Green mussel Perna viridis L.: attachment behaviour and preparation of antifouling surfaces

The green mussel Perna viridis LINNE can be kept in simulated seawater for more than 6 months in good condition. The mussel forms many threads by secreting an adhesive protein from the foot, and attaches with more than 50 byssal threads, which makes most mussels clump together. In order to investigate the preparation of the antifouling surfaces toward green mussels, the attachment of mussels was tested using glass surfaces modified with silane coupling agents, together with non-treated material surfaces such as glass and silicone. The correlation between the attachment percentage and the mean number of the secreted byssus was highly significant, indicating that the mussel selects a favorable surface prior to the secretion of byssus. The relationships between the mussel attachment and the surface chemical parameters (surface free energy (sfe) and its dispersion and polar components) were examined based on a working hypothesis, which we have previously reported. The result of statistical regression test indicated that a certain correlation was found between the dispersion component and the mussel attachment, while the polar component did not correlate to the mussel attachment. The present surface chemical approach provided an additional clue for the preparation of ecologically clean antifouling materials that takes into account the combination of the wettability of both the marine adhesive proteins (MAP) and the modified surfaces.     

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.     

Does rock type account for variation in mussel attachment strength? A test with <Emphasis Type="Italic">Brachidontes rodriguezii</Emphasis> in the southwestern Atlantic

Mussel attachment strength varies in space and time, frequently in association with variations in wave exposure. Yet, it remains uninvestigated whether different rock types can contribute to variation in mussel attachment. Here we compared the attachment strength of the mussel Brachidontes rodriguezii between soft and hard intertidal rock substrates that are typical of coastal Buenos Aires Province, Argentina: Pampean loess cemented by calcium carbonate and orthoquartzite, respectively. Overall comparisons of mussel attachment across natural platforms of either rock type (10 loess sites and 4 orthoquartzite sites) indicated stronger mussel attachment to orthoquartzite. However, mussel attachment strength did not differ when compared across natural loess platforms and introduced orthoquartzite blocks (i.e., groins and revetments) occurring within the same site. Mussels attaching to loess showed more byssal threads than those attaching to orthoquartzite at the same site. These findings suggest, first, that rock type does not influence mussel attachment strength in our study system, secondly, that overall differences in mussel attachment strength with rock type across natural platforms in our study range are due to confounding influences of co-varying factors (e.g., wave exposure) and, finally, that mussels can increase byssus production to counteract potential substrate failure when attaching to soft, friable rock. The latter likely explains the ability of mussels to maintain relatively stable cover across rocks of contrasting hardness.     

Effects of biofilms on zebra mussel postveliger attachment to artificial surfaces

Abstract. The zebra mussel is an introduced fouling organism in North American inland waters. This field study tested whether natural biofilms, formed by covering substrata with a 100-μm mesh that allows microorganisms to attach and films to develop in the absence of postveligers, influenced the attachment of zebra mussel postveligers to artificial surfaces. Low-wettable polycarbonate and wettable glass surfaces were used in the experiments over four field seasons to study biofilm formation (1997–1998) and mussel attachment (1998–2000). The presence of the mesh did not quantitatively affect biofilm development on either substratum as determined by microscopic direct counts and colony-forming units on R2A agar. Natural biofilms on polycarbonate surfaces positively influenced postveliger attachment compared to substrata that initially had no film (ANOVA, p-values ranged from ≤.05 to ≤.001). Biofilms did not influence postveliger attachment to glass surfaces (ANOVA, p>.05). Attachment to both substrata was similar on surfaces with and without previously settled postveligers. Based on these results, we conclude that biofilms can enhance postveliger attachment to some but not all artificial surfaces.     

