Barnacles are the only sessile crustaceans, and their larva, the cyprid, is supremely adapted for attachment to surfaces. Barnacles have a universal requirement for strong adhesion at the point of larval attachment. Selective pressure on the cyprid adhesive has been intense and led to evolution of a tenacious and versatile natural glue. Here we provide evidence that carbohydrate polymers in the form of chitin provide stability to the cyprid adhesive of Balanus amphitrite. Chitin was identified surrounding lipid-rich vesicles in the cyprid cement glands. The functional role of chitin was demonstrated via removal of freshly attached cyprids from surfaces using a chitinase. Proteomic analysis identified a single cement gland-specific protein via its association with chitin and localized this protein to the same vesicles. The role of chitin in cyprid adhesion raises intriguing questions about the evolution of barnacle adhesion, as well as providing a new target for antifouling technologies.

The copepod Lepeophtheirus salmonis is an obligate ectoparasite of salmonids. Salmon lice are major pests in salmon aquaculture and due to its economic impact Lepeophtheirus salmonis is one of the most well studied species of marine parasite. However, there is limited understanding of how increased concentration of pCO2 associated with ocean acidification will impact host-parasite relationships. We investigated the effects of increased pCO2 on growth and metabolic rates in the planktonic stages, rearing L. salmonis from eggs to 12 days post hatch copepodids under three treatment levels: Control (416 µatm), Mid (747 µatm), and High (942 µatm). The pCO2 treatment had a significant effect on oxygen consumption rate with the High treatment animals exhibiting the greatest respiration. The treatments did not have a significant effect on the other biological endpoints measured (carbon, nitrogen, lipid volume, and fatty acid content). The results indicate that L. salmonis have mechanisms to compensate for increased concentration of pCO2and that populations will be tolerant of projected future ocean acidification scenarios. The work reported here also describes catabolism during the lecithotrophic development of L. salmonis, information that is not currently available to parameterize models of dispersal and viability of the planktonic free-living stages.

The proteins derived from the foliated shell layer of the oyster, Crassostrea virginica, are unusually acidic and highly phosphorylated. Here we report the identification of a gene encoding a member of this class of phosphoproteins that we collectively refer to as folian. Using an in silico approach, a virtual probe was constructed from an N-terminal sequence (DEADAGD) determined for a 48 kDa folian phosphoprotein and used to screen an oyster EST databank. A sequence that matched the N-terminus of the 48 kDa protein was found and used to identify the full-length gene from a C. virginica BAC library. The molecular weight of the deduced gene product is 32 kDa and was named folian-cv1. Genomic Southern analysis revealed two variants of the gene. The mature protein is composed of 43.3% Asp, 32.6% Ser, and 9.1% Glu with 37.5% of the amino acids of the protein potentially phosphorylated. The primary sequence of folian-cv1 is organized in blocks, with a short relatively hydrophobic block at the N-terminus and with the remainder containing low complexity regions largely dominated by aspartic acid and serine. Overall, the protein is predicted to be highly disordered. PCR and sequence analyses identified folian-cv1 expression in the mantle and hemocytes. Immuno-histochemical staining of mantle tissue reveals that cells of the shell-facing epithelium and in the periostracal groove secrete a continuous layer of folian-positive material and that folian-positive hemocytes move through the mantle epithelium. The function in shell formation of folian proteins including folian-cv1 is not known. However, based on the complexity of this class of proteins and the two methods of their delivery to the region of shell formation, it is possible they are involved in diverse ways in this process.

Chitin is often reported in molluscan shells, where it likely contributes to the mechanical strength of the biomineral. However, the role of this polysaccharide in relation to the process of shell formation is not well understood. We investigated the deposition of chitin during shell repair in the Eastern oyster, Crassostrea virginica, by inserting stainless steel and glass implants in a region of shell damage. This work documents the time course of deposition of both chitin fibrils and calcium carbonate layers. Chitin was detected by confocal laser scanning microscopy (CLSM) using a chitin-specific fluorescent probe that was produced from clones of a chitin-binding domain. The presence of fibrils was confirmed using electron microscopy of implants. The fibrils' dimensions were reduced after treatment with both acid and bleach, suggesting that chitin interacts with inorganic minerals and other organic components such as proteins and lipids as early as 5 h after shell damage. With CLSM, it was shown that chitin co-localized with the cell membrane, suggesting the importance of cells located on the implants in the process of fibril formation. Using observations from this study as well as those from the literature on chitin synthase production, we propose two cellular models for chitin deposition related to shell formation.

