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.     

Algal antifouling and fouling-release properties of metal surfaces coated with a polymer inspired by marine mussels

The marine antifouling and fouling-release performance of titanium surfaces coated with a bio-inspired polymer was investigated. The polymer consisted of methoxy-terminated poly(ethylene glycol) (mPEG) conjugated to the adhesive amino acid l-3,4-dihydroxyphenylalanine (DOPA) and was chosen based on its successful resistance to protein and mammalian cell fouling. Biofouling assays for the settlement and release of the diatom Navicula perminuta and settlement, growth and release of zoospores and sporelings (young plants) of the green alga Ulva linza were carried out. Results were compared to glass, a poly(dimethylsiloxane) elastomer (Silastic T2) and uncoated Ti. The mPEG-DOPA3 modified Ti surfaces exhibited a substantial decrease in attachment of both cells of N. perminuta and zoospores of U. linza as well as the highest detachment of attached cells under flow compared to control surfaces. The superior performance of this polymer over a standard silicone fouling-release coating in diatom assays and approximately equivalent performance in zoospore assays suggests that this bio-inspired polymer may be effective in marine antifouling and fouling-release applications.     

Poly(ethylene glycol)-containing hydrogel surfaces for antifouling applications in marine and freshwater environments

This work describes the fabrication, characterization, and biological evaluation of a thin protein-resistant poly(ethylene glycol) (PEG)-based hydrogel coating for antifouling applications. The coating was fabricated by free-radical polymerization on silanized glass and silicon and on polystyrene-covered silicon and gold. The physicochemical properties of the coating were characterized by infrared spectroscopy, ellipsometry, and contact angle measurements. In particular, the chemical stability of the coating in artificial seawater was evaluated over a six-month period. These measurements indicated that the degradation process was slow under the test conditions chosen, with the coating thickness and composition changing only marginally over the period. The settlement behavior of a broad and diverse group of marine and freshwater fouling organisms was evaluated. The tested organisms were barnacle larvae (Balanus amphitrite), algal zoospores (Ulva linza), diatoms (Navicula perminuta), and three bacteria species (Cobetia marina, Marinobacter hydrocarbonoclasticus, and Pseudomonas fluorescens). The biological results showed that the hydrogel coating exhibited excellent antifouling properties with respect to settlement and removal.     

Effect of bacterial biofilms formed on fouling-release coatings from natural seawater and Cobetia marina,on the adhesion of two marine algae

Previous studies have shown that bacterial biofilms formed from natural seawater (NSW) enhance the settlement of spores of the green alga Ulva linza, while single-species biofilms may enhance or reduce settlement, or have no effect at all. However, the effect of biofilms on the adhesion strength of algae, and how that may be influenced by coating/surface properties, is not known. In this study, the effect of biofilms formed from natural seawater and the marine bacterium Cobetia marina, on the settlement and the adhesion strength of spores and sporelings of the macroalga U. linza and the diatom Navicula incerta, was evaluated on Intersleek® 700, Intersleek® 900, poly(dimethylsiloxane) and glass. The settlement and adhesion strength of these algae were strongly influenced by biofilms and their nature. Biofilms formed from NSW enhanced the settlement (attachment) of both algae on all the surfaces while the effect of biofilms formed from C. marina varied with the coating type. The adhesion strength of spores and sporelings of U. linza and diatoms was reduced on all the surfaces biofilmed with C. marina, while adhesion strength on biofilms formed from NSW was dependent on the alga (and on its stage of development in the case of U. linza), and coating type. The results illustrate the complexity of the relationships between fouling algae and bacterial biofilms and suggest the need for caution to avoid over-generalisation.     

Gregarious settlement in cypris larvae:the effects of cyprid age and assay duration

Gregarious behaviour of marine larvae is perhaps most clearly associated with finding a suitable habitat in a changeable or restricted environment, or with finding other conspecifics with which to mate. Prior work has shown that in settlement assays using cypris larvae of the barnacle Balanus amphitrite, gregarious interactions significantly affected the interpretation of experiments testing the activity of organic settlement promoters and inhibitors. Other studies have also shown effects of cyprid age and pheromone concentration on settlement behaviour. However, the effects of interactions between gregariousness and these two factors are not known. The aim of this study was to test the hypotheses that i) as cyprids age the effects of gregariousness become less apparent, and ii) as the duration of the experiment increases gregarious effects become more apparent, using cypris larvae of B. amphitrite and Balanus improvisus. Three age classes of cyprids were used at six densities in a fully factorial design. For B. improvisus cyprids significant gregarious effects occurred between 3 or more larvae, and although larval age and experiment duration had significant main effects, there were no interactions between these important factors and gregariousness. For B. amphitrite cyprids significant gregarious effects also occurred with 3 larvae per well, though this effect was strongly dependent upon experiment duration. B. amphitrite cyprid sensitivity to conspecific cues does not change with age, although increasing experiment duration and age interact to increase settlement. Differences between species may be due to different thresholds to conspecific larval cues, or B. improvisus cyprids release much more larval temporary adhesive during exploration.     

