Analysis of the Behaviours Mediating Barnacle Cyprid Reversible Adhesion

When exploring immersed surfaces the cypris larvae of barnacles employ a tenacious and rapidly reversible adhesion mechanism to facilitate their characteristic ‘walking’ behaviour. Although of direct relevance to the fields of marine biofouling and bio-inspired adhesive development, the mechanism of temporary adhesion in cyprids remains poorly understood. Cyprids secrete deposits of a proteinaceous substance during surface attachment and these are often visible as ‘footprints’ on previously explored surfaces. The attachment structures, the antennular discs, of cyprids also present a complex morphology reminiscent of both the hairy appendages used by some terrestrial invertebrates for temporary adhesion and a classic ‘suction cup’. Despite the numerous analytical approaches so-far employed, it has not been possible to resolve conclusively the respective contributions of viscoelastic adhesion via the proteinaceous ‘temporary adhesive’, ‘dry’ adhesion via the cuticular villi present on the disc and the behavioural contribution by the organism. In this study, high-speed photography was used for the first time to capture the behaviour of cyprids at the instant of temporary attachment and detachment. Attachment is facilitated by a constantly sticky disc surface – presumably due to the presence of the proteinaceous temporary adhesive. The tenacity of the resulting bond, however, is mediated behaviourally. For weak attachment the disc is constantly moved on the surface, whereas for a strong attachment the disc is spread out on the surface. Voluntary detachment is by force, requiring twisting or peeling of the bond – seemingly without any more subtle detachment behaviours. Micro-bubbles were observed at the adhesive interface as the cyprid detached, possibly an adaptation for energy dissipation. These observations will allow future work to focus more specifically on the cyprid temporary adhesive proteins, which appear to be fundamental to adhesion, inherently sticky and exquisitely adapted for reversible adhesion underwater.     

Influence of Substratum Wettability on Attachment of Freshwater Bacteria to Solid Surfaces

We studied the attachment of a number of freshwater bacteria from River Sowe, Coventry, England, to test substrata. The attachment of each organism to hydrophobic and hydrophilic surfaces was evaluated, and further studies evaluated the attachment of selected isolates to a number of substrata with a range of water wettabilities. The wettability of each substratum was determined by contact angle measurements and was expressed as the work of adhesion (W_A). No generic pattern of attachment to the test surfaces was found, although the majority of the organisms isolated showed a preference for the hydrophobic surface. A more detailed study of selected isolates showed a relationship between W_A and number of attached cells. Each bacterium attached in maximum numbers to a surface that was characteristic of that organism and that had a W_A between 75 and 105 mJ m⁻².     

Sample Preparation for Single Virion Atomic Force Microscopy and Super-resolution Fluorescence Imaging

Immobilization of virions to glass surfaces is a critical step in single virion imaging. Here we present a technique adopted from single molecule imaging assays which allows adhesion of single virions to glass surfaces with specificity. This preparation is based on grafting the surface of the glass with a mixture of PLL-g-PEG and PLL-g-PEG-Biotin, adding a layer of avidin, and finally creating virion anchors through attachment of biotinylated virus specific antibodies. We have applied this technique across a range of experiments including atomic force microscopy (AFM) and super-resolution fluorescence imaging. This sample preparation method results in a control adhesion of the virions to the surface.     

Mini-review: Barnacle adhesives and adhesion

Barnacles are intriguing, not only with respect to their importance as fouling organisms, but also in terms of the mechanism of underwater adhesion, which provides a platform for biomimetic and bioinspired research. These aspects have prompted questions regarding how adult barnacles attach to surfaces under water. The multidisciplinary and interdisciplinary nature of the studies makes an overview covering all aspects challenging. This mini-review, therefore, attempts to bring together aspects of the adhesion of adult barnacles by looking at the achievements of research focused on both fouling and adhesion. Biological and biochemical studies, which have been motivated mainly by understanding the nature of the adhesion, indicate that the molecular characteristics of barnacle adhesive are unique. However, it is apparent from recent advances in molecular techniques that much remains undiscovered regarding the complex event of underwater attachment. Barnacles attached to silicone-based elastomeric coatings have been studied widely, particularly with respect to fouling-release technology. The fact that barnacles fail to attach tenaciously to silicone coatings, combined with the fact that the mode of attachment to these substrata is different to that for most other materials, indicates that knowledge about the natural mechanism of barnacle attachment is still incomplete. Further research on barnacles will enable a more comprehensive understanding of both the process of attachment and the adhesives used. Results from such studies will have a strong impact on technology aimed at fouling prevention as well as adhesion science and engineering.     