Bugula neritina is a sessile marine bryozoan with a pelagic larval stage. Larvae frequently settle on boat hulls, facilitating the introduction of B. neritina to bays and estuaries worldwide. Adrenergic agonists, such as norepinephrine, inhibit larval settlement in a variety of marine invertebrate species, including B. neritina. Light also inhibits larval settlement of B. neritina, yet the underlying mechanisms by which light and adrenergic compounds exert their effects on larvae are largely unknown. Octopamine is considered the invertebrate analog of norepinephrine, and may be one endogenous compound involved in larval settlement pathways. In this study, we investigated the effects of norepinephrine and the adrenergic antagonist phentolamine on larval settlement, and found that norepinephrine inhibited larval attachment and increased larval swimming behavior, while phentolamine increased larval attachment and decreased larval swimming behavior. We used fluorescent labeling and immunocytochemistry to localize sensory system components, and found that larvae possess adrenergic-like receptors and octopamine-like immunoreactivity. We also exposed larvae to phentolamine in both dark and light conditions, and found that light inhibited larval attachment, but phentolamine blocked those inhibitory effects. Based on these results, we put forth a putative mechanistic explanation for the effects of both light and adrenergic compounds on B. neritina larval settlement behavior. This study sheds light on previously unknown pathways underlying larval settlement behavior of bryozoans, and may aid in the development of effective, non-toxic biofouling control strategies.

Characterizing the first event of biological production of calcium carbonate requires a combination of microscopy approaches. First, intracellular pH distribution and calcium ions can be observed using live microscopy over time. This allows identification of the life stage and the tissue with the feature of interest for further electron microscopy studies. Life stage and tissues of interest are typically higher in pH and Ca signals. Here, using H. elegans, we present a protocol to characterize the presence of calcium carbonate structures in a biological specimen on the scanning electron microscope (SEM), using energy-dispersive X-ray spectroscopy (EDS) to visualize elemental composition, using electron backscatter diffraction (EBSD) to determine the presence of crystalline structures, and using transmission electron microscopy (TEM) to analyze the composition and structure of the material. In this protocol, a focused ion beam (FIB) is used to isolate samples with dimension suitable for TEM analysis. As FIB is a site specific technique, we demonstrate how information from the previous techniques can be used to identify the region of interest, where Ca signals are highest.

Mobile barnacle cypris larvae settle and metamorphose, transitioning to sessile juveniles with morphology and growth similar to that of adults. Because biofilms exist on immersed surfaces on which they attach, barnacles must interact with bacteria during initial attachment and subsequent growth. The objective of this study was to characterize the developing interface of the barnacle and substratum during this key developmental transition to inform potential mechanisms that promote attachment. The interface was characterized using confocal microscopy and fluorescent dyes to identify morphological and chemical changes to the interface and the status of bacteria present as a function of barnacle developmental stage. Staining revealed patchy material containing proteins and nucleic acids, reactive oxygen species amidst developing cuticle, and changes in bacteria viability at the developing interface. We found that as barnacles metamorphose from the cyprid to juvenile stage, proteinaceous materials with the appearance of coagulated liquid were released into and remained at the interface. It stained positive for proteins, including phosphoprotein, as well as nucleic acids. Regions of the developing cuticle and the patchy material itself stained for reactive oxygen species. Bacteria were absent until the cyprid was firmly attached, but populations died as barnacle development progressed. The oxidative environment may contribute to the cytotoxicity observed for bacteria and has the potential for oxidative crosslinking of cuticle and proteinaceous materials at the interface.

