Quantifying the exploratory behaviour of Amphibalanus amphitrite cyprids

The behavioural response of cypris larvae from A. amphitrite (=Balanus amphitrite) exploring three model glass surfaces is quantified by close-range microscopy. Step length and step duration measurements reveal a response to both surface properties and flow. Without flow, 2-day-old cyprids took larger steps with shorter step duration on hydrophilic glass surfaces (bare and NH₂-treated) vs hydrophobic glass (CH₃-treated). These parameters suggest a more detailed, local inspection of hydrophobic surfaces and a more extensive exploration for hydrophilic surfaces. Cyprids under flow took longer steps and exhibited shorter probing times on hydrophobic glass. On hydrophilic glass, cyprids increased their step duration under flow. This active response is attributed to drag and lift forces challenging the cyprids' temporary anchoring to the substratum. Seven-day-old cyprids showed almost no discrimination between the model surfaces. Microscopic-scale observation of cyprid exploration is expected to provide new insights into interactions between cyprids and surfaces.     

Barnacle cyprid motility and distribution in the water column as an indicator of the settlement-inhibiting potential of nontoxic antifouling chemistries

Testing of new coatings to control fouling frequently involves single-species laboratory bioassays. Barnacle cyprids are among the most widely used model organisms in marine biofouling research, and surfaces that inhibit their settlement are considered to be promising candidates for new coating concepts. An analysis of motility parameters (mean velocity and swimming area coefficient) and distribution of cyprids of Balanus amphitrite in different swimming regions in the vicinity of model surfaces (self-assembled monolayers) is presented. The data are correlated with the settlement preferences of cyprids on these surfaces. Cyprids were predominantly found in interfacial regions and the transition frequencies between swimming regions of different depths were determined.     

Functional polymer brushes via surface-initiated atom transfer radical graft polymerization for combating marine biofouling

Dense and uniform polymer brush coatings were developed to combat marine biofouling. Nonionic hydrophilic, nonionic hydrophobic, cationic, anionic and zwitterionic polymer brush coatings were synthesized via surface-initiated atom transfer radical polymerization (SI-ATRP) of 2-hydroxyethyl methacrylate, 2,3,4,5,6-pentafluorostyrene, 2-(methacryloyloxy)ethyl trimethylammonium chloride, 4-styrenesulfonic acid sodium and N,N′-dimethyl-(methylmethacryloyl ethyl) ammonium propanesulfonate, respectively. The functionalized surfaces had different efficacies in preventing adsorption of bovine serum albumin (BSA), adhesion of the Gram-negative bacterium Pseudomonas sp. NCIMB 2021 and the Gram-positive Staphylococcus aureus, and settlement of cyprids of the barnacle Amphibalanus amphitrite (=Balanus amphitrite). The nonionic hydrophilic, anionic and zwitterionic polymer brushes resisted BSA adsorption during a 2 h exposure period. The nonionic hydrophilic, cationic and zwitterionic brushes exhibited resistance to bacterial fouling (24 h exposure) and cyprid settlement (24 and 48 h incubation). The hydrophobic brushes moderately reduced protein adsorption, and bacteria and cyprid settlement. The anionic brushes were least effective in preventing attachment of bacteria and barnacle cyprids. Thus, the best approach to combat biofouling involves a combination of nonionic hydrophilic and zwitterionic polymer brush coatings on material surfaces.     

Bacterial colonisation and settlement of algal spores and barnacle larvae on low surface energy materials

Melt‐coated films of poly(fluoroalkylacrylate)s and poly(fluoroalkylmethacrylate)s have been exposed respectively to bacterial cultures (Pseudomonas spp., Alteromonas sp. NCIMB 1534 and Desulphovibrio alaskensis NCIMB 13491), Enteromorpha zoospores and cyprid larvae of Balanus amphitrite Darwin. In all experiments, settlement and development was considerably less on the films than on controls of glass, poly‐(methylmethacrylate), poly(tetrafluoroethene) (ptfe) and polyester/glassf ibre composite. The settled bacteria were also much more weakly attached to the fluoropo‐lymer films than to the controls. Enteromorpha zoospores tended to settle at surface faults/cracks and also showed some sensitivity to the fluorine content of the materials. Cyprids did not settle on ptfe and could find only occasional sights for attachment on the films. Although the fluoropolymers show promise as fouling‐resistant coatings, improvements in film quality are required.     