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.     

Vascular smooth muscle cells on polyelectrolyte multilayers: hydrophobicity-directed adhesion and growth

Polyelectrolyte multilayer films were employed to support attachment of cultured rat aortic smooth muscle A7r5 cells. Like smooth muscle cells in vivo, cultured A7r5 cells are capable of converting between a nonmotile "contractile" phenotype and a motile "synthetic" phenotype. Polyelectrolyte films were designed to examine the effect of surface charge and hydrophobicity on cell adhesion, morphology, and motility. The hydrophobic nature and surface charge of different polyelectrolyte films significantly affected A7r5 cell attachment and spreading. In general, hydrophobic polyelectrolyte film surfaces, regardless of formal charge, were found to be more cytophilic than hydrophilic surfaces. On the most hydrophobic surfaces, the A7r5 cells adhered, spread, and exhibited little indication of motility, whereas on the most hydrophilic surfaces, the cells adhered poorly if at all and when present on the surface displayed characteristics of being highly motile. The two surfaces that minimized cell adhesion consisted of two varieties of a diblock copolymer containing hydrophilic poly(ethylene oxide) and a copolymer bearing a zwitterionic group AEDAPS, (3-[2-(acrylamido)-ethyldimethyl ammonio] propane sulfonate). Increasing the proportion of AEDAPS in the copolymer decreased the adhesion of cells to the surface. Cells presented with micropatterns of cytophilic and cytophobic surfaces generated by polymer-on-polymer stamping displayed a surface-dependent cytoskeletal organization and a dramatic preference for adhesion to, and spreading on, the cytophilic surface, demonstrating the utility of polyelectrolyte films in manipulating smooth muscle cell adhesion and behavior.     

The Effects of Leaf Roughness,Surface Free Energy and Work of Adhesion on Leaf Water Drop Adhesion

The adhesion of water droplets to leaves is important in controlling rainfall interception, and affects a variety of hydrological processes. Leaf water drop adhesion (hereinafter, adhesion) depends not only on droplet formulation and parameters but also on the physical (leaf roughness) and physico-chemical (surface free energy, its components, and work-of-adhesion) properties of the leaf surface. We selected 60 plant species from Shaanxi Province, NW China, as experimental materials with the goal of gaining insight into leaf physical and physico-chemical properties in relation to the adhesion of water droplets on leaves. Adhesion covered a wide range of area, from 4.09 to 88.87 g/m² on adaxial surfaces and 0.72 to 93.35 g/m² on abaxial surfaces. Distinct patterns of adhesion were observed among species, between adaxial and abaxial surfaces, and between leaves with wax films and wax crystals. Adhesion decreased as leaf roughness increased (r =  −0.615, p = 0.000), but there were some outliers, such as Salix psammophila and Populus simonii with low roughness and low adhesion, and the abaxial surface of Hyoscyamus pusillus and the adaxial surface of Vitex negundo with high roughness and high adhesion. Meanwhile, adhesion was positively correlated with surface free energy (r = 0.535, p = 0.000), its dispersive component (r = 0.526, p = 0.000), and work of adhesion for water (r = 0.698, p = 0.000). However, a significant power correlation was observed between adhesion and the polar component of surface free energy (p = 0.000). These results indicated that leaf roughness, surface free energy, its components, and work-of-adhesion for water played important roles in hydrological characteristics, especially work-of-adhesion for water.     

Functional demands of dynamic biological adhesion: an integrative approach

Climbing organisms are constantly challenged to make their way rapidly and reliably across varied and often novel terrain. A diversity of morphologically and mechanically disparate attachment strategies have evolved across widely distributed phylogenetic groups to aid legged animals in scaling these surfaces, notable among them some very impressive adhesive pads. Despite the differences between, for example, the dry fibrillar pads of geckos and the smooth, secretion-aided pads of stick insects, I hypothesize that they face similar functional demands in their environment. I outline three broad criteria defining dynamic biological adhesion: reusability, reversibility, and substrate tolerance. Organismal adhesive pads must be able to attach repeatedly without significant decline in performance, detach easily at will, and adhere strongly to the broadest possible range of surfaces in their habitat. A survey of the literature suggests that evidence for these general principles can be found in existing research, but that many gaps remain to be filled. By taking a comparative, integrative approach to biological dynamic adhesion, rather than focusing on a few model organisms, investigators will continue to discover new and interesting attachment strategies in natural systems.     

