In situ ATR–FTIR characterization of primary cement interfaces of the barnacle Balanus amphitrite

A method is presented for characterizing primary cement interfaces of barnacles using in situ attenuated total reflection–Fourier transform infrared spectroscopy. Primary cement of the barnacle, Balanus amphitrite (Amphibalanus amphitrite), was characterized without any disruption to the original cement interface, after settling and growing barnacles directly on double sided polished germanium wafers. High-quality IR spectra were acquired of live barnacle cement interfaces, providing a spectroscopic fingerprint of cured primary cement in vivo with the barnacle adhered to the substratum. Additional spectra were also acquired of intact cement interfaces for which the upper portion of the barnacle had been removed leaving only the base plate and cement layer attached to the substratum. This allowed further characterization of primary cement interfaces that were dried or placed in D₂O. The resulting spectra were consistent with the cement being proteinaceous, and allowed analysis of the protein secondary structure and water content in the cement layer. The estimated secondary structure composition was primarily β-sheet, with additional α-helix, turn and unordered components. The cement of live barnacles, freshly removed from seawater, was estimated to have a water content of 20–50% by weight. These results provide new insights into the chemical properties of the undisturbed barnacle adhesive interface.     

Prolonged morphometric study of barnacles grown on soft substrata of hydrogels and elastomers

A long-term investigation of the shell shape and the basal morphology of barnacles grown on tough, double-network (DN) hydrogels and polydimethylsiloxane (PDMS) elastomer was conducted in a laboratory environment. The elastic modulus of these soft substrata varied between 0.01 and 0.47 MPa. Polystyrene (PS) (elastic modulus, 3 GPa) was used as a hard substratum control. It was found that the shell shape and the basal plate morphology of barnacles were different on the rigid PS substratum compared to the soft substrata of PDMS and DN hydrogels. Barnacles on the PS substratum had a truncated cone shape with a flat basal plate while on soft PDMS and DN gels, barnacles had a pseudo-cylindrical shape and their basal plates showed curvature. In addition, a large adhesive layer was observed under barnacles on PDMS, but not on DN gels. The effect of substratum stiffness is discussed in terms of barnacle muscle contraction, whereby the relative stiffness of the substratum compared to that of the muscle is considered as the key parameter.     

An in situ study of the nanomechanical properties of barnacle (Balanus amphitrite) cyprid cement using atomic force microscopy (AFM)

Cyprids are the final planktonic stage in the larval dispersal of barnacles and are responsible for surface exploration and attachment to appropriate substrata. The nanomechanical properties of barnacle (Balanus amphitrite) cyprid permanent cement were studied in situ using atomic force microscopy (AFM). Force curves were recorded from the cement disc continually over the course of its curing and these were subsequently analysed using custom software. Results showed a narrowing of the pull-off force distribution with time, as well as a reduction in molecular stretch length over time. In addition, there was a strong correlation between maximum pull-off force and molecular stretch length for the cement, suggesting 'curing' of the adhesive; some force curves also contained a 'fingerprint' of modular protein unfolding. This study provides the first direct experimental evidence in support of a putative 'tanning' mechanism in barnacle cyprid cement.     

Release characteristics of reattached barnacles to non-toxic silicone coatings

Release mechanisms of barnacles (Amphibalanus amphitrite or Balanus amphitrite) reattached to platinum-cured silicone coatings were studied as a function of coating thickness (210-770 microm), elastic modulus (0.08-1.3 MPa), and shear rate (2-22 microm s(-1)). It was found that the shear stress of the reattached, live barnacles necessary to remove from the silicone coatings was controlled by the combined term (E/t)(0.5) of the elastic modulus (E) and thickness (t). As the ratio of the elastic modulus to coating thickness decreased, the barnacles were more readily removed from the silicone coatings, showing a similar release behavior to pseudobarnacles (epoxy glue). The barnacle mean shear stress ranged from 0.017 to 0.055 MPa whereas the pseudobarnacle mean shear stress ranged from 0.022 to 0.095 MPa.     