Functional polymer brushes via surface-initiated atom transfer radical graft polymerization for combating marine biofouling

Dense and uniform polymer brush coatings were developed to combat marine biofouling. Nonionic hydrophilic, nonionic hydrophobic, cationic, anionic and zwitterionic polymer brush coatings were synthesized via surface-initiated atom transfer radical polymerization (SI-ATRP) of 2-hydroxyethyl methacrylate, 2,3,4,5,6-pentafluorostyrene, 2-(methacryloyloxy)ethyl trimethylammonium chloride, 4-styrenesulfonic acid sodium and N,N'-dimethyl-(methylmethacryloyl ethyl) ammonium propanesulfonate, respectively. The functionalized surfaces had different efficacies in preventing adsorption of bovine serum albumin (BSA), adhesion of the Gram-negative bacterium Pseudomonas sp. NCIMB 2021 and the Gram-positive Staphylococcus aureus, and settlement of cyprids of the barnacle Amphibalanus amphitrite (=Balanus amphitrite). The nonionic hydrophilic, anionic and zwitterionic polymer brushes resisted BSA adsorption during a 2?h exposure period. The nonionic hydrophilic, cationic and zwitterionic brushes exhibited resistance to bacterial fouling (24?h exposure) and cyprid settlement (24 and 48?h incubation). The hydrophobic brushes moderately reduced protein adsorption, and bacteria and cyprid settlement. The anionic brushes were least effective in preventing attachment of bacteria and barnacle cyprids. Thus, the best approach to combat biofouling involves a combination of nonionic hydrophilic and zwitterionic polymer brush coatings on material surfaces.     

Analysis of behavioural rejection of micro-textured surfaces and implications for recruitment by the barnacle Balanus improvisus

Experiments performed in the field and in the laboratory show that the barnacle, Balanus improvisus, preferentially settles on smooth surfaces. Settlement and recruitment of B. improvisus was evaluated on micro-textured surfaces with scales of surface texture ranging from 1 to 100 μm in profile heights. Surface texture with profile heights within a topographic range of 30-45 μm reduced settlement and recruitment by 92% as compared to smooth surfaces. The reduction in recruitment on micro-textured surfaces is best explained by behavioural responses to surface topography. Behavioural experiments show that cyprids have a higher propensity for smooth surfaces than for micro-textured surfaces. Cyprids spend more time exploring smooth surfaces and more time swimming when exposed to micro-textured surfaces. Micro-textured surfaces are more often rejected by cyprids after exploration than smooth surfaces. It is suggested that some scales of surface texture could be exploited to improve future anti-fouling techniques in geographical areas where Balanus improvisus is a severe fouling problem.     

Correlation between surface chemistry and settlement behaviour in barnacle cyprids (Balanus improvisus)