Although space delineation is a well-accepted requirement for biologically controlled biomineralization, the actual location of the mineralizing compartment within marine invertebrates has only recently been determined. We observed that the biomineralization was compartmented within the collar region of the metamorphosing larvae of Hydrodies elegans at its earliest possible time, i.e. at the post-metamorphic stage. We have also found that these highly regulated compartments contained aragonite crystals, as detected by EBSD and confirmed by electron diffraction TEM. Within these compartments, the metamorphosed larvae maintained a pH 9, at the pKa for CaCO3 formation. This model describes how biomineralization is a space delineation event in which calcium carbonate formation is an intracellular phenomenon.

The phylum Mollusca is unmatched in the mastery of cellular-engineered shell microstructures, producing shells that are visually elegant, structurally complex and fracture resistant. Shell formation in the eastern oyster, Crassostrea virginica is a process that involves both hemocytes (blood cells) and the outer mantle epithelial cells (OME). This study reports the secretion of an extracellular matrix (ECM) and cellular activity during shell layer formation by observing folia and prismatic development on different metal alloy surfaces (Ti6Al4V titanium, 7075-T6 aluminum, and 316L stainless steel) implanted under the OME. During prism formation, a close association was observed between the ECM surrounding the prisms, the OME, and hemocytes. The prismatic ECM walls appeared to originate from the OME surface and were auto-fluorescent. During folia formation, an initial infiltration response of hemocytes to the implant surface was observed followed by the formation of a thin translucent ECM membrane onto which exosome-like vesicles, some containing crystals, deposited. These structures progressively organized into well-defined folia mineral. It is proposed that molluscan shell is formed through a series of coordinated events involving cells and cell products whereby both organic and mineral phases are secreted, organized, and simultaneously formed. The coordination of cells and cell products for shell synthesis opens a new realm of cellular control not previously explored in shell formation.

Thoracian barnacles rely heavily upon their ability to adhere to surfaces and are environmentally and economically important as biofouling pests. Their adhesives have unique attributes that define them as targets for bio-inspired adhesive development. With the aid of multi-photon and broadband coherent anti-Stokes Raman scattering microscopies, we report that the larval adhesive of barnacle cyprids is a bi-phasic system containing lipids and phosphoproteins, working synergistically to maximize adhesion to diverse surfaces under hostile conditions. Lipids, secreted first, possibly displace water from the surface interface creating a conducive environment for introduction of phosphoproteins while simultaneously modulating the spreading of the protein phase and protecting the nascent adhesive plaque from bacterial biodegradation. The two distinct phases are contained within two different granules in the cyprid cement glands, implying far greater complexity than previously recognized. Knowledge of the lipidic contribution will hopefully inspire development of novel synthetic bioadhesives and environmentally benign antifouling coatings.

Foliar fertilization is a common practice to deliver nitrogen (N) to turfgrasses. The mechanisms of foliar applied nutrient uptake, particularly the effects of the leaf cuticle layer, have not been clearly characterized in turfgrasses. The objectives of this study were to determine the effect of acute moisture stress on the morphological and compositional components of the cuticle and the resulting effect on foliar-applied N absorption. Creeping bentgrass ( Agrostis stolonifera L.) was irrigated to return 100% or 50% evapotranspiration rate (ET) for 10 days to examine cuticular modifications resulting from acute moisture stress and foliar N uptake with and without a surfactant. Acute water stress increased the total cuticle wax by 11%, mostly as a result of the compound 1-hexacosanol, and increased crystalloid density creating a rougher leaf surface. The 50% ET treatment significantly reduced recovery of 15 N-labeled urea by 14%, which was attributed to the increased total cuticle wax and crystalloid density making the surface less receptive to foliar applications. The surfactant addition to the urea solution increased 15 N-labeled urea recovery by 21% and absorption of 15 N in 50% ET plants to levels consistent with the 100% ET plants. These results suggest that acute moisture stress modifies the cuticle wax load and morphology, thereby hindering foliar absorption; however, a surfactant addition can help to mitigate this effect and increase absorption of N.

