Polydimethylsiloxane Microdomains Formation at the Polythiourethane/Air Interface and Its Influence on Barnacle Release.

In this study, polydimethylsiloxane (PDMS)/polythiourethane (PTU) composite reinforced with tetrapodal shaped micro-nano ZnO particles (t-ZnO) was successfully produced by a versatile, industrially applicable polymer blending process. On the surface of this composite, PDMS is distributed in the form of microdomains embedded in a PTU matrix. The composite inherited not only good mechanical properties originating from PTU but also promising fouling-release (FR) properties due to the presence of PDMS on the surface. It was shown that the preferential segregation of PDMS domains at the polymer/air interface could be attributed to the difference in the surface free energy of PDMS and PTU. The PDMS microdomains at the PTU/air interface significantly reduced the barnacle adhesion strength on the composite. Both the pseudo- and natural barnacle adhesion strength on the composite was approximately 0.1 MPa, similar to that on pure PDMS. The pseudo-barnacle adhesion on reference surfaces AlMg3 and PTU reached approximately 4 and 6 MPa, respectively. Natural barnacles could not be removed intact from AlMg3 and PTU surfaces without breaking the shell, indicating that the adhesion strength was higher than the mechanical strength of a barnacle shell (approximately 0.4 MPa). The integrity of PDMS microdomains was maintained after 12 months of immersion in seawater and barnacle removal. No surface deteriorations were found. In short, the composite showed excellent potential as a long-term stable FR coating for marine applications.

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

The morphology and composition of tissue located within parietal shell canals of the barnacle Amphibalanus amphitrite are described. Longitudinal canal tissue nearly spans the length of side shell plates, terminating near the leading edge of the specimen basis in proximity to female reproductive tissue located throughout the peripheral sub-mantle region, i.e. mantle parenchyma. Microscopic examination of stained longitudinal canal sections reveal the presence of cell nuclei as well as an abundance of micron-sized spheroids staining positive for basic residues and lipids. Spheroids with the same staining profile are present extensively in ovarioles, particularly within oocytes which are readily identifiable at various developmental stages. Mass spectrometry analysis of longitudinal canal tissue compared to tissue collected from the mantle parenchyma reveals a nearly 50% overlap of the protein profile with the greatest number of sequence matches to vitellogenin, a glycolipoprotein playing a key role in vitellogenesis-yolk formation in developing oocytes. The morphological similarity and proximity to female reproductive tissue, combined with mass spectrometry of the two tissues, provides compelling evidence that one of several possible functions of longitudinal canal tissue is supporting the female reproductive system of A. amphitrite, thus expanding the understanding of the growth and development of this sessile marine organism.

Comparative Transcriptomic Analysis Reveals Candidate Genes and Pathways Involved in Larval Settlement of the Barnacle Megabalanus volcano.

Megabalanus barnacle is one of the model organisms for marine biofouling research. However, further elucidation of molecular mechanisms underlying larval settlement has been hindered due to the lack of genomic information thus far. In the present study, cDNA libraries were constructed for cyprids, the key stage for larval settlement, and adults of Megabalanus volcano. After high-throughput sequencing and de novo assembly, 42,620 unigenes were obtained with a N50 value of 1532 bp. These unigenes were annotated by blasting against the NCBI non-redundant (nr), Swiss-Prot, Cluster of Orthologous Groups (COG), and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases. Finally, 19,522, 15,691, 14,459, and 10,914 unigenes were identified correspondingly. There were 22,158 differentially expressed genes (DEGs) identified between two stages. Compared with the cyprid stage, 8241 unigenes were down-regulated and 13,917 unigenes were up-regulated at the adult stage. The neuroactive ligand-receptor interaction pathway (ko04080) was significantly enriched by KEGG enrichment analysis of the DEGs, suggesting that it possibly involved in larval settlement. Potential functions of three conserved allatostatin neuropeptide-receptor pairs and two light-sensitive opsin proteins were further characterized, indicating that they might regulate attachment and metamorphosis at cyprid stage. These results provided a deeper insight into the molecular mechanisms underlying larval settlement of barnacles.