Bacterial Primary Colonization and Early Succession on Surfaces in Marine Waters as Determined by Amplified rRNA Gene Restriction Analysis and Sequence Analysis of 16S rRNA Genes

The nearly universal colonization of surfaces in marine waters by bacteria and the formation of biofilms and biofouling communities have important implications for ecological function and industrial processes. However, the dynamics of surface attachment and colonization in situ, particularly during the early stages of biofilm establishment, are not well understood. Experimental surfaces that differed in their degrees of hydrophilicity or hydrophobicity were incubated in a salt marsh estuary tidal creek for 24 or 72 h. The organisms colonizing these surfaces were examined by using a cultivation-independent approach, amplified ribosomal DNA restriction analysis. The goals of this study were to assess the diversity of bacterial colonists involved in early succession on a variety of surfaces and to determine the phylogenetic affiliations of the most common early colonists. Substantial differences in the representation of different cloned ribosomal DNA sequences were found when the 24- and 72-h incubations were compared, indicating that some new organisms were recruited and some other organisms were lost. Phylogenetic analyses of the most common sequences recovered showed that the colonists were related to organisms known to inhabit surfaces or particles in marine systems. A total of 22 of the 26 clones sequenced were affiliated with the Roseobacter subgroup of the α subdivision of the division Proteobacteria (α-Proteobacteria), and most of these clones were recovered at a high frequency from all surfaces after 24 or 72 h of incubation. Two clones were affiliated with the Alteromonas group of the γ-Proteobacteria and appeared to be involved only in the very early stages of colonization (within the first 24 h). A comparison of the colonization patterns on the test surfaces indicated that the early bacterial community succession rate and/or direction may be influenced by surface physicochemical properties. However, organisms belonging to the Roseobacter subgroup are ubiquitous and rapid colonizers of surfaces in coastal environments.     

Mechanical factors favoring release from fouling release coatings

For some twenty years the marine coatings industry has been intrigued by polymer surfaces with low adhesion to other materials, especially to the biological glues used by marine organisms. Polymers with fouling release surfaces have been made from sundry materials, and their resistance to marine fouling in both static and dynamic tests has been evaluated in the world's oceans. Although the polymer surface property most frequently correlated with bioadhesion is its critical surface tension (γ(?)), resistance to fouling is also influenced by other bulk and surface properties of the polymer. This paper reviews the types of bonding associated with polymeric materials used in fouling resistant coatings, describes the removal process in terms of fracture mechanics, and discusses the importance of surface energy, elastic modulus and coating thickness in the release of biofoulants.     

Influence of substratum hydration and adsorbed macromolecules on bacterial attachment to surfaces.

The attachment of Pseudomonas fluorescens and an Acinetobacter sp. to hydrogel and polystyrene surfaces was investigated to evaluate the influence of adsorbed water and macromolecules on adhesion. With both organisms, there was a decrease in attachment numbers with increasing water content of the hydrogels. There was also a decrease in attachment with a decrease in water contact angle on untreated, tissue culture and sulfonated polystyrene surfaces; however, the attachment numbers were higher than would be expected on the basis of the hydrogel data. With P. fluorescens, attachment to untreated and tissue culture polystyrene was inhibited by bovine serum albumin, Escherichia coli lipopolysaccharide, and the supernatant from spent medium, both when the conditioning substances were added to the suspension of attaching cells and when they were preadsorbed onto the surfaces. Dextran inhibited attachment only when added to the bacterial suspension. Supernatants from centrifuged natural freshwater samples had no effect. Thus, hydration of a surface and the adsorption of macromolecules can reduce bacterial attachment; however, additional factors relating to the chemical composition of the substratum and polymeric stabilization of suspended cells can affect the adhesion interaction and resultant numbers of attached cells.     

