Conditioning of surfaces by macromolecules and its implication for the settlement of zoospores of the green alga Ulva linza

Conditioning, ie the adsorption of proteins and other macromolecules, is the first process that occurs in the natural environment once a surface is immersed in seawater, but no information is available either regarding the conditioning of surfaces by artificial seawater or whether conditioning affects data obtained from laboratory assays. A range of self-assembled monolayers (SAMs) with different chemical terminations was used to investigate the time-dependent formation of conditioning layers in commercial and self-prepared artificial seawaters. Subsequently, these results were compared with conditioning by solutions in which zoospores of the green alga Ulva linza had been swimming. Spectral ellipsometry and contact angle measurements as well as infrared reflection absorption spectroscopy (IRRAS) were used to reveal the thickness and chemical composition of the conditioning layers. The extent that surface preconditioning affected the settlement of zoospores of U. linza was also investigated. The results showed that in standard spore settlement bioassays (45-60 min), the influence of a molecular conditioning layer is likely to be small, although more substantial effects are possible at longer settlement times.     

Effect of bacterial biofilms formed on fouling-release coatings from natural seawater and Cobetia marina,on the adhesion of two marine algae

Previous studies have shown that bacterial biofilms formed from natural seawater (NSW) enhance the settlement of spores of the green alga Ulva linza, while single-species biofilms may enhance or reduce settlement, or have no effect at all. However, the effect of biofilms on the adhesion strength of algae, and how that may be influenced by coating/surface properties, is not known. In this study, the effect of biofilms formed from natural seawater and the marine bacterium Cobetia marina, on the settlement and the adhesion strength of spores and sporelings of the macroalga U. linza and the diatom Navicula incerta, was evaluated on Intersleek® 700, Intersleek® 900, poly(dimethylsiloxane) and glass. The settlement and adhesion strength of these algae were strongly influenced by biofilms and their nature. Biofilms formed from NSW enhanced the settlement (attachment) of both algae on all the surfaces while the effect of biofilms formed from C. marina varied with the coating type. The adhesion strength of spores and sporelings of U. linza and diatoms was reduced on all the surfaces biofilmed with C. marina, while adhesion strength on biofilms formed from NSW was dependent on the alga (and on its stage of development in the case of U. linza), and coating type. The results illustrate the complexity of the relationships between fouling algae and bacterial biofilms and suggest the need for caution to avoid over-generalisation.     

Holographic microscopy provides new insights into the settlement of zoospores of the green alga Ulva linza on cationic oligopeptide surfaces

Interaction of zoospores of Ulva linza with cationic, arginine-rich oligopeptide self-assembled monolayers (SAMs) is characterized by rapid settlement. Some spores settle (ie permanently attach) in a ‘normal’ manner involving the secretion of a permanent adhesive, retraction of the flagella and cell wall formation, whilst others undergo ‘pseudosettlement’ whereby motile spores are trapped (attached) on the SAM surface without undergoing the normal metamorphosis into a settled spore. Holographic microscopy was used to record videos of swimming zoospores in the vicinity of surfaces with different cationic oligopeptide concentrations to provide time-resolved insights into processes associated with attachment of spores. The data reveal that spore attachment rate increases with increasing cationic peptide content. Accordingly, the decrease in swimming activity in the volume of seawater above the surface accelerated with increasing surface charge. Three-dimensional trajectories of individual swimming spores showed a ‘hit and stick’ motion pattern, exclusively observed for the arginine-rich peptide SAMs, whereby spores were immediately trapped upon contact with the surface.     

Antifouling properties of oligo(lactose)-based self-assembled monolayers

The antifouling (AF) properties of oligo(lactose)-based self-assembled monolayers (SAMs), using four different proteins, zoospores of the green alga Ulva linza and cells of the diatom Navicula incerta, were investigated. The SAM-forming alkylthiols, which contained 1, 2 or 3 lactose units, showed significant variation in AF properties, with no differences in wettability. Non-specific adsorption of albumin and pepsin was low on all surfaces. Adsorption of lysozyme and fibrinogen decreased with increasing number of lactose units in the SAM, in agreement with the generally observed phenomenon that thicker hydrated layers provide higher barriers to protein adsorption. Settlement of spores of U. linza followed an opposite trend, being greater on the bulkier, more hydrated SAMs. These SAMs are more ordered for the larger saccharide units, and it is therefore hypothesized that the degree of order, and differences in crystallinity or stiffness between the surfaces, is an important parameter regulating spore settlement on these surfaces.     

