Effects of inter-organism interactions in biofouling on microtopographic surfaces

An extended model of the surface energetic attachment (SEA) model is introduced to study the fouling of marine organisms on microtopographic surfaces, taking into account the excluded volume interaction and the attraction between the organisms. It is shown that the excluded volume interaction leads to changes in the site-typed attachment probabilities which increase with the average spore density on the surface. As a result of these changes, the spore density map is flattened under very high density fouling. The attractive interaction on the other hand leads to aggregation of spores and the average aggregate size increased with the strength of attraction. The model can be mapped to a specific experiment to determine the attachment energy parameters. In contrast to various prior empirical approaches, the extended SEA model is rigorous from the statistical mechanics viewpoint, thus it provides a reliable tool for studying complex attachment behaviors of microorganisms on topographic surfaces.     

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.     

Engineered antifouling microtopographies: the role of Reynolds number in a model that predicts attachment of zoospores of Ulva and cells of Cobetia marina

A correlation between the attachment density of cells from two phylogenetic groups (prokaryotic Bacteria and eukaryotic Plantae), with surface roughness is reported for the first time. The results represent a paradigm shift in the understanding of cell attachment, which is a critical step in the biofouling process. The model predicts that the attachment densities of zoospores of the green alga, Ulva, and cells of the marine bacterium, Cobetia marina, scale inversely with surface roughness. The size and motility of the bacterial cells and algal spores were incorporated into the attachment model by multiplying the engineered roughness index (ERI_II), which is a representation of surface energy, by the Reynolds number (Re) of the cells. The results showed a negative linear correlation of normalized, transformed attachment density for both organisms with ERI_II · Re (R ² = 0.77). These studies demonstrate for the first time that organisms respond in a uniform manner to a model, which incorporates surface energy and the Reynolds number of the organism.     

Disruption-free imaging by Raman spectroscopy reveals a chemical sphere with antifouling metabolites around macroalgae

Investigations of the surface chemistry of marine organisms are essential to understand their chemically mediated interactions with fouling organisms. In this context, the concentration of natural products in the immediate vicinity of algal surfaces, as well as their biological activity, are of particular importance. However, due to lack of appropriate methods, the distribution of compounds within the chemical sphere around marine algae is unknown. This study demonstrates the suitability of confocal resonance Raman microspectroscopy for the determination of metabolites around algal surfaces with a micrometer resolution. The spatial distribution of carotenoids in the diffusion boundary layer of the brown alga Fucus vesiculosus and the green alga Ulva sp. was determined using the disruption-free optical method. A gradient of carotenoids was determined within 0 to 150 μm from the surface of thealgae, thereby demonstrating the release of the non-polar metabolites involved in antifouling processes. Thedifferences in the carotenoid composition of the brown and green algae were reflected in the spectra. Resonance Raman microspectroscopy also allowed visualization of the lateral distribution of fucoxanthin on the algal surface and localization of concentration maxima within a 50 × 50 μm² area. The results from this work show clearly that established dipping techniques suitable for the extraction of the diffusion boundary layer of macroalgae only provide an average of the local strongly variable concentrations of metabolites on algal surfaces.     

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.     

Interactions between microbial biofilms and marine fouling algae: a mini review

Natural and artificial substrata immersed in the marine environment are typically colonized by microorganisms, which may moderate the settlement/recruitment of algal spores and invertebrate larvae of macrofouling organisms. This mini-review summarizes the major interactions occurring between microbial biofilms and marine fouling algae, including their effects on the settlement, growth and morphology of the adult plants. The roles of chemical compounds that are produced by both bacteria and algae and which drive the interactions are reviewed. The possibility of using such bioactive compounds to control macrofouling will be discussed.     

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.     

RAPID NITRATE UPTAKE RATES AND LARGE SHORT‐TERM STORAGE CAPACITIES MAY EXPLAIN WHY OPPORTUNISTIC GREEN MACROALGAE DOMINATE SHALLOW EUTROPHIC ESTUARIES1