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

The radial growth and advancement of the adhesive interface to the substratum of many species of acorn barnacles occurs underwater and beneath an opaque, calcified shell. Here, the time-dependent growth processes involving various autofluorescent materials within the interface of live barnacles are imaged for the first time using 3D time-lapse confocal microscopy. Key features of the interface development in the striped barnacle, Amphibalanus (= Balanus) amphitrite were resolved in situ and include advancement of the barnacle/substratum interface, epicuticle membrane development, protein secretion, and calcification. Microscopic and spectroscopic techniques provide ex situ material identification of regions imaged by confocal microscopy. In situ and ex situ analysis of the interface support the hypothesis that barnacle interface development is a complex process coupling sequential, timed secretory events and morphological changes. This results in a multi-layered interface that concomitantly fulfills the roles of strongly adhering to a substratum while permitting continuous molting and radial growth at the periphery.

Engineered antifouling microtopographies: an energetic model that predicts cell attachment.

We have developed a model for the prediction of cell attachment to engineered microtopographies based on two previous models: the attachment point theory and the engineered roughness index (ERI) model. The new surface energetic attachment (SEA) model is based on both the properties of the cell-material interface and the size and configuration of the topography relative to the organism. We have used Monte Carlo simulation to examine the SEA model's ability to predict relative attachment of the green alga Ulva linza to different locations within a unit cell. We have also compared the predicted relative attachment for Ulva linza, the diatom Navicula incerta, the marine bacterium Cobetia marina, and the barnacle cyprid Balanus amphitrite to a wide variety of microtopographies. We demonstrate good correlation between the experimental results and the model results for all tested experimental data and thus show the SEA model may be used as a powerful indicator of the efficacy for antifouling topographies.

Unusual adhesive production system in the barnacle Lepas anatifera: an ultrastructural and histochemical investigation.

Adhesives that are naturally produced by marine organisms are potential sources of inspiration in the search for medical adhesives. Investigations of barnacle adhesives are at an early stage but it is becoming obvious that barnacles utilize a unique adhesive system compared to other marine organisms. The current study examined the fine structure and chemistry of the glandular system that produces the adhesive of the barnacle Lepas anatifera. All components for the glue originated from large single-cell glands (70-180 µm). Staining (including immunostaining) showed that L-3,4-dihydroxyphenylalanine and phosphoserine were not present in the glue producing tissues, demonstrating that the molecular adhesion of barnacles differs from all other permanently gluing marine animals studied to date. The glandular tissue and adhesive secretion primarily consisted of slightly acidic proteins but also included some carbohydrate. Adhesive proteins were stored in cytoplasmic granules adjacent to an intracellular drainage canal (ICC); observations implicated both merocrine and apocrine mechanisms in the transport of the secretion from the cell cytoplasm to the ICC. Inside the ICC, the secretion was no longer contained within granules but was a flocculent material which became "clumped" as it traveled through the canal network. Hemocytes were not seen within the adhesive "apparatus" (comprising of the glue producing cells and drainage canals), nor was there any structural mechanism by which additions such as hemocytes could be made to the secretion. The unicellular adhesive gland in barnacles is distinct from multicellular adhesive systems observed in marine animals such as mussels and tubeworms. Because the various components are not physically separated in the apparatus, the barnacle adhesive system appears to utilize completely different and unknown mechanisms for maintaining the liquid state of the glue within the body, as well as unidentified mechanisms for the conversion of extruded glue into hard cement.

Presence and distribution of FMRFamide-like immunoreactivity in the cyprid of the barnacle Balanus amphitrite (Cirripedia, Crustacea).

The presence and distribution of FMRFamide-like peptides (FLPs) in the cyprid larvae of the barnacle Balanus amphitrite were investigated using immunohistochemical methods. Barnacles are considered to be one of the most important constituents of animal fouling communities, and the cyprid stage is specialized for settlement and metamorphosis in to the sessile adult condition. FLPs immunoreactive (IR) neuronal cell bodies were detected in both the central and the peripheral nervous system. One bilateral group of neurons somata was immunodetected in the brain, and IR nerve fibers were observed in the neuropil area and optic lobes. Intense immunostaining was also observed in the frontal filament complex: frontal filament tracts leaving the optic lobes and projecting towards the compound eyes, swollen nerve endings in the frontal filament vesicles, and thin nerve endings in the external frontal filament. Thin IR nerve fibers were also present in the cement glands. Two pairs of neuronal cell bodies were immunodetected in the posterior ganglion; some of their axons appear to project to the cirri. FLPs IR neuronal cell bodies were also localized in the wall of the dilated midgut and in the narrow hindgut; their processes surround the gut wall and allow gut neurons to synapse with one another. Our data demonstrated the presence of FLPs IR substances in the barnacle cyprid. We hypothesize that these peptides act as integrators in the central nervous system, perform neuromuscular functions for thoracic limbs, trigger intestinal movements and, at the level of the frontal filament, play a neurosecretory role.