Topographic cues guide the attachment of diatom cells and algal zoospores

Surface topography plays a key role in the colonization of substrata by the colonizing stages of marine fouling organisms. For the innovation of marine antifouling coatings, it is essential to understand how topographic cues affect the settlement of these organisms. In this study, tapered, spiked microstructures and discrete honeycombs of varying feature dimensions were designed and fabricated in order to examine the influence of topography on the attachment of zoospores of the green macroalga Ulva linza and cells of the diatom (microalga) Navicula incerta. Contrasting results were obtained with these two species of algae. Indeed, the preferred location of cells of N. incerta was dominated by attachment point theory, which suggested a positive correlation between the density of cells adhering and the amount of available attachment points, while the settlement of spores of U. linza was mainly regulated by both Wenzel roughness and local binding geometry.     

Charged hydrophilic polymer brushes and their relevance for understanding marine biofouling

The resistance of charged polymers to biofouling was investigated by subjecting cationic (PDMAEMA), anionic (PSPMA), neutral (PHEMA-co-PEG₁₀MA), and zwitterionic (PSBMA) brushes to assays testing protein adsorption; attachment of the marine bacterium Cobetia marina; settlement and adhesion strength of zoospores of the green alga Ulva linza; settlement of barnacle (Balanus amphitrite and B. improvisus) cypris larvae; and field immersion tests. Several results go beyond the expected dependence on direct electrostatic attraction; PSPMA showed good resistance towards attachment of C. marina, low settlement and adhesion of U. linza zoospores, and significantly lower biofouling than on PHEMA-co-PEG₁₀MA or PSBMA after a field test for one week. PDMAEMA showed potential as a contact-active anti-algal coating due to its capacity to damage attached spores. However, after field testing for eight weeks, there were no significant differences in biofouling coverage among the surfaces. While charged polymers are unsuitable as antifouling coatings in the natural environment, they provide valuable insights into fouling processes, and are relevant for studies due to charging of nominally neutral surfaces.     

The performance of aminoalkyl/fluorocarbon/hydrocarbon-modified xerogel coatings against the marine alga Ectocarpus crouaniorum: relative roles of surface energy and charge

The effect of a series of xerogel coatings modified with aminoalkyl/fluorocarbon/hydrocarbon groups on the adhesion of a new test species, the filamentous brown alga Ectocarpus crouaniorum, has been explored, and compared with the green alga Ulva linza. The results showed that E. crouaniorum adhered weakly to the less polar, low wettability coatings in the series, but stronger adhesion was shown on polar, higher surface energy coatings containing aminoalkyl groups. The results from a separate series of coatings tuned to have similar surface energies and polarities after immersion in artificial seawater (ASW), but widely different surface charges, demonstrated that surface charge was more important than surface energy and polarity in determining the adhesion strength of both E. crouaniorum and U. linza on xerogel coatings. No correlation was found between adhesion and contact angle hysteresis. X-ray photoelectron spectroscopy analysis of samples after immersion in ASW confirmed the presence of charged ammonium groups on the surface of the aminoalkylated coatings.     

Antifouling potential of Subtilisin A immobilized onto maleic anhydride copolymer thin films

The proteinaceous nature of the adhesives used by most fouling organisms to attach to surfaces suggests that coatings incorporating proteolytic enzymes may provide a technology for the control of biofouling. In the present article, the antifouling (AF) and fouling release potential of model coatings incorporating the surface-immobilized protease, Subtilisin A, have been investigated. The enzyme was covalently attached to maleic anhydride copolymer thin films; the characteristics of the bioactive coatings obtained were adjusted through variation of the type of copolymer and the concentration of the enzyme solution used for immobilization. The bioactive coatings were tested for their effect on the settlement and adhesion strength of two major fouling species: the green alga Ulva linza and the diatom Navicula perminuta. The results show that the immobilized enzyme effectively reduced the settlement and adhesion strength of zoospores of Ulva and the adhesion strength of Navicula cells. The AF efficacy of the bioactive coatings increased with increasing enzyme surface concentration and activity, and was found to be superior to the equivalent amount of enzyme in solution. The results provide a rigorous analysis of one approach to the use of immobilized proteases to reduce the adhesion of marine fouling organisms and are of interest to those investigating enzyme-containing coating technologies for practical biofouling control.     