In laboratory-based biofouling assays, the influence of physico-chemical surface characteristics on barnacle settlement has been tested most frequently using the model organism Balanus amphitrite (= Amphibalanus amphitrite). Very few studies have addressed the settlement preferences of other barnacle species, such as Balanus improvisus (= Amphibalanus improvisus). This study aimed to unravel the effects of surface physico-chemical cues, in particular surface-free energy (SFE) and surface charge, on the settlement of cyprids of B. improvisus. The use of well-defined surfaces under controlled conditions further facilitates comparison of the results with recent similar data for B. amphitrite. Zero-day-old cyprids of B. improvisus were exposed to a series of model surfaces, namely self-assembled monolayers (SAMs) of alkanethiols with varying end-groups, homogenously applied to gold-coated polystyrene (PS) Petri dishes. As with B. amphitrite, settlement of cyprids of B. improvisus was influenced by both SFE and charge, with higher settlement on low-energy (hydrophobic) surfaces and negatively charged SAMs. Positively charged SAMs resulted in low settlement, with intermediate settlement on neutral SAMs of similar SFE. In conclusion, it is demonstrated that despite previous suggestions to the contrary, these two species of barnacle show similar preferences in response to SFE; they also respond similarly to charge. These findings have positive implications for the development of novel antifouling (AF) coatings and support the importance of consistency in substratum choice for assays designed to compare surface preferences of fouling organisms.     

Effect of cyprid age on the settlement of balanus amphitrite darwin in response to natural biofilms

Larval settlement of the barnacle Balanus amphitrite Darwin (Cirripedia, Balanidae) is influenced by natural biofilms. In previous work by others, discriminatory settlement of aged cyprids has been observed in response to biofilms of different age. This study extends prior work by considering the effect of the age of cyprids on the outcome of settlement assays. Settlement was investigated with 0‐day‐old (newly metamorphosed) and 5‐day‐old cyprids. Biofilms under investigation were developed in the field for periods of 5 d and 1 month, and were subsequently included in laboratory settlement assays with a choice between a filmed and an unfilmed substratum. The bioassay was modified from the conventional horizontal dish design in order to generate a low water surface‐to‐volume ratio, which served to suppress larval entrapment in an organic layer on the water surface. Irrespective of cyprid age, a clear discrimination between a filmed and an unfilmed substrata was observed, and the preference for filmed or unfilmed substratum was dependent on the age of the cyprids. Settlement of 0‐day‐old cyprids was inhibited by a biofilmed substratum whereas induction occurred with aged cyprids. This pattern of settlement was independent of biofilm age. Bacterial abundance on unfilmed substrata in treatments and controls was significantly lower than that on biofilmed surfaces, confirming that bacterial contamination did not change the qualitative option during the assay.     

Cypris Ultrastructure,Metamorphosis and Sex in Seven Families of Parasitic Barnacles (Crustacea: Cirripedia: Rhizocephala)

Cypris morphology in the Rhizocephala differs both between the sexes of individual species and between the species. The large aesthetasc on the third antennular segment is unique to male cyprids of the Lernaeodiscidae, Peltogasteridae and Sacculinidae (suborder Kentrogonida) and Mycetomorpha vancouverensis (suborder Akentrogonida). A fourth segmental aesthetasc, common to both sexes, is much longer in males than in females. A spinous process on the attachment disc is another male-specific character, but is absent altogether in the Lernaeodiscidae. It is argued that these aesthetascs help locate the settlement targets. Except in Mycetomorpha vancourverensis, aesthetascs have been secondarily lost in cyprids of the suborder Akentrogonida, and there are no other morphological means for separating the male and female settling stage. In cyprids of Sylon hippolytes (Sylonidae) and of Mycetomorpha vancouverensis and Thompsonia sp. (Akentrogonidae) the long and slender antennules are probably used in penetration without formation of a kentrogon, as already reported for Clistosaccus paguri (Clistosaccidae). Cyprids of Sylon hippolytes and Clistosaccus paguri are almost morphologically identical.     

