An in vivo study of exocytosis of cement proteins from barnacle Balanus improvisus (D.) cyprid larva.

Barnacles, like many marine invertebrates, cause serious biofouling to marine industrial constructions and hulls of vessels as they attach themselves to such surfaces. Precise biochemical understanding of the underwater adhesion to surfaces requires a detailed characterization of the biology of the control of barnacle cement secretion and the proteins that make up the cement. In this study, we have investigated cement secretion by cyprid larvae of Balanus improvisus (D.) and the morphology of their cement glands. We studied the cement protein organization within cement granules and categorized the granules into four different types according to their size and morphology, before and after stimulation of secretion. In addition, we followed the exocytotic process of cement secretion in vivo and discovered that granules undergo a dramatic swelling during secretion. Such swelling might be due to an increased osmotic activity of granule contents, following a process of hydration. We hypothesize that this hydration is essential for exocytotic secretion and conclude that cement protein exocytosis is a more complex process than previously thought and is similar to exocytotic secretion in vertebrate systems, such as histamine secretion from mast cells and exocrine secretion in the salivary gland and the pancreas.

Evidence for different pharmacological targets for imidazoline compounds inhibiting settlement of the barnacle Balanus improvisus.

We describe the effect of eight different imidazoline/guanidinium compounds on the settlement and metamorphosis of larvae of the barnacle Balanus improvisus. These agents were chosen on the basis of their similar pharmacological classification in vertebrates and their chemical similarity to medetomidine and clonidine, previously described as highly potent settlement inhibitors (nanomolar range). Seven of the tested compounds were found to inhibit settlement in a dose-dependent manner in concentrations ranging from 100 nM to 10 microM without any significant lethal effects. In vertebrate systems these substances have overlapping functions and interact with both alpha-adrenoceptors as well as imidazoline binding sites. Antagonizing experiments using the highly specific alpha(2)-antagonist methoxy-idazoxan or agmatine (the putative endogenous ligand at imidazoline receptors) were performed to discriminate between putative pharmacological mechanisms involved in the inhibition of cyprid settlement. Agmatine was not able to reverse the effect of any of the tested compounds. However, methoxy-idazoxan almost completely abolished the settlement inhibition mediated by guanabenz (alpha(2)-agonist, I(2) ligand), moxonidine (alpha(2)-agonist, I(1) ligand) and tetrahydrozoline (alpha-agonist, I(2) ligand). The actions of cirazoline (alpha(1)-agonist, I(2) ligand) BU 224 (I(2) ligand) and metrazoline (I(2) ligand) were not reversed by treatment with methoxy-idazoxan. These results suggest that the settlement inhibition evoked by the I(2) ligands and alpha(2)-agonists used in this study of the neurologically simple but well-organized barnacle larva is mediated through different physiological targets important in the overall 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.