Adhesion Strength of Settled Spores of the Green Alga Enteromorpha

Strengths of attachment of spores of the green fouling alga Enteromorpha to glass have been measured using a modified water jet apparatus. Surface pressures of ～250 kPa were required to quantitatively remove attached spores after 4 h contact with a surface. The development of adhesive and cohesive strength is highly time-dependent; after 8 h in contact with a surface spores did not detach, even at pressures in excess of 250 kPa. Spores settled in groups are more resistant to detachment than single spores, which suggests that the adaptive value of gregarious settlement behaviour may lie in the greater resistance of groups to detachment forces in a naturally turbulent environment. The interfacial forces exerted as water impinges on the surface and the derivation of adhesion strength values in terms of wall shear stress are discussed and compared with those obtained by other methods. A surface pressure of 250 kPa approximates to 325 Pa wall shear stress. Calculation using the power-law formula predicts that detachment forces of this magnitude are unlikely to be realized at operating speeds for most vessels and that most Enteromorpha spores would not detach from untreated hulls.     

Antifouling Potential of Lubricious,Micro-engineered,PDMS Elastomers against Zoospores of the Green Fouling Alga Ulva (Enteromorpha)

The settlement and release of Ulva spores from chemically modified, micro-engineered surface topographies have been investigated using poly(dimethyl siloxane) elastomers (PDMSe) with varying additions of non-network forming poly(dimethyl siloxane) based oils. The topographic features were based on 5?μm wide pillars or ridges separated by 5, 10, or 20?μm wide channels. Pattern depths were 5 or 1.5?μm. Swimming spores showed no marked difference in settlement on smooth surfaces covered with excess PDMS oils. However, incorporation of oils significantly reduced settlement density on many of the surfaces with topographic features, in particular, the 5?μm wide and deep channels. Previous results, confirmed here, demonstrate preferences by the spores to settle in channels and against pillars with spatial dimensions of 5?μm, 10?μm and 20?μm. The combination of lubricity and pillars significantly reduced the number of attached spores compared to the control, smooth, unmodified PDMSe surfaces when exposed to turbulent flow in a flow channel. The results are discussed in relation to the energy needs for spores to adhere to various surface features and the concepts of ultrahydrophobic surfaces. A factorial, multi-level experimental design was analyzed and a 2nd order polynomial model was regressed for statistically significant effects and interactions to determine the magnitude and direction of influence on the spore density measurements between factor levels.     

Effect of Bacteria Associated with the Green Alga Ulva reticulata on Marine Micro- and Macrofouling

The green alga Ulva reticulata (Forsskal) is often free from biofouling in Hong Kong waters. An early study indicated that bioactive substances from this alga inhibit settlement of the polychaete Hydroides elegans (Haswell). It is also predicted that epibiotic bacteria protect this alga from micro- and macrofouling. In this study, bacterial strains from the surface of U. reticulata were isolated and their inhibitive activities on micro- and macrofouling assayed. The strains were identified by 16S rRNA analysis as belonging to the genera Alteromonas , Pseudoalteromonas and Vibrio . There was no significant effect of these strains or their extracts (aqueous and ethanol) on the growth of five Vibrio strains isolated from natural biofilm. Two bacterial strains ( Alteromonas sp. and Vibrio sp. 3) were non-toxic to the benthic diatom Nitzschia paleacea (Grunow) while the other five strains caused a low level of mortality. No one bacterial strain was toxic to the larvae of H. elegans . Aqueous extract of one of the isolated bacterial species, i.e. Vibrio sp. 2, significantly ( p <0.00001) inhibited the settlement and metamorphosis of H. elegans larvae. The putative antifouling compounds have a molecular weight of >100 kD. On the other hand, biofilm of Pseudoalteromonas sp. 2 and aqueous extract of Vibrio sp. 2 suppressed the settlement of larvae induced by 3-isobutyl-1-methylxanthine (IBMX). Other epibiotic bacteria and their extracts had neither inhibitive nor inductive effects on larval settlement of H. elegans . The results indicate that the antifouling mechanism of U. reticulata may be dependent not only on materials from the macroalga itself but also on the epibiotic bacteria on the algal surface.     