Roughness-dependent removal of settled spores of the green alga Ulva (syn. Enteromorpha) exposed to hydrodynamic forces from a water jet

Topographic features change the hydrodynamic regime over surfaces subjected to flow. Hydrodynamic microenvironments around topographic structures may have consequences for recruitment and removal of propagules of marine benthic organisms. The settlement and adhesion of zoospores from the green alga Ulva linza (syn. Enteromorpha linza) to defined topographies was investigated. A range of topographic size scales (Rz: 25-100 microm) was manufactured from plankton nets, creating patterns with ridges and depressions. The topographic scales span a roughness similar to that of natural substrata and antifouling coatings. Spores were removed from the surfaces by a calibrated water jet. Fewer spores were removed from the smallest topographic structure tested (Rz: 25 microm) compared to both the smooth (Rz: 1) and the roughest (Rz: 100 microm) structures. Zoospores that settled in depressions were less likely to be removed compared to spores on the ridges. The results in terms of the interaction between surface topography and hydrodynamic forces have implications for both natural substrata exposed to wave action and antifouling surfaces on ships' hulls. The possible effects of topography on increasing zoospore adhesion and offering a refuge from hydrodynamic forces are discussed.     

The effects of nitric oxide in settlement and adhesion of zoospores of the green alga Ulva

Previous studies have shown that elevated nitric oxide (NO) reduces adhesion in diatom, bacterial and animal cells. This article reports experiments designed to investigate whether elevated NO reduces the adhesion of zoospores of the green alga Ulva, an important fouling species. Surface-normalised values of NO were measured using the fluorescent indicator DAF-FM DA and parallel hydrodynamic measurements of adhesion strength were made. Elevated levels of NO caused by the addition of the exogenous NO donor SNAP reduced spore settlement by 20% and resulted in lower adhesion strength. Addition of the NO scavenger cPTIO abolished the effects of SNAP on adhesion. The strength of attachment and NO production by spores in response to four coatings (Silastic® T2; Intersleek® 700; Intersleek® 900 and polyurethane) shows that reduced adhesion is correlated with an increase in NO production. It is proposed that in spores of Ulva, NO is used as an intracellular signalling molecule to detect how conducive a surface is for settlement and adhesion. The effect of NO on the adhesion of a range of organisms suggests that NO-releasing coatings could have the potential to control fouling.     

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.     

Charged hydrophilic polymer brushes and their relevance for understanding marine biofouling

The resistance of charged polymers to biofouling was investigated by subjecting cationic (PDMAEMA), anionic (PSPMA), neutral (PHEMA-co-PEG₁₀MA), and zwitterionic (PSBMA) brushes to assays testing protein adsorption; attachment of the marine bacterium Cobetia marina; settlement and adhesion strength of zoospores of the green alga Ulva linza; settlement of barnacle (Balanus amphitrite and B. improvisus) cypris larvae; and field immersion tests. Several results go beyond the expected dependence on direct electrostatic attraction; PSPMA showed good resistance towards attachment of C. marina, low settlement and adhesion of U. linza zoospores, and significantly lower biofouling than on PHEMA-co-PEG₁₀MA or PSBMA after a field test for one week. PDMAEMA showed potential as a contact-active anti-algal coating due to its capacity to damage attached spores. However, after field testing for eight weeks, there were no significant differences in biofouling coverage among the surfaces. While charged polymers are unsuitable as antifouling coatings in the natural environment, they provide valuable insights into fouling processes, and are relevant for studies due to charging of nominally neutral surfaces.     

Species-specific engineered antifouling topographies: correlations between the settlement of algal zoospores and barnacle cyprids

Novel, non-toxic antifouling technologies are focused on the manipulation of surface topography to deter settlement of the dispersal stages of fouling organisms. This study investigated the effect of the aspect ratio (feature height/feature width) of topographical features engineered in polydimethylsiloxane, on the settlement of cyprids of Balanus amphitrite and zoospores of Ulva linza. The correlation of relative aspect ratios to antifouling efficacy was proven to be significant. An increase in aspect ratio resulted in an increase of fouling deterrence for both zoospores and cyprids. The spore density of Ulva was reduced 42% with each unit increase in aspect ratio of the Ulva-specific Sharklet AF topography. Similarly, the number of settled cyprids was reduced 45% with each unit increase in aspect ratio. The newly described barnacle-specific Sharklet AF topography (40 microm feature height, aspect ratio of 2) reduced cyprid settled by 97%. Techniques have been developed to superimpose the smaller Ulva-specific topographies onto the barnacle-specific surfaces into a hierarchical structure to repel both organisms simultaneously. The results for spore settlement on first-generation hierarchical surfaces provide insight for the efficacious design of such structures when targeting multiple settling species.     