A comparison of the antifouling/foul-release characteristics of non-biocidal xerogel and commercial coatings toward micro- and macrofouling organisms

Five non-biocidal xerogel coatings were compared to two commercial non-biocidal coatings and a silicone standard with respect to antifouling (AF)/fouling-release (FR) characteristics. The formation and release of biofilm of the marine bacterium Cellulophaga lytica, the attachment and release of the microalga Navicula incerta, and the fraction removal and critical removal stress of reattached adult barnacles of Amphibalanus amphitrite were evaluated in laboratory assays. Correlations of AF/FR performance with surface characteristics such as wettability, surface energy, elastic modulus, and surface roughness were examined. Several of the xerogel coating compositions performed well against both microfouling organisms while the commercial coatings performed less well toward the removal of microalgae. Reattached barnacle adhesion as measured by critical removal stress was significantly lower on the commercial coatings when compared to the xerogel coatings. However, two xerogel compositions showed release of 89-100% of reattached barnacles. These two formulations were also tested in the field and showed similar results.     

Activity of commercial enzymes on settlement and adhesion of cypris larvae of the barnacle Balanus amphitrite, spores of the green alga Ulva linza, and the diatom Navicula perminuta

Fouling species produce adhesive polymers during the settlement, adhesion and colonization of new surfaces in the marine environment. The present paper tests the hypothesis that enzymes of the appropriate specificity may prevent biofouling by hydrolysing these adhesive polymers. Seventeen commercially available enzyme preparations designed originally for bulk use in a range of end-use applications were tested for their effects on the settlement and/or adhesion of three major fouling species, viz. the green alga Ulva linza, the diatom Navicula perminuta and the barnacle Balanus amphitrite. The serine-proteases were found to have the broadest antifouling potential reducing the adhesion strength of spores and sporelings of U. linza, cells of N. perminuta and inhibiting settlement of cypris larvae of B. amphitrite. Mode-of-action studies on the serine-protease, Alcalase, indicated that this enzyme reduced adhesion of U. linza in a concentration-dependent manner, that spores of the species could recover their adhesive strength if the enzyme was removed and that the adhesive of U. linza and juvenile cement of B. amphitrite became progressively less sensitive to hydrolysis as they cured.     

Factors that influence elastomeric coating performance: the effect of coating thickness on basal plate morphology, growth and critical removal stress of the barnacle Balanus amphitrite

Silicone coatings are currently the most effective non-toxic fouling release surfaces. Understanding the mechanisms that contribute to the performance of silicone coatings is necessary to further improve their design. The objective of this study was to examine the effect of coating thickness on basal plate morphology, growth, and critical removal stress of the barnacle Balanus amphitrite. Barnacles were grown on silicone coatings of three thicknesses (0.2, 0.5 and 2 mm). Atypical ("cupped") basal plate morphology was observed on all surfaces, although there was no relationship between coating thickness and i) the proportion of individuals with the atypical morphology, or ii) the growth rate of individuals. Critical removal stress was inversely proportional to coating thickness. Furthermore, individuals with atypical basal plate morphology had a significantly lower critical removal stress than individuals with the typical ("flat") morphology. The data demonstrate that coating thickness is a fundamental factor governing removal of barnacles from silicone coatings.     

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

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

Species specificity of barnacle settlement-inducing proteins

We previously isolated a larval settlement-inducing protein complex (SIPC) from adult extracts of the barnacle, Balanus amphitrite using a nitrocellulose membrane settlement assay. In the present study, we found that the extracts of other adult barnacles, Megabalanus rosa and Balanus eburneus, also induced the settlement of B. amphitrite cyprids although the inductive activity was slightly lower than that of conspecific extracts. Furthermore, we examined reactivity to anti-SIPC antibody in adult extracts from six species of Japanese barnacles other than B. amphitrite, brine shrimp and eight marine sessile organisms besides barnacles. The results showed that all barnacles examined contained SIPC-like proteins with slightly different molecular weight, while the other animals did not react to the antibody by immunoblot analysis. These findings suggest that species specificity in settlement-inducing proteins of barnacles is not so strict, but these proteins are characteristic to barnacle species.     