Monoclonal antibodies to adhesive cell coat glycoproteins secreted by zoospores of the green alga Enteromorpha

Zoospores of Enteromorpha compressa (L.) Grev. secrete an adhesive cell coat which is involved in their attachment to various substrata. Two monoclonal antibodies (mAbs), designated Ent 1 and Ent 6, were raised against settled zoospores displaying secreted adhesive. Both antibodies labelled specifically the anterior region of the cell containing putative adhesive vesicles. During settlement the antigens recognised by both mAbs were secreted but whereas Ent 6 recognised a fibrillar material released within a few minutes of settlement, Ent 1 recognised components which were associated predominantly with the developing cell wall at later time points. Both mAbs also labelled a Golgi-rich region of settled spores, suggesting that these antigens are also synthesised after settlement. Both mAbs labelled the cell walls of vegetative tissue. Competitive enzyme-linked immunosorbent assay indicated that the two antibodies recognise separate, but overlapping epitopes. In spore settlement assays the Ent 6 immunoglobulin strongly reduced initial adhesion at low concentration whereas the inhibitory effects of Ent 1 occurred at later time points. On analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, (SDS-PAGE) both MAbs recognised a major buffer- and SDS-soluble, polydisperse 110-kDa antigen. The 110-kDa component was present in extracts of zoospores and sporulating tissue, but absent, in soluble form, from vegetative tissue. Deglycosylation of zoospore extract with anhydrous HF and peptide N-glycosidase digestion, showed that the major 110-kDa antigen is an N-linked glycan, and that the epitope is borne by the protein component. Time-course experiments showed that the Ent 6 antigen became progressively insoluble after zoospore attachment. Taken together, the data indicate that the two antibodies recognise separate but closely related antigens which have distinctive roles in adhesion and cell wall development. Received: 8 February 1999 / Accepted: 26 July 1999     

Pharmacological Induction of Larval Settlement and Metamorphosis in the Blue Mussel Mytilus edulis L.

The blue mussel Mytilus edulis L. is an important aquaculture and fouling species in northern seas. Although the general role of chemical cues for settlement of larvae of the blue mussel has been proposed, few studies have focused on induction of settlement and metamorphosis by pharmacological agents. In this study, the induction of larval settlement of the blue mussel by pharmacological compounds was investigated through a series of laboratory experiments with an aim of identifying artificial cues for laboratory bioassay systems in fouling and antifouling research. Gamma-aminobutiric acid (GABA), dihydroxyphenyl L-alanine (DOPA), isobutyl methylxanthine (IBMX) and acetylcholine chloride (ACH) at 10^{m 7}-10^{m 2} M as well as KCl at 10-40 mM K⁺ in excess of the level in normal seawater were tested for their inductive effect on larval settlement. In filtered seawater (FSW) <9% of the larvae settled after 48 h. Elevated K⁺ and GABA levels had no effect on larval settlement and metamorphosis. DOPA at 10^{m 5} M and IBMX at 10^{m 6}-10^{m 4} M induced 41-83% larval settlement and ACH at 10^{m 7}-10^{m 5} M induced < 40% larval settlement. While the highest settlement rates were observed after 48 h exposure to the chemicals, most of the larvae settled within 24 h. Compounds at concentrations of 10^{m 3}-10^{m 2} M were either toxic to larvae or retarded the growth of the post-larvae shell. Juveniles resulting from induction by lower concentrations of chemicals had a very high survival rate, completed metamorphosis and grew as well as the juveniles that metamorphosed spontaneously. IBMX at 10^{m 6}-10^{m 4} M and L-DOPA at 10^{m 5} M are effective agents for induction of settlement and metamorphosis for future studies using juvenile M. edulis.     

Optimising settlement tiles: The effects of surface texture and energy,orientation and deployment duration upon the fouling community

The aim of this study was to produce a set of tiles for field studies on settlement and biofouling with carefully controlled surface characteristics and practical design, and test them under field conditions. Impressions of precisely defined surface textures were made in silicone. Double sided tiles in epoxy, polyester and silanised epoxy resins were cast from the impressions. Tile characteristics tested were texture (R_a = 0, 0.19, 0.62, 1.1, 2.2 mm), surface free energy (60, 52, 24 mN m^{m 2}), and surface orientation (up, down, into, away). Tiles were deployed in the Red Sea for 4 and 7 months. Measures of community cover, dominance and richness were all significantly affected by each of the factors. The tiles proved durable and robust during the 7 month deployment with no observable changes in surface characteristics and none were lost or broken. These settlement tiles have a wide applicability for both biofouling and ecological studies. The field test demonstrated the complexities of the interactions between just four surface characteristics. This study has also underlined the need for multidimensional analysis of fouling communities for applied and basic research.     