The attachment of Enteromorpha zoospores to a bacterial biofilm assemblage

This study has investigated the relationship between bacterial biofilms and the attachment of zoospores of the green macroalga Enteromorpha. Zoospore attachment to glass slides was enhanced in the presence of a bacterial biofilm assemblage, and the number attaching increased with the number of bacteria present. Zoospores also attached to control surfaces, but at lower numbers; glass surfaces conditioned in autoclaved seawater had the same number of zoospores attached as new glass surfaces. The spatial relationship between bacterial cells and attached zoospores was quantified by image analysis. The hypothesis tested was that zoospores attached preferentially to, or in the very close vicinity of, bacterial cells. Spatial microscopic analysis showed that more bacteria were covered by zoospores than would be expected if zoospore attachment was a random process and zoospores appeared to attach to bacterial clusters. The most likely explanation is that zoospores are attracted to bacterial cells growing on surfaces and the presence of a bacterial biofilm enhances their settlement. The possibility is discussed that Enteromorpha zoospores respond to a chemical signal produced by bacteria, i.e. that there may be prokaryote‐eukaryote cell signalling.     

Engineered antifouling microtopographies – correlating wettability with cell attachment

Abstract

Bioadhesion and surface wettability are influenced by microscale topography. In the present study, engineered pillars, ridges and biomimetic topography inspired by the skin of fast moving sharks (Sharklet AF?) were replicated in polydimethylsiloxane elastomer. Sessile drop contact angle changes on the surfaces correlated well (R² = 0.89) with Wenzel and Cassie and Baxter's relationships for wettability. Two separate biological responses, i.e. settlement of Ulva linza zoospores and alignment of porcine cardiovascular endothelial cells, were inversely proportional to the width (between 5 and 20 μm) of the engineered channels. Zoospore settlement was reduced by ～85% on the finer (ca 2 μm) and more complex Sharklet AF? topographies. The response of both cell types suggests their responses are governed by the same underlying thermodynamic principles as wettability.     

Effect of Marine Bacterial Isolates on the Growth and Morphology of Axenic Plantlets of the Green Alga Ulva linza

The green marine macroalga, Ulva linza, adopts an “atypical” form when grown in the absence of bacteria. Twenty unique strains of periphytic bacteria, isolated from three species of Ulva, were identified by 16S rDNA sequencing. These isolates were assessed for their effect on the growth and morphological development of axenic plantlets of U. linza. Results showed that the effect of bacterial strains was strain- but not taxon-specific. Thirteen isolates returned the aberrant morphology to normal and of these, five also significantly increased growth rate. One isolate increased growth, but had no effect on morphology. Biofilms of some of these isolates stimulated the settlement of Ulva zoospores but there was no correlation between bacterial isolates that stimulated zoospore settlement and those that initiated changes in morphology and/or growth of the cultured alga.     

The performance of hybrid titania/silica-derived xerogels as active antifouling/fouling-release surfaces against the marine alga Ulva linza: in situ generation of hypohalous acids

Mixed titania/silica xerogels were prepared using titanium tetraisopropoxide (TTIP) and tetraethoxy orthosilicate (TEOS). Xerogel properties were modified by incorporating n-octyltriethoxysilane (C8). The xerogels catalyze the oxidation of bromide and chloride with hydrogen peroxide (H₂O₂) to produce hypohalous acids at pH 7 and pH 8. The antifouling/ fouling-release performance of a TTIP/C8/TEOS xerogel in the presence and absence of H₂O₂ was evaluated for the settlement of zoospores of the marine alga Ulva linza and for the removal of sporelings (young plants). In the absence of H₂O₂, differences in the settlement of zoospores and removal of sporelings were not significant relative to a titanium-free C8/TEOS xerogel. Addition of H₂O₂ gave a significant reduction in zoospore settlement and sporeling removal relative to the C8/TEOS xerogel and relative to peroxide-free conditions. The impact of TTIP on xerogel characteristics was evaluated by comprehensive contact angle analysis, scanning electron microscopy, and X-ray photoelectron spectroscopy.     