vasa-related genes and their expression in stem cells of colonial parasitic rhizocephalan barnacle Polyascus polygenea (Arthropoda: Crustacea: Cirripedia: Rhizocephala).

vasa (vas)-related genes are members of the DEAD-box protein family and are expressed in the germ cells of many Metazoa. We cloned vasa-related genes (PpVLG, CpVLG) and other DEAD-box family related genes (PpDRH1, PpDRH2, CpDRH, AtDRHr) from the colonial parasitic rhizocephalan barnacle Polyascus polygenea, the non-colonial Clistosaccus paguri (Crustacea: Cirripedia: Rhizocephala), and the parasitic isopodan Athelgis takanoshimensis (Crustacea: Isopoda). The colonial Polyascus polygenea, a parasite of the coastal crabs Hemigrapsus sanguineus and Hemigrapsus longitarsis was used as a model object for further detailed investigations. Phylogenetic analysis suggested that PpVLG and CpVLG are closely related to vasa-like genes of other Arthropoda. The rest of the studied genes form their own separate branch on the phylogenetic tree and have a common ancestry with the p68 and PL10 subfamilies. We suppose this group may be a new subfamily of the DEAD-box RNA helicases that is specific for parasitic Crustacea. We found PpVLG and PpDRH1 expression products in stem cells from stolons and buds of internae, during asexual reproduction of colonial P. polygenea, and in germ cells from sexually reproducing externae, including male spermatogenic cells and female oogenic cells.

Antifouling activity of synthesized peptide analogs of the sponge metabolite barettin.

Barettin (cyclo [(6-bromo-8-en-tryptophan) arginine]), a diketopiperazine isolated from the marine sponge Geodia barretti, is a potent inhibitor of barnacle larvae settlement with an EC50-value of 0.9 microM. In the present study, 14 analogs of barettin and its structural congener dipodazine were synthezised and tested for their ability to inhibit larval settlement. Two of the analogs have an intact barettin skeleton. The remaining analogs have a dipodazine skeleton (a diketopiperazine where arginine is replaced with glycine). Six of the tested synthetic analogs displayed significant settlement inhibition with the most potent inhibitor being benzo[g]dipodazine, which displayed even stronger activity than barettin (EC50-value 0.034 microM). The effect of benzo[g]dipodazine was also shown to be readily reversible, when cyprids were transferred to filtered seawater (FSW).

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.

Presence and distribution of serotonin immunoreactivity in the cyprids of the barnacle Balanus amphitrite.

In this work, the presence and distribution of serotonin in the cyprid of the barnacle Balanus amphitrite were investigated by immunohistochemical methods. Serotonin-like immuno-reactive neuronal cell bodies were detected in the central nervous system only. Various clusters of immunoreactive neuronal cell bodies are distributed in the brain (protocerebrum, deutocerebrum, optical lobes), and at least, four pairs of neuronal cell bodies were detected in the centrally positioned neuropil of the posterior ganglion. Rich plexuses of immunoreactive nerve fibers in the neuropil area were also observed. Furthermore, bundles of strongly immunoreactive nerve fibers surrounding the gut wall were localized, and immunoreactive nerve terminals in the antennules and compound eyes were observed. These data demonstrate the presence of a serotonin-like immunoreactive substance in the barnacle cyprids; furthermore, its immunolocalization in the cephalic nerve terminals allows us to postulate the involvement of this bioactive molecule in substrate recognition during the settlement process.

Impact of polymer surface affinity of novel antifouling agents.

