Drag-reducing riblets with fouling-release properties: development and testing.

The manufacture and preliminary testing of a drag-reducing riblet texture with fouling-control properties is presented. The commercial fouling-release product Intersleek® 1100SR was modified to manufacture riblet-textured coatings with an embossing technology. Hydrodynamic drag measurements in a Taylor-Couette set-up showed that the modified Intersleek® riblets reduced drag by up to 6% compared to a smooth surface. Barnacle settlement assays demonstrated that the riblets did not substantially reduce the ability of Intersleek® 1100SR to prevent fouling by cyprids of Balanus amphitrite. Diatom adhesion tests revealed significantly higher diatom attachment on the riblet surface compared to smooth Intersleek® 1100SR. However, after exposure to flow, the final cell density was similar to the smooth surface. Statically immersed panels in natural seawater showed an increase of biofilm cover due to the riblets. However, the release of semi-natural biofilms grown in a multi-species biofilm culturing reactor was largely unaffected by the presence of a riblet texture.

Attachment of Algal Cells to Zwitterionic Self-Assembled Monolayers Comprised of Different Anionic Compounds.

The influence of zwitterionic self-assembled monolayers on settlement and removal of algae was studied. The monolayers were constructed either from zwitterionic thiols or from solutions of positively and negatively charged thiols. The cationic component was composed of quaternary ammonium terminated thiols and the anionic component contained sulfate or carboxylate termination. During assembly, all surfaces showed a strong tendency for equilibration of the surface charge. Settlement and adhesion assays with zoospores of Ulva linza and the diatom Navicula incerta, and field tests of the initial surface colonization revealed the relevance of charge equilibration for the biological inertness of the prepared surfaces.

Resistance of Amphiphilic Polysaccharides against Marine Fouling Organisms.

Amphiphilic coatings are promising candidates for fouling-release applications. As hydrophilic components, polysaccharides are interesting and environmentally benign building blocks. We used covalently coupled alginic acid (AA) and hyaluronic acid (HA) and postmodified them with a hydrophobic fluorinated amine. The surfaces showed good stability under marine conditions and fluorination led to a decreased uptake of Ca(2+) ions after modification. In single species settlement assays (bacteria, diatoms, barnacle cypris larvae), the modification decreased the settlement density and/or the adhesion strength of many of the tested species. Field studies supported findings of the laboratory experiments, as hydrophobic modification of AA and HA decreased diatom colonization.

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.

Three dimensional tracking of exploratory behavior of barnacle cyprids using stereoscopy.

Surface exploration is a key step in the colonization of surfaces by sessile marine biofoulers. As many biofouling organisms can delay settlement until a suitable surface is encountered, colonization can comprise surface exploration and intermittent swimming. As such, the process is best followed in three dimensions. Here we present a low-cost transportable stereoscopic system consisting of two consumer camcorders. We apply this novel apparatus to behavioral analysis of barnacle larvae (≈800 µm length) during surface exploration and extract and analyze the three-dimensional patterns of movement. The resolution of the system and the accuracy of position determination are characterized. As a first practical result, three-dimensional swimming trajectories of the cypris larva of the barnacle Semibalanus balanoides are recorded in the vicinity of a glass surface and close to PEG2000-OH and C(11)NMe(3)(+)Cl(-) terminated self-assembled monolayers. Although less frequently used in biofouling experiments due to its short reproductive season, the selected model species [Marechal and Hellio (2011), Int Biodeterior Biodegrad, 65(1):92-101] has been used following a number of recent investigations on the settlement behavior on chemically different surfaces [Aldred et al. (2011), ACS Appl Mater Interfaces, 3(6):2085-2091]. Experiments were scheduled to match the availability of cyprids off the north east coast of England so that natural material could be used. In order to demonstrate the biological applicability of the system, analysis of parameters such as swimming direction, swimming velocity and swimming angle are performed.

Tensile and dynamic mechanical analysis of the distal portion of mussel (Mytilus edulis) byssal threads.

Dynamic mechanical analysis was used to record the behaviour of hydrated and dehydrated byssal threads under tensile stress and during dynamic thermal cycling. Fresh byssi, and byssi aged two weeks prior to testing, were used to further study the effects of age on the mechanical properties of this material. It was found that while older threads demonstrated increased stiffness, age did not necessarily affect their ultimate tensile strength. Dehydration had a more pronounced effect on thread stiffness and also increased the ultimate strength of the material. In their dry state, byssal threads displayed multiple yield points under tension and these, it is suggested, could equate to different phases within the bulk of the material. Dynamic analysis revealed glass transition (Tg) and ecologically relevant operational temperatures for byssi, where their modulus (E') remained constant. These discoveries are related to the ecological function of byssal threads and to the emerging field of biomimetics.

Mussel (Mytilus edulis) byssus deposition in response to variations in surface wettability.