Model simulations of conditions suitable for the establishment of Enteromorpha sp. (Chlorophyta) macroalgal mats

Floating mats of algae regularly appear in shallow bays in many coastal areas around the world. This paper presents a model that simulates the growth of ephemeral macroalgae in shallow bays, and interprets the growth according to physical and biological prerequisites. Model runs emphasized the role of internal nutrient cycling to provide the enhanced nutrient availability needed to ensure abundant growth of macroalgae. Thus, the presence of macroalgae, as well as long water residence times, influenced the nutrient cycling in the bay to support further growth of macroalgal mats.     

A simple method for the freeze-preservation of zoospores of the green macroalga Enteromorpha intestinalis

Settled zoospores of the green macroalga Enteromorpha intestinalis were subjected to several different freezing and storing treatments at both cryogenic and non-cryogenic temperatures after which their viability was assessed using a spore germination bioassay. Three different cooling rates were tested: slow cooling at –1°C min⁻¹ and –0.5°C min⁻¹ to end temperatures in the range –20°C to –40°C, and a two-step procedure whereby the spores were frozen to –30°C at a rate of –1°C min⁻¹ prior to immersion in liquid nitrogen at –196°C. Spore viability was also investigated using the cryoprotectants glycerol and dimethyl suphoxide (DMSO), a reduced saline medium and various storage times. In the majority of experiments, the use of a cryoprotectant during the freezing process significantly increased the viability of the spores, with DMSO affording slightly greater protection than glycerol. All treatments produced high viabilities (ranging from 75.3–100.0%) after 5-min storage at the different end temperatures. However, progressively longer storage up to 7 days generally resulted in a marked reduction in viability. This was with the exception of spores frozen in a reduced saline medium; a medium of 75% seawater and either 5 or 10% DMSO greatly increased spore viability, with values of > 40% recorded for spores stored at –20°C for up to 5 weeks. This revised version was published online in July 2006 with corrections to the Cover Date.     

Photosynthetic Eukaryotes of Freshwater Wetland Biofilms: Adaptations and Structural Characteristics of the Extracellular Matrix in the Green Alga, Cosmarium reniforme (Zygnematophyceae, Streptophyta)

ABSTRACT. Cosmarium reniforme (Zygnematophyceae, Streptophyta) is a green alga that is commonly found in biofilms of wetlands of the Adirondack region, NY (USA). Two distinctive characteristics that are critical to this alga's survival in a benthic biofilm are its elaborate cell morphology and extracellular matrix (ECM). In this study, ultrastructural, immunocytochemical, and experimental methodologies were employed in order to elucidate the cellular characteristics that are critical for survival in a biofilm. The ECM consists of a thick, outwardly lobed cell wall (CW), which contains a patterned network of structurally complex pores. Each pore consists of a narrow channel, terminating internally at a bulb that invaginates localized regions of the plasma membrane. The outer region of the pore contains arabinogalactan protein-like and extensin epitopes that are likely involved in adhesion mechanisms of the cell. External to the CW is the extracellular polymeric substance that is employed in ensheathment of the cell to the substrate and in gliding motility. The architectural design/biochemical make-up of the CW and a secretory system that encompasses the coordinated activities of the endomembrane and cytomotile/cytoskeletal systems provide the organism with effective mechanisms to support life within the biofilm complex.     

Evidence for the Presence of Chlorophyllide b in the Green Alga Scenedesmus obliquus in vivo

Chlorophyllide b could be extracted from the wild type of Scenedesmus obliquus and its pigment mutant C-2A'. Its identity was proved by absorption and fluorescence spectroscopy and by a positive hydroxylamine test. Chlorophyllide b could be transformed into pheophorbide b and methylpheophorbide b. The formation of chlorophyllide b from chlorophyll b by dephytylation with chlorophyllase could be ruled out. The stimulation of chlorophyllide b biosynthesis with o-phenanthroline, as described in the literature, could not be confirmed under physiological conditions.     

Natural Synchronisation for the Study of Cell Division in the Green Unicellular Alga Ostreococcus tauri

Ostreococcus tauri (Prasinophyceae) is a marine unicellular green alga which diverged early in the green lineage. The interest of O. tauri as a potential model to study plant cell division is based on its key phylogenetic position, its simple binary division, a very simple cellular organisation and now the availability of the full genome sequence. In addition O. tauri has a minimal yet complete set of cell cycle control genes. Here we show that division can be naturally synchronised by light/dark cycles and that organelles divide before the nucleus. This natural synchronisation, although being only partial, enables the study of the expression of CDKs throughout the cell cycle. The expression patterns of OtCDKA and OtCDKB were determined both at the mRNA and protein levels. The single OtCDKA gene is constantly expressed throughout the cell cycle, whereas OtCDKB is highly regulated and expressed only in S/G2/M phases. More surprisingly, OtCDKA is not phosphorylated at the tyrosine residue, in contrast to OtCDKB which is strongly phosphorylated during cell division. OtCDKA kinase activity appears before the S phase, indicating a possible role of this protein in the G1/S transition. OtCDKB kinase activity occurs later than OtCDKA, and its tyrosine phosphorylation is correlated to G2/M, suggesting a possible control of the mitotic activity. To our knowledge this is the first organism in the green lineage which showed CDKB tyrosine phosphorylation during cell cycle progression.     