Dual-functional biomimetic materials: nonfouling poly(carboxybetaine) with active functional groups for protein immobilization

We introduce a dual-functional biocompatible material based on zwitterionic poly(carboxybetaine methacrylate) (polyCBMA), which not only highly resists protein adsorption/cell adhesion, but also has abundant functional groups convenient for the immobilization of biological ligands, such as proteins. The dual-functional properties are unique to carboxybetaine moieties and are not found in other nonfouling moieties such as ethylene glycol, phosphobetaine, and sulfobetaine. The unique properties are demonstrated in this work by grafting a polyCBMA polymer onto a surface or by preparing a polyCBMA-based hydrogel. PolyCBMA brushes with a thickness of 10-15 nm were grafted on a gold surface using the surface-initiated atom transfer radical polymerization method. Protein adsorption was analyzed using a surface plasmon resonance sensor. The surface grafted with polyCBMA very largely prevented the nonspecific adsorption of three test proteins, that is, fibrinogen, lysozyme, and human chorionic gonadotropin (hCG). The immobilization of anti-hCG on the surface resulted in the specific binding of hCG while maintaining a high resistance to nonspecific protein adsorption. Transparent polyCBMA-based hydrogel disks were decorated with immobilized fibronectin. Aortic endothelial cells did not bind to the polyCBMA controls, but appeared to adhere well and spread on the fibronectin-modified surface. With their dual functionality and biomimetic nature, polyCBMA-based materials are very promising for their applications in medical diagnostics, biomaterials/tissue engineering, and drug delivery.     

Photon-modulated changes of cell attachments on poly(spiropyran-co-methyl methacrylate) membranes

Spiropyran is a photoresponsive molecule, and nonionic spiropyran is reversibly changed by UV irradiation to a hydrophilic polar, zwitterionic merocyanine isomer, and back again by visible light irradiation. A copolymer of nitrobenzospiropyran and methyl methacrylate, poly(NSP-co-MMA) was used as a material with a photosensitive surface. UV irradiation of the photosensitive surface of poly(NSP-co-MMA)-coated glass plates decreased the water contact angles (11 +/- 1 degrees ) and increased diameter of a water drop relative to the unexposed surface. Light-induced detachment of platelets and mesenchymal stem (KUSA-A1) cells on poly(NSP-co-MMA)-coated glass plates was observed upon simple- and patterned-light irradiation, whereas no light-induced detachment of platelets and mesenchymal stem cells was observed on poly(methyl methacrylate)-coated glass plates. This is a result of the change from a closed nonpolar spiropyran to the polar zwitterionic merocyanine isomer induced by UV irradiation. Light-induced detachment of fibrinogen adsorbed on poly(NSP-co-MMA) coated glass plates was also observed in this investigation.     

THE ACTION OF COPPER IN ANTIFOULING PAINTS

Experiments on the number of barnacle cyprids found on plane surfaces and on the surface of small pits coated with compositions containing different amounts of cuprous oxide show that the presence of copper does not deter the cyprids from attempting to settle. Copper-oxide paints do not inhibit settlement appreciably; their efficiency as antifouling agents is caused by the toxic action of copper on cyprids and young spat after initial attachment.
The cementing of the shell to the substratum in young barnacles which have survived metamorphosis appears to be hindered by the presence of copper paints so that they are easily dislodged.     

Effects of surface charge and Gibbs surface energy on the settlement behaviour of barnacle cyprids (Balanus amphitrite)

Gibbs surface energy has long been considered to be an important parameter in the design of fouling-resistant surfaces for marine applications. Rigorous testing of the hypothesis that settlement is related to Gibbs surface energy however has never been accomplished, due mainly to practical limitations imposed by the necessary combination of surface engineering and biological evaluation methods. In this article, the effects of surface charge and Gibbs surface energy on the settlement of cyprids of an important fouling barnacle, Balanus amphitrite, were evaluated. Settlement assays were conducted on a range of self-assembled monolayers (SAMs) (CH₃-, OH-, COOH-, N(CH₃)₃ ⁺-, NH₂-terminated), presented in gold-coated polystyrene well plates, varying in terms of their surface charge and Gibbs surface energy. Contrary to contemporary theory, settlement was not increased by high-energy surfaces, rather the opposite was found to be the case with cyprids settling in greater numbers on a low-energy CH₃- SAM compared to a high-energy OH- SAM. Settlement was also greater on negatively-charged SAMs, compared to neutral and positively-charged SAMs. These findings are discussed in the context of data drawn from surfaces that varied in multiple characteristics simultaneously, as have been used previously for such experiments. The finding that surface charge, rather than total surface energy, may be responsible for surface selection by cyprids, will have significant implications for the design of future fouling-resistant materials.     