Exopolysaccharide production and attachment strength of bacteria and diatoms on substrates with different surface tensions

Attachment strength and exopolysaccharide (EPS) production of Pseudomonas sp. (bacteria) and the diatom Amphora coffaeformis were studied on six different substrata with surface tensions between 19 and 64.5 mN m^–1. Test panels of the materials were exposed to bacterial cultures between 3 and 120 hours, and to diatom cultures between 48 and 72 hours. Exopolysaccharide production by surface-associated cells was measured using the phenol sulfuric acid method. Attachment studies were run by exposing test panels to laminar flow pressure using a radial flow chamber. Highest EPS production by bacteria and diatoms was recorded on substrata with surface tensions above 30 mN m^–1. Lowest EPS production occurred on substrata between 20 and 25 mN m^–1. Highest EPS production and strongest adhesion was found on polycarbonate (33.5 mN m^–1). Both test organisms improved their attachment strength with exposure time on most materials. However, amounts of produced EPS and improvement of attachment indicated that mechanisms other than polysaccharide production are more important on substrata with low surface tensions (<25 mN m^–1). Simply producing more polysaccharides is not sufficient to overcome weak attachment on materials with low surface tensions. For example, adhesion of Pseudomonas sp. and A. coffaeformis on polytetrafluorethylene/perfluor-copolymer (PFA; 22 mN m^–1). and glass (64.5 mN m^–1. was equally strong although EPS production was much higher on glass than on PFA. This is somewhat surprising for A. coffaeformis because polysaccharide production has been considered the most important attachment mechanism of A. coffaeformis.     

Adhesion of Biodegradative Anaerobic Bacteria to Solid Surfaces

In order to exploit the ability of anaerobic bacteria to degrade certain contaminants for bioremediation of polluted subsurface environments, we need to understand the mechanisms by which such bacteria partition between aqueous and solid phases, as well as the environmental conditions that influence partitioning. We studied four strictly anaerobic bacteria, Desulfomonile tiedjei, Syntrophomonas wolfei, Syntrophobacter wolinii, and Desulfovibrio sp. strain G11, which theoretically together can constitute a tetrachloroethylene- and trichloroethylene-dechlorinating consortium. Adhesion of these organisms was evaluated by microscopic determination of the numbers of cells that attached to glass coverslips exposed to cell suspensions under anaerobic conditions. We studied the effects of the growth phase of the organisms on adhesion, as well as the influence of electrostatic and hydrophobic properties of the substratum. Results indicate that S. wolfei adheres in considerably higher numbers to glass surfaces than the other three organisms. Starvation greatly decreases adhesion of S. wolfei and Desulfovibrio sp. strain G11 but seems to have less of an effect on the adhesion of the other bacteria. The presence of Fe³⁺ on the substratum, which would be electropositive, significantly increased the adhesion of S. wolfei, whereas the presence of silicon hydrophobic groups decreased the numbers of attached cells of all species. Measurements of transport of cells through hydrophobic-interaction and electrostatic-interaction columns indicated that all four species had negatively charged cell surfaces and that D. tiedjei and Desulfovibrio sp. strain G11 possessed some hydrophobic cell surface properties. These findings are an early step toward understanding the dynamic attachment of anaerobic bacteria in anoxic environments.     

Adhesion of anaerobic microorganisms to solid surfaces and the effect of sequential attachment on adhesion characteristics

The attachment of three anaerobic microorganisms, Desulfomonile tiedjei, Syntrophomonas wolfei, and Desulfovibrio sp. strain G11, was investigated to determine if the presence of one species could influence the adhesion of another species to glass surfaces. The results indicated that the numbers and distribution of attached cells of one species could be influenced considerably by the presence of another species and the order in which the test species were exposed to the surface. D. tiedjei was found to detach readily from surfaces when it was not the primary colonizer. The attachment of Desulfovibrio G11 as the primary colonizer appeared to be stabilized by exposure to another test species. Under certain experimental conditions the test organisms formed close associations with each other on the surfaces. These findings demonstrate that the characteristics of anaerobic community biofilms can be determined by both the adhesion characteristics of the individual species and the interactions among those microorganisms.     

Influence of substratum hydration and adsorbed macromolecules on bacterial attachment to surfaces

The attachment of Pseudomonas fluorescens and an Acinetobacter sp. to hydrogel and polystyrene surfaces was investigated to evaluate the influence of adsorbed water and macromolecules on adhesion. With both organisms, there was a decrease in attachment numbers with increasing water content of the hydrogels. There was also a decrease in attachment with a decrease in water contact angle on untreated, tissue culture and sulfonated polystyrene surfaces; however, the attachment numbers were higher than would be expected on the basis of the hydrogel data. With P. fluorescens, attachment to untreated and tissue culture polystyrene was inhibited by bovine serum albumin, Escherichia coli lipopolysaccharide, and the supernatant from spent medium, both when the conditioning substances were added to the suspension of attaching cells and when they were preadsorbed onto the surfaces. Dextran inhibited attachment only when added to the bacterial suspension. Supernatants from centrifuged natural freshwater samples had no effect. Thus, hydration of a surface and the adsorption of macromolecules can reduce bacterial attachment; however, additional factors relating to the chemical composition of the substratum and polymeric stabilization of suspended cells can affect the adhesion interaction and resultant numbers of attached cells.     