Mussels (Mytilus edulis) are economically important in their role as an aquaculture species and also with regard to marine biofouling. They attach tenaciously to a wide variety of submerged surfaces by virtue of collagenous attachment threads termed 'byssi'. The aim of this study was to characterize the spreading of the byssal attachment plaque, which mediates attachment to the surface, on a range of surfaces in response to changes in wettability. To achieve this, well characterized self-assembled monolayers of omega-terminated alkanethiolates on gold were used, allowing correlation of byssal plaque spreading with a single surface characteristic--wettability. The present results were inconsistent with those from previous studies, in that there was a positive correlation between plaque size and surface wettability; a trend which is not explained by conventional wetting theory for a three-phase system. A recent extension to wetting theory with regard to hydrophilic proteins is discussed and the results of settlement assays are used to attempt reconciliation of these results with those of similar previous studies and, also, with recent data presented for the spreading of Ulva linza spore adhesive.

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.

Effects of 4-n-nonylphenol and 17beta-oestradiol on early development of the barnacle Elminius modestus.

Pollutants that are present in the aquatic environment and cause abnormal endocrine function in wildlife populations have been termed endocrine disrupting chemicals (EDCs). The impacts of these chemicals on the reproduction and development of vertebrates has been shown to be significant in both field studies and laboratory experiments. Over the past decade the number of investigations into the impacts of EDCs that affect reproductive and sexual characteristics (reproductive EDCs) has increased and evidence of their potency is evident in numerous wildlife species and through data from in vitro tests. However, little information is available on whether chemicals which act as EDCs in vertebrate species affect aquatic invertebrates. The case of imposex in archeogastropods following exposure to tributyltin (TBT) is a notable exception. Moreover, a number of studies have shown that development, fecundity and reproductive output of some aquatic invertebrates are affected significantly by exposure to pollutants. In order to determine whether external signs of exposure to vertebrate EDCs can be observed and monitored in invertebrate species, we exposed larvae of the barnacle Elminius modestus to environmentally realistic concentrations of the xeno-oestrogen, 4-n-nonylphenol (NP), and the natural oestrogen, 17beta-oestradiol (E(2)). Early life stages (nauplii and cyprids) were also exposed in the laboratory to determine whether there were effects on the timing of larval development and settlement. Ovary development and size of juveniles was measured following chronic exposure. Exposure to NP in the concentration range 0.01-10 µg l(-1) resulted in disruption of the timing of larval development. Similar results were obtained with E(2). Pulse exposures showed that the timing of exposure is critical and exposures for a period of 12 months caused long-term effects. A linear, concentration-dependent response was not evident.

Identification of methyl farnesoate in the cypris larva of the barnacle, Balanus amphitrite, and its role as a juvenile hormone.

Previous investigations have shown that insect juvenile hormone (JH) and its analogues induce precocious metamorphosis of barnacle cypris larvae. In the present study, methyl farnesoate (MF; structurally identical to JH III, except for the absence of an epoxide group) has been shown to have a concentration-dependent effect on the development of cyprids of the barnacle Balanus amphitrite. Analysis of cypris extracts by gas chromatography-mass spectrometry with selected ion monitoring (GC-MS-SIM) confirmed the presence of endogenous MF. These data provide evidence that MF functions as a juvenilizing hormone in barnacle cyprids, an effect that hitherto has not been noted.

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.

Eicosanoid biosynthesis in an advanced deuterostomate invertebrate, the sea squirt (Ciona intestinalis).

The eicosanoid generating potential of tunic, branchial basket, intestine, ovary and tadpole larvae from the sea squirt, Ciona intestinalis, was examined using a combination of reverse phase high performance liquid chromatography, gas chromatography-mass spectrometry and enzyme immunoassay. All organs examined synthesized the lipoxygenase products 12-hydroxyeicosapentaenoic acid (12-HEPE) and 8-HEPE implying that both 8- and 12-lipoxygenase activity are widely distributed in this species. In addition, tunic and branchial basket generated significant amounts of 8,15-diHEPE and smaller amounts of 8,15-dihydroxyeicosatetraenoic acid (8,15-diHETE), while tunic alone generated small amounts of conjugated tetraene-containing material with a UV chromophore and mass ion characteristic of a lipoxin-like compound. The broad range lipoxygenase inhibitors, esculetin and nordihydroguaiaretic acid, both caused a significant dose dependent inhibition of 12-HEPE and 8,15-diHEPE biosynthesis in tunic, while the specific 5-lipoxygenase inhibitor, REV-5901, and the specific 5-lipoxygenase activating protein inhibitor, MK-866, had no observable effect on the lipoxygenase profile of this tissue. Tunic, branchial basket, intestine and ovary all generated significant amounts of prostaglandin (PG) E and PGF immunoreactive material and smaller amounts of thromboxane B immunoreactive material as measured by enzyme immunoassay. The non-specific cyclooxygenase (COX) inhibitor, indomethacin, the selective COX-1 inhibitors, resveratrol and valerylsalicylate, and the specific COX-2 inhibitors, NS-398, etolodac and DFU (5,5-dimethyl-3-(3-fluorophenyl)-4-(4-methylsulphonyl) phenyl-2(5H)-furanone) all caused a significant dose dependent inhibition of the biosynthesis of PGE immunoreactive material. However, the specific COX-2 inhibitors were most effective, perhaps implying that a COX-2-like enzyme may be present in this species.