Isolation of Protoplasts from the Coccal Green Alga Eremosphaera viridis De Bary for Patch-clamp Measurements

A method for enzymatic isolation of protoplasts from the unicellular green alga Eremosphaera viridis for patch-clamp measurements is described. Viable protoplasts with “patch-clean” plasma membranes could only be isolated when combining high enzyme concentrations and long incubation times. In whole-cell recordings the protoplasts exhibited electrical properties similar to those measured in intact cells. Taken together with the protoplasts' ability for rapid deplasmolysis after transfer into hypotonic solution, this indicates the viability of the isolated protoplasts.     

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.     

Biosorption and Reuse Potential of a Blue Green Alga for the Removal of Hazardous Reactive Dyes from Aqueous Solutions

The adsorption potential of a nonliving alga Nostoc comminutum for the removal of hazardous dyes from aqueous solutions was assessed. The algal biomass in its native form exhibited the highest dye removal efficiency at a pH of 1 and at a contact time of 50 min. The effect of various chemical pretreatments was studied in order to assess the role of surface chemistry and the underlying mechanism. Combined with Fourier transform infrared (FTIR) studies, it was concluded that the carboxylic and amine groups played a dominant role in the dye binding process. pH studies further revealed that besides the electrostatic mechanism, other physical interactions might be operative, which is also revealed from the Dubinin-Radushkevich (D-R) isotherm model. Kinetic studies indicated that the adsorption process followed the second-order kinetics and particle diffusion mechanisms were operative. Thermodynamic studies revealed that the adsorption of two dyes onto the algal biomass was feasible, spontaneous, and exothermic under the studied conditions. Microwave irradiation was proposed as a green method for the regeneration and reuse of the biomass. A 16% loss in the regeneration efficiency of the biosorbent was observed, which reveals its stability and reuse potential. Column experiments with real textile wastewater established the practicality of the developed system. Finally, a comparative study revealed the efficacy of the biosorbent with other previously reported biosorbents for dye removal.     

Nucleotide Diversity of the Colorless Green Alga Polytomella parva (Chlorophyceae,Chlorophyta): High for the Mitochondrial Telomeres,Surprisingly Low Everywhere Else*

ABSTRACT. Silent‐site nucleotide diversity data (π_silent) can provide insights into the forces driving genome evolution. Here we present π_silent statistics for the mitochondrial and nuclear DNAs of Polytomella parva, a nonphotosynthetic green alga with a highly reduced, linear fragmented mitochondrial genome. We show that this species harbors very little genetic diversity, with the exception of the mitochondrial telomeres, which have an excess of polymorphic sites. These data are compared with previously published π_silent values from the mitochondrial and nuclear genomes of the model species Chlamydomonas reinhardtii and Volvox carteri, which are close relatives of P. parva, and are used to understand the modes and tempos of genome evolution within green algae.     

Atomic Basis for the Species-specific Inhibition of αV Integrins by Monoclonal Antibody 17E6 Is Revealed by the Crystal Structure of αVβ3 Ectodomain-17E6 Fab Complex

The function-blocking, non-RGD-containing, and primate-specific mouse monoclonal antibody 17E6 binds the αV subfamily of integrins. 17E6 is currently in phase II clinical trials for treating cancer. To elucidate the structural basis of recognition and the molecular mechanism of inhibition, we crystallized αVβ3 ectodomain in complex with the Fab fragment of 17E6. Protein crystals grew in presence of the activating cation Mn²⁺. The integrin in the complex and in solution assumed the genuflected conformation. 17E6 Fab bound exclusively to the Propeller domain of the αV subunit. At the core of αV-Fab interface were interactions involving Propeller residues Lys-203 and Gln-145, with the latter accounting for primate specificity. The Propeller residue Asp-150, which normally coordinates Arg of the ligand Arg-Gly-Asp motif, formed contacts with Arg-54 of the Fab that were expected to reduce soluble FN10 binding to cellular αVβ3 complexed with 17E6. This was confirmed in direct binding studies, suggesting that 17E6 is an allosteric inhibitor of αV integrins.