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.     

Lateral and Perpendicular Interaction Forces Involved in Mobile and Immobile Adhesion of Microorganisms on Model Solid Surfaces

Gliding and near-surface swimming of microorganisms are described as a mobile form of microbial adhesion that need not necessarily be reversible. It is argued that the reversibility of microbial adhesion depends on the depth of the secondary interaction minimum, calculated from the forces between an organism and a substratum acting in a direction perpendicular to the substratum surface. The mobility of adhering microorganisms depends on lateral interactions between the organisms. On ideally homogeneous and smooth model surfaces, only mobile adhesion occurs because the multibody, lateral interactions are weak compared with the thermal or Brownian motion energy of the organisms. Minor chemical or structural heterogeneities, which exist on all real-life surfaces, yield a lateral interaction on adhering microorganisms. This causes their immobilization, which helps to explain the physicochemical nature of microbial gliding or near-surface swimming. Moreover, these lateral interaction energies are one order of magnitude smaller than the Lifshitz-Van der Waals, electrostatic, and acid-base forces acting perpendicular to substratum surfaces that are responsible for adhesion. Received: 2 April 1998 / Accepted: 26 May 1998     

Adhesion of anaerobic microorganisms to solid surfaces and the effect of sequential attachment on adhesion characteristics

The attachment of three anaerobic microorganisms, Desulfomonile tiedjei, Syntrophomonas wolfei, and Desulfovibrio sp. strain G11, was investigated to determine if the presence of one species could influence the adhesion of another species to glass surfaces. The results indicated that the numbers and distribution of attached cells of one species could be influenced considerably by the presence of another species and the order in which the test species were exposed to the surface. D. tiedjei was found to detach readily from surfaces when it was not the primary colonizer. The attachment of Desulfovibrio G11 as the primary colonizer appeared to be stabilized by exposure to another test species. Under certain experimental conditions the test organisms formed close associations with each other on the surfaces. These findings demonstrate that the characteristics of anaerobic community biofilms can be determined by both the adhesion characteristics of the individual species and the interactions among those microorganisms.     

Characterization of spore surfaces from a Geobacillus sp. isolate by pH dependence of surface charge and infrared spectra

Aims: The surfaces of spores from a Geobacillus sp. isolated from a milk powder production line were examined to obtain fundamental information relevant to bacterial spore adhesion to materials. Materials and Results: The surfaces of spores were characterized using transmission electron microscopy and infrared spectroscopy. Thin sections of spores stained with ruthenium red revealed an exosporium with a hair‐like nap around the spores. Attenuated total reflection infrared spectra of the spores exposed to different pH solutions on a ZnSe prism revealed that pH‐sensitive carboxyl and phosphodiester groups associated with proteins and polysaccharides contributed to the spore’s negative charge which was revealed by our previous zeta potential measurements on the spores. Lowering the pH to the isoelectric point of spores resulted in an increase in intensity of all spectral bands, indicating that the spores moved closer to the zinc selenide (ZnSe) surface as the charged surface groups were neutralized and the spore surface polymers compressed. The attachment of spores to stainless steel was threefold higher at pH 3 compared with pH 7. Conclusions: This research showed that spore attachment to surfaces is influenced by electrostatic interactions, surface polymer conformation and associated steric interactions. Significance and Impact of the Study: The adhesion of thermophilic spores is largely controlled by functional groups of surface polymers and polymer conformation.     

Marine antifouling laboratory bioassays: an overview of their diversity

In aquatic environments, biofouling is a natural process of colonization of submerged surfaces, either living or artificial, involving a wide range of organisms from bacteria to invertebrates. Antifouling can be defined as preventing the attachment of organisms onto surfaces. This article reviews the laboratory bioassays that have been developed for studying the control of algae and invertebrates by epibiosis (chemical ecology) and the screening of new active compounds (natural products and biocides) to inhibit settlement or adhesion, ie fouling-release coatings. The assays utilize a range of organisms (mainly marine bacteria, diatoms, algae, barnacles). The main attributes of assays for micro- and macroorganisms are described in terms of their main characteristics and depending on the biological process assessed (growth, adhesion, toxicity, behavior). The validation of bioassays is also discussed.