beaufort, NC 28516, USA. Electronic address:

Plastics are common and pervasive anthropogenic debris in marine environments. Floating plastics provide opportunities to alter the abundance, distribution and invasion potential of sessile organisms that colonize them. We selected plastics from seven recycle categories and quantified settlement of (i) bryozoans Bugula neritina (Linnaeus, 1758) in the lab and in the field, and of (ii) barnacles Amphibalanus (= Balanus) amphitrite (Darwin, 1854) in the field. In the laboratory we cultured barnacles on the plastics for 8 weeks and quantified growth, mortality, and breaking strength of the side plates. In the field all recyclable plastics were settlement substrata for bryozoans and barnacles. Settlement depended on the type of plastic. Fewer barnacles settled on plastic surfaces compared to glass. In the lab and in the field, bryozoan settlement was higher on plastics than on glass. In static laboratory rearing, barnacles growing on plastics were initially significantly smaller than on glass. This suggested juvenile barnacles were adversely impacted by materials leaching from the plastics. Barnacle mortality was not significantly different between plastic and glass surfaces, but breaking strength of side plates of barnacles on polyvinyl chloride (PVC) and polycarbonate (PC) were significantly lower than breakage strength on glass. Plastics impact marine ecosystems directly by providing new surfaces for colonization with fouling organisms and by contaminants shown previously to leach out of plastics and impact biological processes.
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http://dx.doi.org/10.1016/j.envpol.2016.08.047DOI Listing
November 2016

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

Biofouling 2016 10;32(9):1017-28

a Department of Biomedical Engineering , Duke University , Durham , NC , USA.

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.
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http://dx.doi.org/10.1080/08927014.2016.1209186DOI Listing
October 2016

Localization of Phosphoproteins within the Barnacle Adhesive Interface.

Biol Bull 2016 06;230(3):233-42

Department of Oral Biology, McGowan Institute for Regenerative Medicine, University of Pittsburgh, 505 SALKP, 335 Sutherland Drive, Pittsburgh, Pennsylvania 15213;

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.
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http://dx.doi.org/10.1086/BBLv230n3p233DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6377941PMC
June 2016

Imaging Active Surface Processes in Barnacle Adhesive Interfaces.

Langmuir 2016 Jan 6;32(2):541-50. Epub 2016 Jan 6.

Duke University Marine Lab , Beaufort, North Carolina 28516, United States.

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.
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http://dx.doi.org/10.1021/acs.langmuir.5b03286DOI Listing
January 2016

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

Environ Sci Technol 2016 Jan 29;50(2):924-31. Epub 2015 Dec 29.

Duke Marine Laboratory, Nicholas School of the Environment, Duke University , Beaufort, North Carolina 28516, United States.

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.
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http://dx.doi.org/10.1021/acs.est.5b02781DOI Listing
January 2016

Modification of Silicone Elastomer Surfaces with Zwitterionic Polymers: Short-Term Fouling Resistance and Triggered Biofouling Release.

ACS Appl Mater Interfaces 2015 Nov 16;7(46):25586-91. Epub 2015 Nov 16.

Department of Chemical and Biomolecular Engineering, North Carolina State University , Raleigh, North Carolina 27695, United States.

We present a method for dual-mode-management of biofouling by modifying surface of silicone elastomers with zwitterionic polymeric grafts. Poly(sulfobetaine methacrylate) was grafted from poly(vinylmethylsiloxane) elastomer substrates using thiol-ene click chemistry and surface-initiated, controlled radical polymerization. These surfaces exhibited both fouling resistance and triggered fouling-release functionality. The zwitterionic polymers exhibited fouling resistance over short-term (∼hours) exposure to bacteria and barnacle cyprids. The biofilms that eventually accumulated over prolonged-exposure (∼days) were easily detached by applying mechanical strain to the elastomer substrate. Such dual-functional surfaces may be useful in developing environmentally and biologically friendly coatings for biofouling management on marine, industrial, and biomedical equipment because they can obviate the use of toxic compounds.
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http://dx.doi.org/10.1021/acsami.5b09199DOI Listing
November 2015

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

Biofouling 2015 ;31(3):265-74

a Department of Biomedical Engineering , Duke University , Durham , NC, USA.