Comparing the low‐salinity tolerance of Ulva species distributed in different environments

The green macroalgal genus Ulva (Ulvales, Ulvophyceae, Chlorophyta) is distributed worldwide from marine to freshwater environments. Comparative analyses of hyposalinity tolerance among marine, brackish, and freshwater Ulva species were performed by fluorescein diacetate viability counts. The subtidal marine species Ulva sp., collected from a depth of 30 m, showed the poorest tolerance to low salinity. This species died in 5 practical salinity units (PSU) artificial seawater or freshwater within 1 day. Its closely related species U. linza L. (an intertidal species) and U. prolifera Müller (a brackish species) showed varying tolerances to low salinity. After 7 days of freshwater exposure, the viability of U. linza L. decreased to approximately 20%, while U. prolifera Müller showed nearly 100% viability. The freshwater species U. limnetica Ichihara et Shimada, not yet found in coastal areas, was highly viable in seawater.     

Antialgal activity of poly(2-(dimethylamino)ethyl methacrylate) (PDMAEMA) brushes against the marine alga Ulva

Marine biofouling has detrimental effects on the environment and economy, and current antifouling coatings research is aimed at environmentally benign, non-toxic materials. The possibility of using contact-active coatings is explored, by considering the antialgal activity of cationic poly(2-(dimethylamino)ethyl methacrylate) (PDMAEMA) brushes. The antialgal activity was investigated via zoospore settlement and sporeling growth assays of the marine algae Ulva linza and U. lactuca. The assay results for PDMAEMA brushes were compared to those for anionic and neutral surfaces. It was found that only PDMAEMA could disrupt zoospores that come into contact with it, and that it also inhibits the subsequent growth of normally settled spores. Based on the spore membrane properties, and characterization of the PDMAEMA brushes over a wide pH range, it is hypothesized that the algicidal mechanisms are similar to the bactericidal mechanisms of cationic polymers, and that further development could lead to successful contact-active antialgal coatings.     

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.     

Parasexual fusion products in green algae: Enteromorpha and Ulvaria (Ulvales,Chlorophyta)

Enteromorpha linza and Ulvaria oxysperma in North Carolina reproduce exclusively by asexual zoospores. Calcofluor white staining indicated that newly released zoospores lack significant cellulose cell wall material, making them suitable for treatment as protoplasts in a parasexual fusion process using high pH-Ca²⁺, PEG and centrifugation. Presumptive fusion products were identified by their larger size, twin chloroplasts and eyespots, and presence of fluorescence labelled and unlabelled portions. Parasexual fusion and karyogamy were confirmed by elevated levels of nuclear DNA in fusion cell germlings. In addition, aceto-orcein staining of fusion cell products revealed a diploid chromosome complement of 2N = 20 in Enteromorpha linza. Fusion cells were isolated by killing the more numerous adjacent unfused zoospores with 2-3 min exposure to blue light (410–490 nm). Unexposed fusion cells could be readily distinguished and recovered by micropipette at the 10-day stage.Center for Marine Science Research UNC-W Contribution No. 008.     

Growth ofEnteromorpha linza in sewage effluent and sewage effluent-seawater mixtures

Enteromorpha linza (L.) J. Ag. was grown in full strength sewage effluent, various combinations of sewage effluent and seawater, and in natural seawater. It was found that full strength sewage effluent with a salinity of 14 supported best growth of the alga. After a 12 day cultivation period, growth ofE. linza in full strength sewage effluent and 75% sewage effluent- seawater mixture showed 3.5-fold and 2-fold increase in fresh weight over that grown in natural seawater; respectively. Uptake of PO _inf4 ^sup3– -P, NH₃-N and NO _inf3 ^sup– -N by cells ofE. linza was extremely efficient in all tested media. Data obtained from the experiments indicated that inorganic nitrogen rather than phosphorus was the limiting nutrient factor for growth ofE. linza in full strength sewage effluent and in other sewage effluent- seawater mixtures. NH₃-N at concentrations above 4.5 ppm was found to inhibit uptake of NO _inf3 ^sup– -N in the same culture medium by the algal cells. The fact that sewage grownE. linza contained comparatively much higher protein content (30.2% dry weight) than that grown in natural seawater (12.5% dry weight) leads to the conclusion that sewage grownE. linza could serve as an economically feasible feed for livestock in Hong Kong where the sewage is characterized by having a salinity of approximately 14. It is proposed that this multicellular green alga is a suitable algal species to serve the dual function of wastewater purification through the production of algal protein from sewage effluent having high salinities.     