Variation among families for characteristics of the adhesive plaque in the barnacle Balanus amphitrite

A quantitative genetics approach was used to examine variation in the characteristics of the adhesive plaque of the barnacle Balanus amphitrite Darwin attached to two silicone substrata. Barnacles settled on silicone polymer films occasionally form thick, soft adhesive plaques, in contrast to the thin, hard plaques characteristic of attachment to other surfaces. The proportion of barnacles producing a thick adhesive plaque was 0.31 for Veridian, a commercially available silicone fouling-release coating, and 0.18 for Silastic T-2, a silicone rubber used for mold-making. For both materials, significant variation among maternal families in the proportion of barnacles producing a thick adhesive plaque was observed, which suggests the presence of genetic variation, or maternal environmental effects, for this plaque characteristic. For the Veridian coating, barnacles expressing the thick adhesive plaque also exhibited significantly reduced tenacity. This represents the first reported case for potential genetic control of intraspecific phenotypic variation in the physical characteristics and tenacity of the adhesive of a fouling invertebrate.     

Identification of the aromatic L-amino acid decarboxylase (AADC) gene and its expression in the attachment and metamorphosis of the barnacle, Balanus amphitrite

Serotonin and dopamine are involved in the attachment and metamorphosis of cypris larvae of barnacles. Aromatic L-amino acid decarboxylase (AADC) gene, the product of which catalyzes the synthesis of serotonin and dopamine from L-5-hydroxytryptophan and L-3,4-dihydroxyphenylalanine, respectively, was characterized. A DNA clone containing part of an AADC sequence was obtained from the genomic DNA library of the barnacle, Balanus amphitrite. This clone had four putative exons consisting of 226 amino acids with an identity of 63.2% and a similarity of 92.1% with human AADC. Northern blot analysis showed that AADC mRNA was expressed at all stages of barnacles: naupliar larvae, cypris larvae and adult barnacles. Two inducers of larval attachment and metamorphosis; that is, serotonin and extract of adult barnacles, obviously increased the expression of AADC mRNA at an early cypris larval stage. These results suggest that intracellular biosynthesis of serotonin, or dopamine, or both is at least partly involved in the control of the attachment and metamorphosis of cypris larvae.     

The rigidity of bacterial flagellar filaments and its relation to filament polymorphism.

We determined and correlated the rigidity of Salmonella typhimurium, Escherichia coli, and Rhizobium lupini flagellar filaments representing various structural and polymorphic states (plain, complex, straight, superhelical, and right- and left-handed). Persistence length, from which the filament's rigidity and other parameters (Young's modulus, bending force constant, buckling persistence length, flexural deformation, and flexural time) were derived, was determined from electron micrographs of isolated, negatively stained filaments. Outer diameters and radii of strong intersubunit connectivity were determined from three-dimensional image reconstructions and radial mass density profiles from scanning transmission electron microscopy. All filaments appear to be highly rigid with no evident correlation with their helical sense or superhelicity. The complex filament of R. lupini is rigid to the extent that it becomes brittle. The overall flexibility of the flagellum seems to stem mainly from the hook and not from the filament. Polymorphism is probably related to the propelling properties and hydrodynamic shape of the filament rather than to its rigidity.     

Salinity effects on the growth,mortality and shell strength of Balanus amphitrite from the Salton Sea,California

The Salton Sea, the largest lake in California, has a salinity of around 43 g l-1 that is increasing by about 0.4 g l-1 y-1. A 15 month microcosm experiment was conducted to determined the effects of salinity (30, 39, 48, 57, and 65 g l-1) and tilapia ( Oreochromis mossambicus) on an assemblage of benthic and planktonic Salton Sea algae and invertebrates, including the barnacle Balanus amphitrite. Eleven months after the microcosms were established, acrylic plates containing newly settled B. amphitrite collected at the Salton Sea were placed in the microcosms to determine the effects of salinity on their growth and shell strength. The Brody-Bertalanffy growth model was fitted to the B. amphitrite growth data. Growth was fastest at 48 g l-1 and slowest at 65 g l-1. B. amphitrite grown at 39–48 g l-1 were the largest and required the greatest force to break, but the strength of the barnacle shell material declined steadily as the salinity increased. However, B. amphitrite at the higher salinities were shorter and had thicker walls relative to their diameters, which may have increased their structural stability. The effects of salinity on the mortality of adult B. amphitrite was determined in laboratory aquaria set up at 43, 60, 70, 75, 80, 90, and 100 g l-1. Salinities were achieved in two ways: by salt addition and by evaporation. Calculated 12-day LC50 values were 83 g l-1 when salinities were achieved through salt addition and 89 g l-1 when salinities were achieved through evaporation. Differences in B. amphitrite mortality between the two methods illustrate the importance of producing experimental salinity levels carefully. B. amphitrite is expected to become extinct within the Salton Sea when the salinity reaches 70–80 g l-1 and to show marked declines in abundance at salinities as low as 50 g l-1. This revised version was published online in July 2006 with corrections to the Cover Date.     