CsgA production by Escherichia coli O157:H7 alters attachment to abiotic surfaces in some growth environments

The role of curli expression in attachment of Escherichia coli O157:H7 to glass, Teflon, and stainless steel (SS) was investigated through the creation of csgA knockout mutants in two isolates of E. coli O157:H7. Attachment assays using epifluorescence microscopy and measurements of the force of adhesion of bacterial cells to the substrates using atomic force microscopy (AFM) force mapping were used to determine differences in attachment between wild-type (wt) and csgA-negative (ΔcsgA) strains following growth in four different media. The hydrophobicity of the cells was determined using contact angle measurements (CAM) and bacterial adhesion to hydrocarbons (BATH). The attachment assay results indicated that ΔcsgA strains attached to glass, Teflon, and SS surfaces in significantly different numbers than their wt counterparts in a growth medium-dependent fashion (P < 0.05). However, no clear correlation was seen between attachment numbers, surface type, or growth medium. No correlation was seen between BATH and CAM results (R(2) < 0.70). Hydrophobicity differed between the wt and ΔcsgA in some cases in a growth medium- and method-dependent fashion (P < 0.05). AFM force mapping revealed no significant difference in the forces of adhesion to glass and SS surfaces between wt and ΔcsgA strains (P > 0.05) but a significantly greater force of adhesion to Teflon for one of the two wt strains than for its ΔcsgA counterpart (P < 0.05). This study shows that CsgA production by E. coli O157:H7 may alter attachment behavior in some environments; however, further investigation is required in order to determine the exact relationship between CsgA production and attachment to abiotic surfaces.     

Multiple drug resistance and strength of attachment to surfaces in Pseudomonas aeruginosa isolates

Aims: To investigate the presence of a relationship between the strength of attachment of Pseudomonas aeruginosa to stainless steel surfaces and their observed multiple drug resistance. Methods and Results: Multiple drug resistance of clinical and environmental isolates of Ps. aeruginosa was evaluated using disc diffusion method. The blot succession technique was used to quantify the strength of attachment of Ps. aeruginosa isolates. Different multiple drug–resistant Ps. aeruginosa isolates exhibited variable attachment strength. Although the highest multiple drug–resistant clinical isolate was shown to have the least attachment strength among clinical isolates, a weak correlation was found between attachment strength and multiple resistance among our investigated Ps. aeruginosa isolates. Conclusions: There is a weak correlation between multiple drug resistance and strength of attachment to stainless steel surfaces. Significance and Impact of the Study: Even low‐resistant Ps. aeruginosa could have the potential of attaching firmly to surfaces and forming biofilm.     

Role of curli fimbriae in mediating the cells of enterohaemorrhagic Escherichia coli to attach to abiotic surfaces

AIMS: The objectives of this study were to evaluate the role of curli in assisting the cells of enterohaemorrhagic Escherichia coli (EHEC) in attaching to abiotic surfaces and to determine the influence of cell-surface contact time on the efficiency of the attachment. METHODS AND RESULTS: Three pairs of EHEC cultures, each with a curli-expressing and a noncurli-expressing variant (O111:H- 7-57C+ and O157:H7 5-9C-, O157:H7 5-11C+ and 5-11C-, as well as O103:H2 7-52C+ and 7-52C-), were allowed to interact with polystyrene, glass, stainless steel and rubber surfaces at 28 degrees C for 24 h (short-term attachment) or 7 days (long-term attachment). The quantities of the cells that attached to the surfaces were measured daily in the long-term attachment study, and in 4 h intervals in the short-term attachment study. Quantification of the cells that attached to the surfaces was accomplished with a crystal violet binding assay. The results of the long-term attachment study indicated that 7-57C+ attached to the polystyrene and glass surfaces more efficiently (P < 0.05) than did 5-9C-. The curli-expressing variant of 5-11 possessed a better ability to adhere to the polystyrene and glass surfaces than did its noncurli-expressing counterpart (P < 0.05). The differences in attachment between 7-52C+ and 7-52C- on polystyrene and stainless steel surfaces were statistically significant (P < 0.05). However, the attachment of the pair on the glass surfaces was statistically insignificant (P > 0.05). In addition, the two members of all three EHEC pairs attached equally well to rubber surfaces (P > 0.05). In the short-term attachment study, only the pair of 7-52 attached differently on glass and stainless steel surfaces (P < 0.05). CONCLUSIONS: These results suggest that curli could be an important cell surface component to mediate the attachment of some EHEC cells to certain abiotic surfaces. Cell-surface contact time could have a significant influence on EHEC attachment to abiotic surfaces. SIGNIFICANCE AND IMPACT OF THE STUDY: The study signifies a possible role of curli in assisting the cells of EHEC in attaching to food-contact surfaces. It underlines the importance of cleaning and sanitizing food-contact surfaces regularly and thoroughly, and of identifying chemical agents that can effectively remove the attached EHEC cells from these surfaces.     