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.
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http://dx.doi.org/10.1080/08927014.2015.1035651DOI Listing
July 2015

Growth and development of the barnacle Amphibalanus amphitrite: time and spatially resolved structure and chemistry of the base plate.

Biofouling 2014 ;30(7):799-812

a Chemistry Division , Naval Research Laboratory , Washington , DC , USA.

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.
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http://dx.doi.org/10.1080/08927014.2014.930736DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4159999PMC
April 2015

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

Nat Commun 2014 Jul 11;5:4414. Epub 2014 Jul 11.

Biological Sciences, Clemson University, 132 Long Hall, Clemson, South Carolina 29634, USA.

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.
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http://dx.doi.org/10.1038/ncomms5414DOI Listing
July 2014

Confocal microscopy-based goniometry of barnacle cyprid permanent adhesive.

J Exp Biol 2013 Jun 21;216(Pt 11):1969-72. Epub 2013 Feb 21.

School of Marine Science and Technology, Newcastle University, Newcastle upon Tyne NE1 7RU, UK.

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.
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http://dx.doi.org/10.1242/jeb.084913DOI Listing
June 2013

Bioinspired surfaces with dynamic topography for active control of biofouling.

Adv Mater 2013 Mar 6;25(10):1430-4. Epub 2013 Jan 6.

Department of Biomedical Engineering, Duke University, Durham, NC 27708, USA.

Dynamic change of the surface area and topology of elastomers is used as a general, environmentally friendly approach for effectively detaching micro- and macro-fouling organisms adhered on the elastomer surfaces. Deformation of elastomer surfaces under electrical or pneumatic actuation can debond various biofilms and barnacles. The bio-inspired dynamic surfaces can be fabricated over large areas through simple and practical processes. This new mechanism is complementary with existing materials and methods for biofouling control.
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http://dx.doi.org/10.1002/adma.201203374DOI Listing
March 2013

Barnacle Balanus amphitrite adheres by a stepwise cementing process.

Langmuir 2012 Sep 11;28(37):13364-72. Epub 2012 Jul 11.

Chemistry Division, Code 6176, U.S. Naval Research Laboratory, Washington, DC 20375-5342, USA.

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.
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http://dx.doi.org/10.1021/la301695mDOI Listing
September 2012

The effects of model polysiloxane and fouling-release coatings on embryonic development of a sea urchin (Arbacia punctulata) and a fish (Oryzias latipes).

Aquat Toxicol 2012 Apr 13;110-111:162-9. Epub 2012 Jan 13.

Duke University Marine Laboratory, Nicholas School of the Environment, Duke University, Beaufort, NC, United States.

In recent decades attention has focused on the development of non-toxic fouling-release coatings based on silicone polymers as an alternative to toxic antifouling coatings. As fouling-release coatings gain market share, they will contribute to environmental contamination by silicones. We report effects of eight model polysiloxane and three commercial foul-release coatings on embryonic development of sea urchins and fish, Japanese medaka. We used model coatings because they have known composition and commercially available components and molecules leaching from these coatings have been partially characterized. The commercial fouling-release coatings are purported to be non-toxic and components are proprietary. Our goal was to expose embryos of well studied model animals to the coatings to determine if the complex mixtures leaching from the coatings impact development. Urchins were chosen because development is rapid and embryos can enter the non-slip layer over surfaces. Medaka was chosen because the female deposits the sticky eggs onto the anal fin and then scrapes them off onto surfaces. Embryos were confined in water over coatings in 24 well plates. Fresh model coatings had no effect on urchin development while commercial fouling-release coatings inhibited development. Fish embryos had delayed hatching, increased mortality of hatchlings and dramatically decreased ability of hatchlings to inflate the swim bladder and reduced hatching success on all coatings. After one-month immersion of coatings in running seawater to simulate initial application in the marine environment, sea urchin embryos died when placed over model silicones. Effects of the commercial coatings were reduced but included retarded development. Effects on fish embryos over leached coating were reduced compared to those of fresh coating and included decreased hatching success, decreased hatchling survival and inability to inflate the swim bladder for commercial coatings. These findings suggest, similar to medical conclusions, compounds leaching from silicone coatings can impact development and the topic deserves study.
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http://dx.doi.org/10.1016/j.aquatox.2012.01.005DOI Listing
April 2012