Biological adhesives are materials of particular interest in the fields of bio-inspired technology and antifouling research. The adhesive of adult barnacles has received much attention over the years; however, the permanent adhesive of the cyprid - the colonisation stage of barnacles - is a material about which very little is presently known. We applied confocal laser-scanning microscopy to the measurement of contact angles between the permanent adhesive of barnacle cyprid larvae and self-assembled monolayers of OH- and CH3-terminated thiols. Measurement of contact angles between actual bioadhesives and surfaces has never previously been achieved and the data may provide insight into the physicochemical properties and mechanism of action of these functional materials. The adhesive is a dual-phase system post-secretion, with the behaviour of the components governed separately by the surface chemistry. The findings imply that the cyprid permanent adhesion process is more complex than previously thought, necessitating broad re-evaluation of the system. Improved understanding will have significant implications for the production of barnacle-resistant coatings as well as development of bio-inspired glues for niche applications.

The strategy of decorating antibiofouling hyperbranched fluoropolymer-poly(ethylene glycol) (HBFP-PEG) networks with a settlement sensory deterrent, noradrenaline (NA), and the results of biofouling assays are presented. This example of a dual-mode surface, which combines both passive and active modes of antibiofouling, works in synergy to improve the overall antibiofouling efficiency against barnacle cyprids. The HBFP-PEG polymer surface, prior to modification with NA, was analyzed by atomic force microscopy, and a significant distribution of topographical features was observed, with a nanoscopic roughness measurement of 110 ± 8 nm. NA attachment to the surface was probed by secondary ion mass spectrometry to quantify the extent of polymer chain-end substitution with NA, where a 3- to 4-fold increase in intensity for a fragment ion associated with NA was observed and 39% of the available sites for attachment were substituted. Cytoskeletal assays confirmed the activity of tethered NA on adhering oyster hemocytes. Settlement assays showed deterrence toward barnacle cyprid settlement, while not compromising the passive biofouling resistance of the surface. This robust strategy demonstrates a methodology for the incorporation of actively antibiofouling moieties onto a passively antibiofouling network.

The present study assessed the toxic effects of stable aqueous colloidal suspensions of gallic-acid-stabilized C70 fullerene on Daphnia magna. The suspensions were stabilized through noncovalent surface modification with gallic acid. In addition to whole-organism responses, changes in antioxidative processes in D. magna were quantified. Acute toxicity was observed with 96LC50 for C70-gallic acid of 0.4 +/- 0.1mg/LC70. Daphnia magna fecundity was significantly reduced in 21-d bioassays at C70-gallic aqcid concentrations below quantifiable limits. Antioxidant enzyme activities of glutathione peroxidase and superoxide dismutase as well as lipid peroxidation suggested that exposed organisms experienced oxidative stress. Microscopic techniques used to determine cellular toxicity via apoptosis proved unsuccessful. Environ. Toxicol. Chem. 2012;31:215220. (C) 2011 SETAC

Exploration of substrata prior to cementation and metamorphosis is a critical step in the life cycle of Cirripedes. To this end, barnacle cyprid larvae have evolved sophisticated sensory mechanisms to investigate chemical as well as topographical composition of the substratum. Since barnacles are major biofoulers, the sensory structures of Balanus amphitrite cyprids have been described in detail using ultrastructural techniques like SEM and TEM. Settlement assays have shown that cyprid settlement is deterred by adrenergic receptor agonists and antagonists, yet the exact location of these receptors on cyprid sensory structures is unknown. Here, using a fluorescent adrenergic receptor antagonist (Bodipy FL-Prazosin) with the aid of confocal microscopy we describe the putative locations of adrenergic-like receptors in B. amphitrite cyprids. Our results indicate that postaxial setae 2 and 3 of the 2nd and 3rd segments respectively, the sub-terminal setae of the 4th antennular segment and the cement gland secretory cells seem to bear the adrenergic-like receptors. Using optical and electron microscopy we further studied the effects of noradrenaline (NA), an adrenergic receptor agonist, on cyprid settlement and metamorphosis. NA treated cyprids forgo the typical settlement behavior and metamorphose into juvenile barnacles without cementing to the substratum. Cyprid-barnacle metamorphosis in NA treated cyprids is also prolonged as compared to untreated barnacles. The key role played by adrenergic-like receptors during surface sensing, cementation and metamorphosis is discussed.