Antifouling character of 'active' hybrid xerogel coatings with sequestered catalysts for the activation of hydrogen peroxide

Halide-permeable xerogel films prepared from sols containing 50 mol% aminopropyltriethoxysilane (APTES)/50 mol% tetraethoxysilane (TEOS) or 10 mol% APTES/90 mol% TEOS and 0.015 M selenoxide or telluride catalyst in the sol gave reduced settlement of cypris larvae of the barnacle Balanus amphitrite and larvae of the tubeworm Hydroides elegans in the presence of artificial seawater (ASW) and hydrogen peroxide (5-100 microM) relative to glass controls. Settlement of Ulva zoospores was lower on both the 50 mol% APTES/50 mol% TEOS and 10 mol% APTES/90 mol% TEOS xerogel formulations in comparison with glass controls with or without the added catalyst. The 50 mol% APTES/50 mol%TEOS xerogel containing telluride catalyst gave reduced settlement of Ulva zoospores in the presence of 100 microM H(2)O(2) in ASW compared with the same coating without added peroxide. Scanning electron microscopy and XPS data suggest that exposure to H(2)O(2) does not lead to chemical or morphological changes on the xerogel surface.     

Quantification of laminarialean zoospores in seawater by real‐time PCR

Laminarialean plants undergo heteromorphic alternation of generations between the macroscopic diploid sporophyte and the microscopic haploid gametophyte. The generations change through the formation and release of asexual or sexual reproductive cells. It is difficult to monitor the release and diffusion of zoospores into the environment. Furthermore, even if zoospores can be detected, species identification remains difficult. This study attempted to develop a sensitive and fast identification method for laminarialean zoospores using quantitative PCR. In addition, we aimed to estimate the density of zoospores in natural seawater. Specific primers for Saccharina japonica and Undaria pinnatifida were designed and used to estimate the quantity of zoospores in seawater. DNA samples (environmental DNA) were collected from seawater once or twice each month for 2 years at the same area, and seasonal variations in the release of zoospores was monitored. The estimated maturation period based on the number of released zoospores in this study was comparable with those of the previously reported maturation periods of S. japonica and U. pinnatifida sporophytes. This supports the validity of our method in estimating zoospore release from laminarialean plants. The method will be a useful tool for ecological studies on these commercially relevant species.     

THE SECRET LIFE OF KELPS: PLANKTONIC PROCESSES AND POPULATION DYNAMICS

Understanding spatio‐temporal variability in recruitment is vital to studies of kelp population dynamics. Research on settlement and post‐settlement processes has suggested that arrival of kelp zoospores to suitable substrate is important in limiting kelp recruitment, yet the role of planktonic processes in kelp population dynamics has not been studied due to difficulties in sampling and identifying zoospores. I developed a method to estimate kelp zoospore abundance from in situ plankton samples and used it to study various processes regulating the availability of giant kelp (Macrocystis pyrifera) zoospores for settlement. My studies focused on (1) identifying temporal scales over which zoospore abundance is most variable, (2) describing physical and biological processes that regulate this variability, and (3) determining the relationship between zoospore abundance and settlement. I found that short‐term variability in zoospore abundance (<24 hrs) was not due to changes in supply but rather dispersion, caused by oscillating hydrodynamic forces (e.g. waves). Long‐term variability in zoospore abundance, however, was best explained by the size and density of reproductive adult plants, with zoospore abundance being most variable at the scale of days to months. Changes in adult reproductive condition caused rapid changes in zoospore abundance suggesting that the supply of kelp zoospores is sensitive to environmental regulation of adult physiology. Thus, unlike with marine animals, these results indicate that variability in kelp propagule supply, over scales most likely to affect subsequent settlement and recruitment, is more tightly coupled to demographic and reproductive mechanisms than to physical transport processes.