We quantified the effects of initial macroalgal tissue nitrogen (N) status (depleted and enriched) and varying pulses of nitrate (NO₃^?) concentration on uptake and storage of nitrogen in Ulva intestinalis L. and Ulva expansa (Setch.) Setch. et N. L. Gardner using mesocosms modeling shallow coastal estuaries in Mediterranean climates. Uptake of NO₃^? (μmol · g dry weight [dwt]^?1 · h^?1) was measured as loss from the water after 1, 2, 4, 8, 12, and 24 h and storage as total tissue nitrogen (% dwt) and nitrate (ppm). Both species of algae exhibited a high affinity for NO₃^? across all N pulses and initial tissue contents. There was greater NO₃^? removal from the water for depleted than enriched algae across all time intervals. In the low‐N‐pulse treatment, U. intestinalis and U. expansa removed all measurable NO₃^? within 8 and 12 h, respectively, and in the medium and high treatments, removal was high and then decreased over time. Maximum mean uptake rates of nitrate were greater for U. expansa (～300 μmol · g dwt^?1 · h^?1) than U. intestinalis (～100 μmol · g dwt^?1 · h^?1); however, uptake rates were highly variable over time. Overall, U. expansa uptake rates were double those of U. intestinalis. Maximum tissue NO₃^? for U. expansa was >1,000 ppm, five times that of U. intestinalis, suggesting that U. expansa has a greater storage capacity in this cellular pool. These results showed that opportunistic green algae with differing tissue nutrient histories were able to efficiently remove nitrate from the water across a wide range of N pulses; thus, both are highly adapted to proliferate in estuarine environments with pulsed nutrient supplies.     

GLYCOCONJUGATE ORGANIZATION OF ENTEROMORPHA (=ULVA) FLEXUOSA AND ULVA FASCIATA (CHLOROPHYTA) ZOOSPORES1

Ecologically successful algae that colonize natural and artificial substrates in the marine environment have distinct strategies for opportunistic dispersal and settlement. The objective of this research was to visualize molecular architecture of zoospores from Enteromorpha (=Ulva) flexuosa (Wulfen) J. Agardh and Ulva fasciata Delile that coexist but alternate in dominance on an intertidal bench. Multiple fluorescent lectins were used to stabilize and probe for diverse zoospore glycoconjugates (GC) that could be involved in cell and substrate interactions. Results from epifluorescence microscopy showed distinct cellular and extracellular polymeric substance (EPS) domains of GC relative to settlement morphologies. Glycoconjugates were similar for both species with (1) α‐d mannose and/or glucose moieties localized on flagella, the anterior domes and anterior regions, the plasma membranes, and EPS; (2) α‐fucose localized on flagella and anterior regions; (3) N or α,ß‐N acetylglucosamine localized on flagella, the anterior regions, and EPS; and (4) varied N‐acetylgalactosamine and/or galactose moieties localized on each domain for both species excluding the plasma membranes. Some differences in lectin binding were observed for each species at the flagella, the anterior domes, and the plasma membranes. Glycoconjugate distributions shifted with morphological changes that followed initial adhesion. TEM of E. flexuosa zoospore stages following carbohydrate‐stabilizing fixations and gold‐conjugated lectin probes resolved GC with α‐d mannose and/or glucose, and/or N‐acetylglucosamine at the plasma membrane, ER and diverse vesicles of the anterior pole, EPS, and discontinuous regions or knobs associated with flagellar surfaces. The distinct distribution and diversity of zoospore GC may be central to recognition and attachment on diverse substrata by these algae.     

DURATIONS OF GAMETE MOTILITY AND CONJUGATION ABILITY OF ULVA COMPRESSA (ULVOPHYCEAE)1

The present study was designed to develop a technique for crossing and to gain insight into how sexual reproduction contributes to the maintenance of local populations of Ulva compressa L. To examine the durations of gamete motility and conjugation ability, freshly released gametes were incubated for various periods of time prior to mixing both mating types. The conjugation ability of the gametes gradually declined after being released from the thalli when the gametes were incubated without mixing with the opposite mating type. The ability to conjugate decreased by half after 6 h, although most of the gametes remained motile. The gametes released 4 h later had the same level of conjugation ability when mixed immediately after releasing. When the mature thalli were wrapped in a moist paper towel to prevent gametes from being released, the gametes were preservable for 7 h without a significant decrease in their conjugation ability. Conjugation occurred soon after mixing gametes of both mating types and reached a plateau after 30 s. However, conjugation rates did not exceed a rate of ～70%, even though freshly released gametes were used. Interestingly, a portion of the gametes newly conjugated 30 min after mixing both mating types, and conjugation rates reached a second plateau at ～90%. Gametes with delayed conjugation are provided some period of time that allows them to be transported away and increases their chances of mating with more distant populations, thus contributing to the maintenance of genetic variation.