中国沿海无柄蔓足类研究进展

无柄蔓足类属节肢动物门甲壳纲,是海洋生态系统和污损生物群落中极为重要的组成部分,在中国海域分布着6科25属110种,主要种类为纹藤壶(Balanus amphitrite amphitrite)、网纹藤壶(B.reticulatus)、高峰星藤壶(Chirona amaryllis)、泥藤壶(Balanus uliginosus)、白脊藤壶(B.albicostatus)、三角藤壶(B.trigonus)、红巨藤壶(Megabalanus rosa)、钟巨藤壶(M.tintinnabulum tintinnabulum)、白条地藤壶(Euraphia withersi)、鳞笠藤壶(Tetraclita squamosa squamosa),其中纹藤壶在黄、渤海为优势种,网纹藤壶则在热带和亚热带海区占优势;泥藤壶多出现在沿海河口的咸淡水交汇处;三角藤壶、红巨藤壶和钟巨藤壶等种类分布于盐度较高的海域。环境因子可对无柄蔓足类的生长发育、繁殖附着、分布状况及形态特征等产生显著影响。幼虫发育阶段要经历6期无节幼虫和1期金星幼虫,青岛大扁藻(Platymonas helgolandica)、牟式角毛藻(Chaetoceros muelleri)和亚心形扁藻(Platymonas subcordiformis)均是幼虫培养较为理想的饵料;金星幼虫可在4—8℃下保存1周左右。藤壶胶粘物由蛋白亚基聚合而成,其初生胶和次生胶组成基本相似。无柄蔓足类不仅是开展防除测试和生态科学研究的理想材料,而且还应进一步分析其在海洋生态系统中的地位和作用,并从分子水平探讨幼虫附着机理、胶粘物作用机制、种类相互关系与系统发生史。     

Surface elastic modulus of barnacle adhesive and release characteristics from silicone surfaces

The properties of barnacle adhesive on silicone surfaces were studied by AFM indentation, imaging, and other tests and compared to the barnacle shear adhesion strength. A multilayered structure of barnacle adhesive plaque is proposed based on layered modulus regions measured by AFM indentation. The fracture of barnacles from PDMS surfaces was found to include both interfacial and cohesive failure of barnacle adhesive plaque, as determined by protein staining of the substratum after forced barnacle release from the substrate. Data for freshly released barnacles showed that there was a strong correlation between the mean Young's modulus of the outermost (softest) adhesive layer (E<0.3 MPa) and the shear strength of adhesion, but no correlation for other higher modulus regions. Linear, quadratic, and Griffith's failure criterion (based on rough estimate of crack length) regressions were used in the fit, and showed significance.     

Dual-curing resin cement with colour indicator for adhesively cemented restorations to dental tissues: Change of colour by curing and some physical properties