Immunological studies on the settlement-inducing protein complex (SIPC) of the barnacle Balanus amphitrite and its possible involvement in larva-larva interactions.

Immunological investigation has revealed that a settlement-inducing protein complex (SIPC), which induces cypris settlement of the barnacle Balanus amphitrite, is synthesized during larval development and accumulates in the cypris larva. We previously purified the SIPC from adult B. amphitrite, which was active when bound to a substratum. The SIPC is a glycoprotein of high molecular mass, consisting of three major subunits of 76, 88 and 98 kDa with lentil lectin (LCA)-binding sugar chains. In the present study, we prepared antiserum against each LCA-binding subunit of SIPC, and performed immunoblot analyses. Immunoblotting of adult extracts showed that anti-76-kDa antibody reacted only with the 76-kDa protein, whereas anti-88-kDa and anti-98-kDa antibodies reacted with both the 88-kDa and the 98-kDa proteins. Immunoblotting of larval extracts indicated that reactivity of the 76-kDa protein to anti-76-kDa antiserum increased during larval development and cyprid extracts reacted strongly. Moreover, by using immunostaining we found that the SIPC was contained in 'footprints' of cyprids, which have been shown to act as a settlement-inducing pheromone, and is secreted onto the antennular attachment discs. The results suggest that the SIPC (or SIPC-like proteins) is involved in both adult-larva and larva-larva interactions during settlement of the barnacle B. amphitrite.

Signal transduction in barnacle settlement: Calcium re-visited.

The search for marine natural product antifoulants is being hampered by problems associated with conventional settlement assays. Yet it has been recognised that the study of how chemical cues are perceived by fouling organisms may offer clues to settlement inhibitors and may identify novel biochemical assays for antifoulants based on signal transduction pathways. Here the role of calcium in barnacle settlement is re-examined. A requirement for calcium in settlement of the cypris larva of Balanus amphitrite has been confirmed; settlement was inibited in low calcium, and calcium-free, seawater. Although 10 mM (19.27 mM) excess calcium had no effect and higher concentrations were inhibitory, a 5 mM excess stimulated settlement. Stimulation is proposed to be effected by an increase in intracellular calcium. The release of calcium from intracellular pools with thapsigargin (but not cyclopiazonic acid) induced settlement and an antagonist of intracellular calcium, TMB-8, generally inhibited settlement. Nevertheless, the calcium ionophore A23187 did not induce settlement at the concentrations tested. Consequently, the relative importance of external calcium and intracellular pools to increased intracellular calcium has yet to be determined. Pharmacological manipulations of calcium channels with organic and inorganic channel blockers strongly indicate calcium channel involvement in barnacle settlement. The data are summarised in an hypothetical scheme for signal transduction at settlement and are compared to those obtained for other marine invertebrate larvae.

The problems of water pollution: an overview.

Most potentially toxic chemicals eventually find their way into waterways. The most common source of this pollution is from the air. More than 50 percent of the chemical pollution of the Great Lakes is believed to come from airborne pollutants, and the main sources of this pollution are smokestacks (energy plants, nuclear or conventional; trash-to-steam incinerators; industrial factories, chemical and wood pulp) and road traffic exhaust. Chemicals may reach waterways directly through partially filtered or untreated sewage and waterway traffic, both industrial and pleasure; or they may be deposited into the soil and reach rivers and estuaries through leakage (PCBs on cables, landfills, and regular or low-level nuclear waste) or locally from fields (fertilizers, herbicides, phosphates, and other chemicals.

Induction of egg spawning in Gibbula umbilicalis (da Costa) by an homogenate of the cerebral ganglia.

A putative egg-spawning neurohormone (ESH) has been demonstrated in extracts of the cerebral ganglia from the prosobranch gastropod, Gibbula umbilicalis, using an in vivo bioassay. The active principle is not sex-specific, although spawning in males cannot be induced with injections of ganglion extracts. A partial characterization of the ESH indicates that it is a stable peptide. ESH activity could be detected in the bioassay at a level of 0.625 cerebral ganglion equivalent in a dilution series ranging down from 5.0 ganglion equivalents, with 1.25 ganglion equivalents being the most potent dose. Egg release could be induced for only a short period of the year in vivo, but this period corresponded well with that of natural spawning in the field.