Adhesion of the marine fouling diatom amphora coffeaeformis to non‐solid gel surfaces

Diatom adhesion to different gel surfaces was tested under different shear conditions, using the fouling marine diatom Amphora coffeaeformis as test organism. Four polymers were selected to obtain a test matrix containing gels with different surface charge as well as different surface energies, viz. agarose, alginate, chitosan and chemically modified polyvinylalcohol (PVA‐SbQ). Three experimental systems were applied to obtain different shear rates. Experimental system 1 consisted of gels cast in a cell culturing well plate for comparing initial adhesion as well as long term biofilm development in the absence of shear. In experimental system 2, microscope slide based test surfaces were tested in aquaria under low shear conditions. A rotating annular biofilm reactor was used to obtain high and controlled shear rates. At high shear rates A. coffeaeformis cells adhered better to the charged polymer gels (alginate and chitosan) than to the low charged polymer gels (agarose and PVA‐SbQ). In the system where shear was absent A. coffeaeformis cells developed a biofilm on agarose equivalent to the charged polymer gels, while adhesion to PVA‐SbQ remained low at all shear rates. It is concluded that non‐solid surfaces did not represent an obstacle to settling and growth of this organism. As observed for solid surfaces, low charge density led to reduced attachment, particularly at high shear.     

Attachment of Vibrio alginolyticus to Glass Surfaces Is Dependent on Swimming Speed

The attachment of Vibrio alginolyticus to glass surfaces was investigated with special reference to the swimming speed due to the polar flagellum. This bacterium has two types of flagella, i.e., one polar flagellum and numerous lateral flagella. The mutant YM4, which possesses only the polar flagellum, showed much faster attachment than the mutant YM18, which does not possess flagella, indicating that the polar flagellum plays an important role. The attachment of YM4 was dependent on Na⁺ concentration and was specifically inhibited by amiloride, an inhibitor of polar flagellum rotation. These results are quite similar to those for swimming speed obtained under the same conditions. Observations with other mutants showed that chemotaxis is not critical and that the flagellum does not act as an appendage for attachment. From these results, it is concluded that the attachment of V. alginolyticus to glass surfaces is dependent on swimming speed.     

Restoring mussel beds in highly dynamic environments by lowering environmental stressors

Restoration of coastal ecosystem engineers that trap sediment and dampen waves has proven to be difficult, especially in the wave‐exposed and eroding areas where they are needed the most. Environmental stressors, such as hydrodynamic stress and predation, can only be overcome if transplanted organisms are able to establish self‐facilitating feedbacks. We investigate if the artificial lowering of multiple environmental stressors can be used to give transplanted juveniles the opportunity to form a self‐sustainable system and thereby increase their long‐term survival on wave‐exposed and eroding shores. We designed a large field experiment using juvenile mussels (Mytilus edulis) as model species on a wave‐exposed tidal flat in the Oosterschelde estuary (the Netherlands). We tested if the environmental stress caused by a high predation pressure and wave‐driven dislodgement could be reduced by a combination of artificial structures such as fences (to exclude predatory crabs), attachment substrates (such as coir‐net or oyster shells), and breakwaters. Despite a low overall mussel survival (29%), we found that under strong hydrodynamic conditions, experimental fences and attachment substrates increased the retention of transplanted mussel seed. However, modification of local hydrodynamic conditions using breakwaters did not improve mussel coverage preservation. Overall, this study highlights the potential of using techniques that lower multiple environmental stressors to create a window of opportunity for establishment in highly dynamic ecosystems.     