The study was aimed to investigate a color indicator containing dual curing resin composite luting cement and to plot the color change to the time of solidification of the cement. In addition some physical properties were studied. Specimens were made of a dual-cure resin cement (Maxcem Elite™ Chroma, Kerr, Orange, CA, USA) and polymerized by autopolymerization only, or with light initiated polymerization. A spectrophotometer was used to quantify the color change of the cement as plotted with the curing time. The efficacy of the curing process was studied by measuring water sorption and the ultimate flexural properties of the cement. The results showed that the flexural strength of cement after autopolymerization was 27.3 MPa and after light initiated polymerization 48.1 MPa. Young’s modulus of bending was 2089.3 MPa and 3781.5 MPa respectively for the same cement samples. Water sorption after two weeks for the autopolymerization cement samples was −1.12 wt% and for the light initiated polymerization samples 0.56 wt%. Non-parametric Spearman’s correlation was measured for autopolymerized cement samples between variables for color and solidification load (N), which showed a strong correlation between curing process and color change (p < 0.05). There was a correlation between the color change and degree of monomer conversion of the dual curing resin composite luting cement which contained a color indicator system for polymerization reaction. The study also suggested that autopolymerization only resulted in suboptimal polymerization of the cement. By additional light curing considerably higher flexural properties were obtained.     

The effect of butenolide on behavioral and morphological changes in two marine fouling species, the barnacle Balanus amphitrite and the bryozoan Bugula neritina

Butenolide [5-octylfuran-2(5H)-one] is a very promising antifouling compound. Here, the effects of butenolide on larval behavior and histology are compared in two major fouling organisms, viz. cypris larvae of Balanus amphitrite and swimming larvae of Bugula neritina. Butenolide diminished the positive phototactic behavior of B. amphitrite (EC50=0.82 μg ml(-1)) and B. neritina (EC50=3 μg ml(-1)). Its effect on the attachment of cyprids of B. amphitrite was influenced by temperature, and butenolide increased attachment of larvae of B. neritina to the bottom of the experimental wells. At concentrations of 4 μg ml(-1) and 10 μg ml(-1), butenolide decreased attachment of B. amphitrite and B. neritina, respectively, but the effects were reversible within a certain treatment time. Morphologically, butenolide inhibited the swelling of secretory granules and altered the rough endoplasmic reticulum (RER) in the cement gland of B. amphitrite cyprids. In B. neritina swimming larvae, butenolide reduced the number of secretory granules in the pyriform-glandular complex.     

Surface elastic modulus of barnacle adhesive and release characteristics from silicone surfaces

The properties of barnacle adhesive on silicone surfaces were studied by AFM indentation, imaging, and other tests and compared to the barnacle shear adhesion strength. A multilayered structure of barnacle adhesive plaque is proposed based on layered modulus regions measured by AFM indentation. The fracture of barnacles from PDMS surfaces was found to include both interfacial and cohesive failure of barnacle adhesive plaque, as determined by protein staining of the substratum after forced barnacle release from the substrate. Data for freshly released barnacles showed that there was a strong correlation between the mean Young's modulus of the outermost (softest) adhesive layer (E< 0.3 MPa) and the shear strength of adhesion, but no correlation for other higher modulus regions. Linear, quadratic, and Griffith's failure criterion (based on rough estimate of crack length) regressions were used in the fit, and showed significance.     

Uptake of the neurotransmitter histamine into the eyes of larvae of the barnacle (Balanus amphitrite)

The photoreceptors of adult barnacles use histamine as their neurotransmitter and take up (3)H-histamine selectively from the extracellular medium. We assayed for the uptake of (3)H-histamine into the eyes of the free-swimming (nauplius) and settling (cyprid) larval stages of Balanus amphitrite. The extracellular space of nauplii proved permeable to dyes below about 800 molecular weight (MW), indicating that (3)H-histamine (MW 111) introduced into seawater would have access to internal structures. (3)H-Histamine was taken up into nauplii by a process with a K(D) of 0.32 microM. Uptake was antagonized by chlorpromazine, which also blocks uptake of (3)H-histamine into adult photoreceptors. In autoradiographs of serial sections of nauplii and cyprids incubated in (3)H-histamine, the ocelli and compound eyes were labeled; other structures in the animal were not. No eyes or other structures were labeled with (3)H-serotonin, a related amine whose transporter commonly transports histamine as well. These experiments show that a histamine-specific transporter similar to that found in the adult is expressed in all of the eyes of barnacle larvae. In the ocelli, where photoreceptors and pigment cells may be distinguished in the light microscope, label was unexpectedly concentrated far more over the pigment cells than over the photoreceptors.