Field-based video observations of wild barnacle cyprid behaviour in response to textural and chemical settlement cues

Many marine invertebrate larvae respond behaviourally to environmental settlement cues, yet behaviours are often only inferred from settlement patterns or are limited to laboratory studies. The behaviour of wild cypris larvae of Semibalanus balanoides L. was filmed on settlement tiles in the field. Tiles were of five different textures with a nested treatment of crude conspecific adult extract (AE). The effects of texture and AE on eleven defined behaviours were analysed. Texture affected the gross and net exploratory distances, velocity, acceleration and time spent exploring. AE attracted more cyprids during the first minute of immersion and increased the time spent on surfaces. Relatively few arrivals that either travel far and fast, or exit the surface rapidly, may indicate a lower chance of settlement. An increase in time spent on a surface may increase the probability of being in contact with the surface when the sign stimulus to settle occurs.     

Influence of oil shale on intertidal organisms: Effect of oil shale extract on settlement of the barnacle Balanus balanoides (L.)

As part of a study to investigate the effect of oil seeps on intertidal organisms, oil extracts of Blackstone oil shale from Kimmeridge on the Dorset coast were used in laboratory experiments to test their effect on the settlement of the barnacle Balanus balanoides (L.). Thin films of oil extract painted on the surface of pits in slate panels had no effect on cyprid settlement when applied up to a surface density of 2.8 g · m^?2, representing a thickness of 3.3 μm. Larger surface densities of oil stimulated cyprids to settle in far greater numbers than on unoiled panels. The maximum effect was obtained at a surface density of between 14.0 and 56.0 g · m ^?2, representing a thickness of 16.5 μm and 66.0 μm. With higher concentration of oil in the pits, stimulation to settle was reduced although cyprid settlement was still encouraged at a surface density of oil of 112g · m^?2 or 132 μm thickness.The unfractionated crude oil shale extract was a less powerful stimulus for barnacle settlement than a partially purified solution of the integumental protein arthropodin, another strong settlement inducer for barnacle cyprids.     

Effects of surface-active block copolymers with oxyethylene and fluoroalkyl side chains on the antifouling performance of silicone-based films

Block copolymers made from a poly(dimethyl siloxane) (Si) and a poly(meth)acrylate carrying oxyethylene (EG) or fluoroalkyl (AF) side chains were synthesized and incorporated as surface-active components into a silicone matrix to produce cross-linked films with different surface hydrophilicity/phobicity. Near-edge X-ray absorption fine structure (NEXAFS) studies showed that film surfaces containing Si-EG were largely populated by the siloxane, with the oxyethylene chains present only to a minor extent. In contrast, the fluorinated block was selectively segregated to the polymer–air interface in films containing Si-AF as probed by NEXAFS and X-ray photoelectron spectroscopy (XPS) analyses. Such differences in surface composition were reflected in the biological performance of the coatings. While the films with Si-EG showed a higher removal of both Ulva linza sporelings and Balanus amphitrite juveniles than the silicone control, those with Si-AF exhibited excellent antifouling properties, preventing the settlement of cyprids of B. amphitrite.     

A novel biofilm channel for evaluating the adhesion of diatoms to non-biocidal coatings

Laboratory assessment of the adhesion of diatoms to non-toxic fouling-release coatings has tended to focus on single cells rather than the more complex state of a biofilm. A novel culture system based on open channel flow with adjustable bed shear stress values (0–2.4?Pa) has been used to produce biofilms of Navicula incerta. Biofilm development on glass and polydimethylsiloxane elastomer (PDMSe) showed a biphasic relationship with bed shear stress, which was characterised by regions of biofilm stability and instability reflecting cohesion between cells relative to the adhesion to the substratum. On glass, a critical shear stress of 1.3–1.4?Pa prevented biofilm development, whereas on PDMS, biofilms continued to grow at 2.4?Pa. Studies of diatom biofilms cultured on zwitterionic coatings using a bed shear stress of 0.54?Pa showed lower biomass production and adhesion strength on poly(sulfobetaine methacrylate) compared to poly(carboxybetaine methacrylate). The dynamic biofilm approach provides additional information to supplement short duration laboratory evaluations.     