Poly(dimethyl siloxane) (PDMS) network blends of amphiphilic acrylic copolymers with poly(ethylene glycol)-fluoroalkyl side chains for fouling-release coatings. II. Laboratory assays and field immersion trials

Amphiphilic copolymers containing different amounts of poly(ethylene glycol)-fluoroalkyl acrylate and polysiloxane methacrylate units were blended with a poly(dimethyl siloxane) (PDMS) matrix in different proportions to investigate the effect of both copolymer composition and loading on the biological performance of the coatings. Laboratory bioassays revealed optimal compositions for the release of sporelings of Ulva linza, and the settlement of cypris larvae of Balanus amphitrite. The best-performing coatings were subjected to field immersion tests. Experimental coatings containing copolymer showed significantly reduced levels of hard fouling compared to the control coatings (PDMS without copolymer), their performance being equivalent to a coating based on Intersleek 700?. XPS analysis showed that only small amounts of fluorine at the coating surface were sufficient for good antifouling/fouling-release properties. AFM analyses of coatings under immersion showed that the presence of a regular surface structure with nanosized domains correlated with biological performance.     

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。     

Microbial biofilms facilitate adhesion in biofouling invertebrates

Much interest has focused on the role of microbial layers--biofilms--in stimulating attachment of invertebrates and algae to submerged marine surfaces. We investigated the influence of biofilms on the adhesion strength of settling invertebrates. Larvae of four species of biofouling invertebrate were allowed to attach to test surfaces that were either clean or coated with a natural biofilm. Measuring larval removal under precisely controlled flow forces, we found that biofilms significantly increased adhesion strength in the ascidian Phallusia nigra, the polychaete tubeworm Hydroides elegans, and the barnacle Balanus amphitrite at one or more developmental stages. Attachment strength in a fourth species, the bryozoan Bugula neritina, was neither facilitated nor inhibited by the presence of a biofilm. These results suggest that adhesive strength and perhaps composition may vary across different invertebrate taxa at various recruitment stages, and mark a new path of inquiry for biofouling research.     

Adrenoceptor compounds prevent the settlement of marine invertebrate larvae: Balanus amphitrite (Cirripedia), Bugula neritina (Bryozoa) and Hydroides elegans (Polychaeta)

The effects of the neurotransmitter blockers idazoxan and phentolamine on the larval settlement of three marine invertebrate species belonging to three different phyla were investigated by using in vitro concentration-response bioassays. Since neurotransmitters are known to influence metamorphic transitions in invertebrate larvae, neurotransmitter blockers were tested to evaluated their sublethal effects on larvae. The alpha-adrenergic antagonists idazoxan and phentolamine inhibited settlement of Balanus amphitrite (Cirripedia), Bugula neritina (Bryozoa) larvae, and larvae of the polychaete Hydroides elegans (Polychaeta) in a concentration-and taxon-dependent manner. At concentrations of 10(-3) M of both agents, larvae of all three species became immobile and subsequently died within 24 h. While cumulative settlement rates were observed after 48 h for B. amphitrite and H. elegans, and after 5 h for B. neritina, >90% of the larvae that settled did so within 24 h for the first two species and within 1 h for B. neritina. The tendency of the hydrophobic idazoxan and phentolamine to accumulate at solid surfaces most probably contributes to their successful inhibition of larval settlement. This ability makes them particularly attractive as candidates for the development of slow-release carriers in antifouling paints.     

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.     

Nature and perception of barnacle settlement pheromones

It is now almost 50 years since the gregarious settlement of barnacles and its chemical basis was first described. Although originally noted for Elminius modestus, mechanistic studies of gregariousness have focused on two species, Semibalanus balanoides and Balanus amphitrite. By virtue of its ease of study and its economic importance as a fouling organism, the latter species has assumed increasing importance in recent years. This paper will provide an overview of studies on settlement pheromones and their perception. An adult glycoprotein, arthropodin (now known as settlement-inducing protein complex or SIPC), was once thought to be the sole pheromone involved in the induction of cypris larval settlement. At least two other pheromones are now known to be involved, a waterborne cue originating from the adult and the cypris temporary adhesive. The latter is related, immunologically, to SIPC. In keeping with many other examples of chemical communication, the available evidence suggests that barnacle settlement induction involves receptor-ligand interactions and a signal transduction pathway(s) that translates into attachment and metamorphosis. Similar findings have been reported for some, but not all, marine invertebrate larvae examined thus far and the implications for antifoulant development are discussed.     