The attachment of nematocytes from the primitive invertebrate Hydra to fibronectin is specific and RDG-dependent

The transient attachment of cells to components of the extracellular matrix is an important step in the complex molecular mechanisms involved in amoeboid cell locomotion. We have analyzed the attachment of nematocytes from the freshwater cnidarian Hydra to flbronectin which is a constituent of the mesoglea, the extracellular matrix, of the polyps. The percentage of attaching cells increased gradually in a concentration-dependent manner and reached a plateau value at a fibronectin concentration of 50 μg/ml. Attachment was inhibited by exposure of the fibronectin-coated surfaces to antibodies against the cell binding domain of fibronectin or by incubating the cells with peptides containing the recognition sequence Arg-Gly-Asp (RGD) known from vertebrate cells. This, together with data obtained by affinity chromatography, indicates that RGD-dependent binding to fibronectin, mediated by a receptor which possibly belongs to the integrin family, already occurs in Hydra, a member of an evolutionary low invertebrate phylum.     

<Emphasis Type="Italic">N</Emphasis>-vanillylnonanamide tested as a non-toxic antifoulant,applied to surfaces in a polyurethane coating

The potential on N-vanillylnonanamide (NVN) in preventing the attachment of Pseudomonas stutzeri and a Bacillus cereus-group strain was investigated. NVN up to 852 μM was not toxic, nor was it an energy source for either organism. Microbial attachment assays were carried out on glass and polylysine slides. with NVN being dispersed in or applied to the surfaces using a polyurethane coating. NVN at 205 μM inhibited Bacillus adhesion on glass slides by 48% and the percentage did not significantly increase at 852 μM. NVN blended into or sprayed onto the coating at 205 μmol/kg did not prevent adhesion. The compound is therefore not useful as an antifouling product under the tested coating conditions.     

Recruitment and distribution of Dreissena polymorpha (Bivalvia) on substrates of different shape and orientation

Dreissena polymorpha recruitment on artificial substrates was studied in the Włocławek Dam Reservoir (the Vistula River, Poland). Densities on downstream and upstream vertical surfaces of plastic plates differed significantly from each other, with the former settled by more individuals. Vertical and horizontal plates, as well as upper and lower horizontal surfaces were settled similarly. In another experiment mussels settled on flat, convex and concave glass surfaces, directed upstream or downstream. Among the upstream surfaces, the concave ones were the most densely settled. No significant differences in mussel recruitment on various downstream surfaces were found. Thus, substrate shape influenced mussels only when they were exposed to water flow. Mussels were aggregated (Lloyd index > 1) along all the edges of the horizontal plates and along the upper edge of the vertical ones. Such distribution was probably caused by the post‐settlement movement of metamorphosed individuals. (© 2005 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Effects of zebra mussel attachment on the foraging behaviour of a larval dragonfly,Macromia illinoiensis

1. Larvae of Macromia illinoiensis Walsh are often colonised by the zebra mussel, Dreissena polymorpha Pallas, a recent invader to North America. To determine how mussel attachment affects an individual's foraging behaviour, we quantified capture of Hexagenia limbata Hexes mayfly prey and the distance moved by newly‐molted final instars before and after an individual's colonisation with zebra mussels. 2. In night trials, larvae sprawled above the sand, and caught more mayflies than individuals in daytime trials, but the estimated distance travelled did not differ. When resting under a layer of sand with only its eyes exposed during the day, an individual could capture a mayfly prey using a sit‐and‐wait ambush strategy. When sprawled above the sand, some larvae caught prey that rested on their legs. 3. When mussel‐free, individuals captured more prey than they did when carrying zebra mussels, although mussel attachment per se did not affect the estimated distance that a larva moved. 4. During day trials, but not night ones, the increasing mussel load of colonised individuals decreased prey capture and the distance moved in an apparent step‐wise function. Although the number of mussels carried did not differ, night foragers carried a heavier load. Independent of time of the day, the distance an individual travelled when mussel‐free was predictive of the number of prey it caught when colonised, suggesting that the greater general activity of some individuals helped mitigate negative effects that mussel attachment had on prey capture. 5. Our results add to a growing number of negative effects of zebra mussel colonisation on sprawling and hiding dragonfly larvae. Although the impact of these costs on dragonfly populations remains to be determined, a decrease in this guild of predators whose life cycle spans aquatic and terrestrial habitats might have cascading effects across ecosystems.     