Individual and Coupled Effects of Echinoderm Extracts and Surface Hydrophobicity on Spore Settlement and Germination in the Brown Alga Hincksia irregularis

Marine substrata possess cues that influence the behavior of fouling organisms. Initial adhesion of fouling algal zoospores to surfaces is also theorized to depend primarily upon interactions between substrata and spore cell bodies and flagellar membranes. In an effort to identify cues and surface characteristics that influence spore settlement and early development, the effects of bioactive echinoderm extracts, surface charge, and surface hydrophobicity were examined individually and in tandem on zoospore settlement and germination in Hincksia irregularis. Experiments utilizing 96-well plastic culture plates confirmed that spore settlement and germination were significantly affected by surface charge and hydrophobicity as well as by echinoderm metabolites, both individually and in tandem. Spore settlement rates in the dark over 30?min were >?400% higher on hydrophobic surfaces than on positively and negatively charged surfaces. Spore germling numbers were >?300% higher on hydrophobic surfaces than on positively and negatively charged surfaces when spores were allowed to settle in the light for 30?min and the settled spores allowed to subsequently germinate for 24?h. Spore germling numbers were consistently >?25% higher on hydrophobic surfaces than on positively and negatively charged surfaces when equal numbers of spores were allowed to completely settle in the light and subsequently germinate for 24?h. H. irregularis germ tube lengths were also significantly longer on positively charged plates than on negatively charged plates. All echinoderm extracts tested had significant effects on germination and settlement at levels below those of estimated ecological concentrations. Short-term (30?min) exposure and subsequent germination experiments indicated that higher concentrations of extracts had rapid toxic effects on algal spores. Synchronous effects of echinoderm extracts and plate charge upon spore settlement varied considerably and did not show a strong dose response relationship. Long-term (24?h) exposure of spores to echinoderm extracts had dosage dependent effects on germination and spore survival. The results of this study indicate that H. irregularis spores possess the capacity for complex responses to their environment, utilizing combined cues of surface charge, surface energy and biochemistry to determine where to settle and germinate. These responses may aid spores in the detection of suitable substrata and conditions for settlement in the marine environment.     

Sex determination in the androdioecious barnacle Scalpellum scalpellum (Crustacea: Cirripedia)

How androdioecy (coexistence of hermaphrodites and males) is maintained is still poorly understood. Therefore, sex determination was studied in the androdioecious barnacle Scalpellum scalpellum L. First, 247 cypris larvae from seven broods were investigated for sexual dimorphism in larval morphology and found to be all identical. Second, experiments with cyprids showed that males and hermaphrodites differ distinctly in morphology as soon as 4–5 days after settlement. Third, 14 252 cyprids were allowed to settle on the bottom of their culture cages, and all surviving larvae developed into hermaphrodites and none into dwarf males. Fourth, larvae settled in hermaphrodite receptacles (i.e. future males) were removed at increasing intervals after settlement to study if the male and hermaphrodite sexual expressions are fixed or plastic. All larvae became dwarf males if allowed to stay there for more than 8 h after settlement. But if removed within 3 h after settlement, half of them developed into hermaphrodites. We conclude that an environmental sex determination mechanism operates in S. scalpellum. Together with a 1:1 hermaphrodite/male ratio observed in previously reported experiments offering a free choice of settlement, we suggest that all larvae are potential hermaphrodites, but only 50% can settle in hermaphrodite receptacles and yield males.