In a previous study we found two agents, the alpha(2)-agonist medetomidine ((+/-)-4-[1-(2,3-dimethylphenyl)ethyl]-1H-imidazole) and the alpha(2)-agonist clonidine (2-(2,6-dichloroanilino)-2-imidazoline), that specifically and efficiently impede settlement of the barnacle Balanus improvisus, one of the most serious biofouling organisms in Swedish waters. Medetomidine, but not clonidine, is known to adsorb to solid polystyrene (PS) surfaces in the presence of salt, a feature that is of particular interest in attempts to develop an efficient antifouling surface. We show that medetomidine, but not clonidine, has a significant ability to adsorb to untreated (hydrophobic) PS in two different incubation media: filtered seawater (FSW) and deionized water (mQ). At negatively charged (hydrophilic) PS, medetomidine displays a strong interaction with the surface in both incubation media. At the hydrophilic PS, clonidine also displays a significant interaction with the surface when incubated in mQ and a weaker, but not significant, interaction when incubated in FSW. By studying the effects of time, incubation media, and pH on the adsorption of medetomidine and clonidine, we suggest that medetomidine is associated to hydrophobic PS by means of hydrophobic interactions, while the adsorption of medetomidine and clonidine to hydrophilic PS contains elements of electrostatic interaction. Using time-of-flight secondary ion mass spectroscopy (TOF-SIMS) we detected only weak signals from medetomidine on the hydrophobic PS surfaces, while strong medetomidine signals were observed on hydrophilic PS. This suggests that the adsorbed medetomidine, to a greater extent, desorbed from the hydrophobic rather than from the hydrophilic PS surfaces during exposure to vacuum. The strong surface affinity of medetomidine on both types of surfaces and the preserved antifouling activity are valuable features in designing a marine coating.

Pressure generation in a contracting myocyte.

The central hypothesis of this investigation is that a shortening myocyte generates a time-varying transmural pressure, or intracellular pressure. A mathematical model was formulated for a single myocyte, consisting of a fluid-filled cylindrical shell with axially arranged contractile filaments, to quantitate the fiber-fluid interaction. In this model, the intracellular pressure mediates the interaction between myofilament force, cell shortening, and the mechanical properties of the sarcolemma. Shortening of myofibrils, which are embedded in the fluid-filled myocytes, deforms the myocyte, thereby altering its transmural fluid pressure. This increase in transmural pressure counteracts fiber shortening, hence constituting an internal load to shortening. The shortening of the myocyte is accompanied by thickening, due to the incompressible nature of its contents. Consequently, the overall contractile performance of the cell is integrally linked to the generation of intracellular pressure. The model manifests a positive transmural pressure during shortening, but not without shortening. The pressure in the myocyte, therefore, is not a direct function of the force generated, but rather of shortening. Intracellular pressure was measured through a fluid-filled glass micropipette (5 mu ID) employing a servo-nulling pressure transducer in a standard micropuncture technique. Measured intracellular pressure in a contracting isolated skeletal myocyte of the giant barnacle is observed to be dynamically related to shortening, but not to tension without shortening. The relation between the force of contraction, cell shortening, and intracellular pressure was assessed during both isotonic and isometric contractions. The results support the prediction that isometric, or nondeforming, contractions will not develop intracellular pressure and identify a reason for relengthening of the myocytes during relaxation.

Shell formation and calcium transport in the barnacle Chthamalus fragilis.

The mantle epithelium of the barnacle Chthamalus fragilis (Darwin) exhibits several ultrastructural features which may serve to regulate the calcification process. At the basis-mural plate and intermural plate junctions where rapid shell growth occurs, cells are characterized by long apical cytoplasmic projections and large intercellular spaces. These features may increase the functional surface area of the epithelium and enable more rapid deposition of calcium. The cells underlying the general shell surfaces contain numerous electron-dense inclusion bodies and show frequent cellular disintegration near the growing shell interface. Release of the granular contents of these inclusion bodies has been observed in both disintegrating and non-disintegrating cells. X-ray microanalysis revealed significantly higher calcium levels in the inclusion bodies than in the surrounding cytoplasm. This suggests a calcium transport role for these inclusion bodies. Cellular debris produced as a result of the disintegration of the mantle cells near the shell may play some role in the formation of the organic matrix of the shell. The presence of large numbers of mitochondria and well-developed apical microvilli in the cells of the inner mantle epithelium suggest that these cells serve to transport calcium into the mantle from the ambient sea water.

Cytoplasmic solvent structure of single barnacle muscle cells studied by electron spin resonance.

A free radical probe was introduced into single barnacle muscle cells, and its freedom of motion inferred from the spin resonance spectra. The probe reported an average local viscosity of 5-10 cp compared with 1 cp for pure water. From a comparison of the temperature dependence of the probe's tumbling rate in model aqueous systems and in the muscle we concluded that in the muscle the probe was undergoing fast exchange between sites of different mobility. Thus 10 cp must be taken as an upper limit for the viscosity of most cell water.