Adhesion of bacteria from mixed cell suspension to solid surfaces

The attachment of four species of bacteria to solid surfaces was investigated to determine whether the attachment of one species of bacterium could be influenced by the presence of other attaching or attached species. Three types of experiment were done: (i) attachment of bacteria from suspensions containing two species (termed simultaneous attachment) was compared to attachment of each species in pure culture, (ii) the attachment of one species of bacterium to surfaces already colonized by a second species (termed sequential attachment) was compared to attachment of the bacteria to clean, uncolonized surfaces, and (iii) bacteria were allowed to attach to a surface already colonized by a second strain, and their effect on the stabilization of adhesion of the initial colonizing strain was determined. The bacteria were Acinetobacter calcoaceticus, a Staphylococcus sp., a coryneform (isolates from a canning factory), and Staphylococcus aureus. The surfaces were tin plate, glass, and nylon. The attachment of each species was either increased, decreased or not affected by the simultaneous or sequential attachment of another species. The results depended upon the species combination, the surface composition, and the sequence of attachment. The detachment of a primary colonizing species was either increased, decreased or not affected by the subsequent attachment of a second species, depending on the species combination and surface. The results demonstrate that bacterial attachment to a surface can be influenced by the composition of the attaching population and can differ considerably from the attachment of the component species in pure culture. This has implications for the control and removal of biofilms in food processing plants, as well as a wider significance for the composition and dynamics of biofilms in industrial and natural environments.Abbreviation PYE Peptone/yeast extract medium     

Attachment force of the beetle Cryptolaemus montrouzieri (Coleoptera, Coccinellidae) on leaflet surfaces of mutants of the pea Pisum sativum (Fabaceae) with regular and reduced wax coverage

This study represents an investigation of surface-related plant–insect interactions. Surface micro-morphology of leaflets in pea (Pisum sativum) with wild-type crystalline surface waxes (waxy) and with reduced crystalline surface waxes (glossy) caused by a mutation (wel) were studied using various microscopy techniques. The free surface energy of these plant surfaces was estimated using contact angles of droplets of three different liquids. The morphological study of the attachment system in the ladybird beetle Cryptolaemus montrouzieri was combined with measurements of attachment (traction) forces, generated by beetles on these plant substrates. Differences were found in wax crystal shape, dimensions, and density between the adaxial and abaxial surfaces of waxy and glossy plants. The crystalline wax was not completely eliminated in the glossy plant: it was only slightly reduced on the adaxial side and underwent greater changes on the abaxial side. The free surface energy for both surfaces of both pea types was rather low with strongly predominating dispersion component. Insects generated low traction forces on all intact plant surfaces studied, except the abaxial surface of the glossy plant, on which the force was greater. After being treated with chloroform, all the surfaces allowed much higher traction forces. It is demonstrated that the difference in the crystal length and density of the epicuticular wax coverage within the observed range did not influence wettability of surfaces, but affected insect attachment. The reduction in insect attachment force on plant surfaces, covered with the crystalline wax, is explained by the decrease of the real contact area between setal tips of beetles and the substrate. Handling editor: Lars Chittka.     

Factors influencing the attachment strength of Dreissena polymorpha (Bivalvia)

Abstract

The effects of several factors (shell length, exposure time, substratum orientation in space, illumination, temperature, conspecifics) upon the attachment strength (measured with a digital dynamometer) of the freshwater, gregarious bivalve Dreissena polymorpha were studied under laboratory conditions. A rapid increase in attachment strength was observed on resocart (a thermosetting polymer based on phenol-formaldehyde resin, with paper as filler) substrata during the first 4-d exposure, after which it stabilised at ca 1 N. The attachment strength increased also with mussel size. Mussel adhesion on variously oriented surfaces (vertical, upper horizontal and lower horizontal) was similar. Illumination inhibited attachment strength, as expected for a photophobic species, but only after a 2-d exposure. After 6 d, no effects of light were detected. Thus, illumination seemed to influence the attachment rate, rather than the final strength. The optimum temperature for mussel attachment was 20 – 25°C. At lower and higher temperatures (5 – 15°C and 30°C), their adhesion strength decreased. The presence of conspecifics stimulated mussel attachment strength.     