Adhesion Strength of Settled Spores of the Green Alga Enteromorpha

Strengths of attachment of spores of the green fouling alga Enteromorpha to glass have been measured using a modified water jet apparatus. Surface pressures of ～250 kPa were required to quantitatively remove attached spores after 4 h contact with a surface. The development of adhesive and cohesive strength is highly time-dependent; after 8 h in contact with a surface spores did not detach, even at pressures in excess of 250 kPa. Spores settled in groups are more resistant to detachment than single spores, which suggests that the adaptive value of gregarious settlement behaviour may lie in the greater resistance of groups to detachment forces in a naturally turbulent environment. The interfacial forces exerted as water impinges on the surface and the derivation of adhesion strength values in terms of wall shear stress are discussed and compared with those obtained by other methods. A surface pressure of 250 kPa approximates to 325 Pa wall shear stress. Calculation using the power-law formula predicts that detachment forces of this magnitude are unlikely to be realized at operating speeds for most vessels and that most Enteromorpha spores would not detach from untreated hulls.     

Forces involved in adhesion of Bacillus cereus spores to solid surfaces under different environmental conditions

H usmark , U. & R önner , U. 1990. Forces involved in adhesion of Bacillus cereus spores to solid surfaces under different environmental conditions. Journal of Applied Bacteriology 69 , 557–562.
The adhesion of Bacillus cereus spores (NCTC 2599) to hydrophobic and hydro-philic glass surfaces was studied when environmental conditions were varied. The spores were exposed in media of different polarities as well as different pH and ionic concentrations. With increasing ethanol concentrations, the polarity of the medium was decreased and the predominant force of attraction was found to be hydrophobic. The spore surface was uncharged at a pH around 3, at which value the spore was most adhesive to both hydrophobic and hydrophilic glass. This could be attributable to the absence of electrostatic repulsion. An increased ionic concentration of the bulk increased the degree of adhesion especially to the hydrophilic surfaces. This indicates the suppression of a solvation barrier at high ionic concentrations, when the polymers of the spore surface become dehydrated.     

Anomalous settlement behavior of Ulva linza zoospores on cationic oligopeptide surfaces

Identification of settlement cues for marine fouling organisms opens up new strategies and methods for biofouling prevention, and enables the development of more effective antifouling materials. To this end, the settlement behaviour of zoospores of the green alga Ulva linza onto cationic oligopeptide self-assembled monolayers (SAMs) has been investigated. The spores interact strongly with lysine- and arginine-rich SAMs, and their settlement appears to be stimulated by these surfaces. Of particular interest is an arginine-rich oligopeptide, which is effective in attracting spores to the surface, but in a way which leaves a large fraction of the settled spores attached to the surface in an anomalous fashion. These 'pseudo-settled' spores are relatively easily detached from the surface and do not undergo the full range of cellular responses associated with normal commitment to settlement. This is a hitherto undocumented mode of settlement, and surface dilution of the arginine-rich peptide with a neutral triglycine peptide demonstrates that both normal and anomalous settlement is proportional to the surface density of the arginine-rich peptide. The settlement experiments are complemented with physical studies of the oligopeptide SAMs, before and after extended immersion in artificial seawater, using infrared spectroscopy, null ellipsometry and contact angle measurements.     

Expression of calmodulin and myosin light chain kinase during larval settlement of the Barnacle Balanus amphitrite

Barnacles are one of the most common organisms in intertidal areas. Their life cycle includes seven free-swimming larval stages and sessile juvenile and adult stages. The transition from the swimming to the sessile stages, referred to as larval settlement, is crucial for their survivor success and subsequent population distribution. In this study, we focused on the involvement of calmodulin (CaM) and its binding proteins in the larval settlement of the barnacle, Balanus ( = Amphibalanus) amphitrite. The full length of CaM gene was cloned from stage II nauplii of B. amphitrite (referred to as Ba-CaM), encoding 149 amino acid residues that share a high similarity with published CaMs in other organisms. Quantitative real-time PCR showed that Ba-CaM was highly expressed in cyprids, the stage at which swimming larvae are competent to attach and undergo metamorphosis. In situ hybridization revealed that the expressed Ba-CaM gene was localized in compound eyes, posterior ganglion and cement glands, all of which may have essential functions during larval settlement. Larval settlement assays showed that both the CaM inhibitor compound 48/80 and the CaM-dependent myosin light chain kinase (MLCK) inhibitor ML-7 effectively blocked barnacle larval settlement, whereas Ca(2+)/CaM-dependent kinase II (CaMKII) inhibitors did not show any clear effects. The subsequent real-time PCR assay showed a higher expression level of Ba-MLCK gene in larval stages than in adults, suggesting an important role of Ba-MLCK gene in larval development and competency. Overall, the results suggest that CaM and CaM-dependent MLCK function during larval settlement of B. amphitrite.