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

ACS Nano 2012 Feb 25;6(2):1503-12. Epub 2012 Jan 25.

Department of Chemistry, Texas A&M University, College Station, Texas 77842, United States.

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.
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http://dx.doi.org/10.1021/nn204431mDOI Listing
February 2012

Compounds from silicones alter enzyme activity in curing barnacle glue and model enzymes.

PLoS One 2011 Feb 17;6(2):e16487. Epub 2011 Feb 17.

MSC Division, Duke University Marine Laboratory, Nicholas School of the Environment, Beaufort, North Carolina, United States of America.

Background: Attachment strength of fouling organisms on silicone coatings is low. We hypothesized that low attachment strength on silicones is, in part, due to the interaction of surface available components with natural glues. Components could alter curing of glues through bulk changes or specifically through altered enzyme activity.

Methodology/principal Findings: GC-MS analysis of silicone coatings showed surface-available siloxanes when the coatings were gently rubbed with a cotton swab for 15 seconds or given a 30 second rinse with methanol. Mixtures of compounds were found on 2 commercial and 8 model silicone coatings. The hypothesis that silicone components alter glue curing enzymes was tested with curing barnacle glue and with commercial enzymes. In our model, barnacle glue curing involves trypsin-like serine protease(s), which activate enzymes and structural proteins, and a transglutaminase which cross-links glue proteins. Transglutaminase activity was significantly altered upon exposure of curing glue from individual barnacles to silicone eluates. Activity of purified trypsin and, to a greater extent, transglutaminase was significantly altered by relevant concentrations of silicone polymer constituents.

Conclusions/significance: Surface-associated silicone compounds can disrupt glue curing and alter enzyme properties. Altered curing of natural glues has potential in fouling management.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0016487PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3040736PMC
February 2011

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

Langmuir 2010 May;26(9):6549-56

U.S. Naval Research Laboratory, Code 6176, Washington, District of Columbia 20375-5342, USA.

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.
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http://dx.doi.org/10.1021/la9041309DOI Listing
May 2010

Barnacle cement: a polymerization model based on evolutionary concepts.

J Exp Biol 2009 Nov;212(Pt 21):3499-510

Duke University Marine Laboratory, Nicholas School of the Environment, Beaufort, NC 28516, USA.

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.
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http://dx.doi.org/10.1242/jeb.029884DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2762877PMC
November 2009

Nanoscale structures and mechanics of barnacle cement.

Biofouling 2009 ;25(3):263-75

Department of Chemistry, University of Toronto, Toronto, Canada.

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.
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http://dx.doi.org/10.1080/08927010802688095DOI Listing
March 2009

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

Biofouling 2008 ;24(3):177-84

Department of Civil and Environmental Engineering, University of Wisconsin-Madison, Madison, WI 53706, USA.

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).
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http://dx.doi.org/10.1080/08927010801975725DOI Listing
July 2008

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

Biofouling 2008 ;24(2):109-18

U.S. Naval Research Laboratory, Washington, District of Columbia, USA.

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.
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http://dx.doi.org/10.1080/08927010701882112DOI Listing
June 2008