Effectiveness of Antifouling Coatings and Water Flow in Controlling Attachment of the Nuisance Mussel Limnoperna fortunei

Various antifouling substrata were tested for their effectiveness in inhibiting attachment of Limnoperna fortunei. Field experiments revealed that surface properties affected the antifouling capabilities of nontoxic substrata. Antifouling capabilities were observed for three silicone resin-based coatings with smooth surfaces (<30 w m roughness) and low surface free energy of the hydrogen bonding force component. A further three silicone resin-based coatings tested, as well as other types of nontoxic coatings, did not show any antifouling capabilities. The percentages of juvenile mussels that attached in laboratory experiments correlated with the settling densities of mussels in the field experiments better than those based on adult mussels. This suggests that laboratory experiments with juveniles may be effective as short term preliminary assays to select promising materials/coatings for longer term field experiments. Mussel abundance surveys within a water transmission pipe and pipe current velocity simulation revealed that less infestation was observed in the areas with a wall-vicinity fluid velocity of >1.3 m s m 1 . Conversely, pipe surfaces with flows of <1.2 m s m 1 were heavily fouled by L. fortunei .     

The impact of the sea anemone Actinothoe sphyrodeta on Mytilus galloprovincialis mussel cultivation (Galicia,Spain)

Abstract

Marine mussel aggregations act as a substratum and refuge for many fouling species. Mussel cultivation in Galicia, Spain, is carried out on hanging ropes in subtidal systems. The fauna associated with this cultivation includes a large number of invertebrates that compete for space or food with the mussels, or use their clusters as a refuge from predators or water turbulence. Outbreaks of the epibiont anemone Actinothoe sphyrodeta have been reported in cultivated Galician mussels since 2013, but their impact has not been investigated rigorously. Here, the temporal and spatial variability of Actinothoe sphyrodeta on mussel shells throughout one year is presented. Sampling of mussel size, weight and byssus attachment strength allowed mussel tenacity (attachment strength relative to size) to be calculated. A higher presence of Actinothoe sphyrodeta correlated with lower mussel tenacity and greater biomass losses, suggesting that this species could be an economically important biofouling component.     

THE ROLE OF NITRIC OXIDE IN DIATOM ADHESION IN RELATION TO SUBSTRATUM PROPERTIES1

Adhesion of raphid diatoms to surfaces, mediated by the secretion of extracellular polymeric substances (EPS), is an important strategy for growth and survival. Diatom biofilms are also important in the context of biofouling. Diatoms exhibit selectivity in adhering to surfaces, but little is understood about how they perceive the properties of a substratum and translate that perception into altered adhesion properties. In this study, we demonstrate that Seminavis robusta Danielidis et D. G. Mann, like many other pennate diatoms, adheres more strongly to hydrophobic surfaces (such as silicone elastomer foul‐release coatings) than to hydrophilic surfaces. To explore the cellular mechanisms that may underlie this selectivity, we tested the hypothesis that diatoms may perceive a hydrophilic surface as unconducive to adhesion through a form of stress response involving nitric oxide (NO) production. Single‐cell imaging with the fluorescent indicator DAF‐FM DA (4‐amino‐5‐methylamino‐2′,7′‐difluorofluorescein diacetate), revealed NO levels that were 4‐fold higher in cells adhered to a hydrophilic surface (acid‐washed glass) compared with a hydrophobic surface (polydimethylsiloxane elastomer, PDMSE). Elevated levels of NO caused by the addition of the NO donor S‐nitroso‐N‐acetylpenicillamine (SNAP) did not affect growth, but cells showed reduced adhesion strength to both glass and PDMSE. Addition of the nitric oxide synthase inhibitor NG‐monomethyl‐l ‐arginine (NMMA) caused a small but significant increase in adhesion strength. Overall, the results suggest that NO acts as a signal of the wettability properties of substrata for Seminavis.