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.     

Protein- and Metal-dependent Interactions of a Prominent Protein in Mussel Adhesive Plaques

The adhesive plaques of Mytilus byssus are investigated increasingly to determine the molecular requirements for wet adhesion. Mfp-2 is the most abundant protein in the plaques, but little is known about its function. Analysis of Mfp-2 films using the surface forces apparatus detected no interaction between films or between a film and bare mica; however, addition of Ca²⁺ and Fe³⁺ induced significant reversible bridging (work of adhesion W_ad ≈ 0.3 mJ/m² to 2.2 mJ/m²) between two films at 0.35 m salinity. The strongest observed Fe³⁺-mediated bridging approaches the adhesion of oriented avidin-biotin complexes. Raman microscopy of plaque sections supports the co-localization of Mfp-2 and iron, which interact by forming bis- or tris-DOPA-iron complexes. Mfp-2 adhered strongly to Mfp-5, a DOPA-rich interfacial adhesive protein, but not to another interfacial protein, Mfp-3, which may in fact displace Mfp-2 from mica. In the presence of metal ions or Mfp-5, Mfp-2 adhesion was fully reversible. These results suggest that plaque cohesiveness depends on Mfp-2 complexation of metal ions, particularly Fe³⁺ and also by Mfp-2 interaction with Mfp-5 at the plaque-substratum interface.     

Spatial distribution of proteins in the quagga mussel adhesive apparatus

The invasive freshwater mollusc Dreissena bugensis (quagga mussel) sticks to underwater surfaces via a proteinacious ‘anchor’ (byssus), consisting of a series of threads linked to adhesive plaques. This adhesion results in the biofouling of crucial underwater industry infrastructure, yet little is known about the proteins responsible for the adhesion. Here the identification of byssal proteins extracted from freshly secreted byssal material is described. Several new byssal proteins were observed by gel electrophoresis. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry was used to characterize proteins in different regions of the byssus, particularly those localized to the adhesive interface. Byssal plaques and threads contain in common a range of low molecular weight proteins, while several proteins with higher mass were observed only in the plaque. At the adhesive interface, a plaque-specific ~8.1 kDa protein had a relative increase in signal intensity compared to the bulk of the plaque, suggesting it may play a direct role in adhesion.     

Mapping chemical gradients within and along a fibrous structural tissue, mussel byssal threads

The byssal thread of a mussel is an extraorganismic connective tissue that exhibits a striking end-to-end gradient in mechanical properties and thus provides a unique opportunity for studying how gradients are made. Mfp-1 (Mytilus foot protein-1) is a conspicuous component of the protective outer cuticle of byssal threads given its high 3,4-dihydroxyphenylalanine (Dopa) content at 10-15 mol %. Amino acid analysis of mfp-1 extracted from successive foot sections of Mytilus galloprovincialis reveals a post-translationally mediated gradient with highest Dopa levels present in mfp-1 from the accessory gland near the tip of the foot decreasing gradually toward the base. The Dopa content of successive segments of byssal threads decreases from the distal to the proximal end and thus reflects the trend of mfp-1 in the foot. Inductively coupled plasma analysis indicates that certain metal ions including iron follow the trend in Dopa along the thread. Energy-dispersive x-ray spectrometry showed that iron, when present, was concentrated in the cuticle of the threads but sparse in the core. The axial iron gradient appears most closely correlated with the Dopa gradient. The direct incubation of mussels and byssal threads in Fe(3+) supplemented seawater showed that byssal threads are unable to sequester iron from the seawater. Instead, particulate/soluble iron is actively taken up by mussels during filter feeding and incorporated into byssal threads during their secretion. Our results suggest that mussels may exploit the interplay between Dopa and metals to tailor the different parts of threads for specific mechanical properties.     

Probing the adhesive footprints of Mytilus californianus byssus

California mussels Mytilus californianus owe their tenacity to a holdfast known as the byssus, a fibrous extracellular structure that ends distally in flattened adhesive plaques. The "footprints" of freshly secreted plaques deposited onto glass coverslips were shown by matrix-assisted laser desorption ionization time of flight mass spectrometry to consist chiefly of proteins ranging in mass from 5200 to 6700 Da. These proteins, variants of a family known as mcfp3 (M. californianus foot protein 3), were purified from acetic acid/urea extracts of plaques and foot tissue. Mcfp3 appears to sort into fast and slow electrophoretic variants. Both are rich in Gly and Asn and exhibit post-translational hydroxylation of Tyr and Arg to Dopa and 4-hydroxyarginine, respectively, with the fast variant containing more than twice as much Lys + Arg. Both the slow and fast variants were partially sequenced from the N terminus, and the complete sequences of 12 variants were deduced from cDNA using degenerate oligonucleotides, PCR, and rapid amplification of cDNA ends. Mcfp3s are highly polar molecules and contain up to 28 mol % Dopa, which remains intact and may be crucial for adhesion to metal and mineral surfaces.     

The effects of natural biofilms on the reattachment of young adult zebra mussels to artificial substrata

This laboratory study examined the effects of natural biofilms on the reattachment of young adult zebra mussels, Dreissena polymorpha, in Petri dishes. Natural biofilms were developed in glass and polystyrene Petri dishes using water samples collected at various times of the year. Biofilms were developed over 1, 3, 8, and 14 d. Controls were clean glass and polystyrene Petri dishes. Zebra mussels collected from the field (< or = 10 mm, ventral length) were placed in the dishes and their reattachment by byssal threads was recorded after 1 d. Zebra mussels reattached to the dish surface or the shells of other mussels in the dish, or remained unattached. The data indicate that reattachment to clean glass was greater than to clean polystyrene (p < or = 0.05, ANOVA), but there were no consistent differences between reattachment to filmed polystyrene and filmed glass dish surfaces. Zebra mussels in control and filmed glass dishes reattached in higher percentages to the dish surface compared to the shells of other mussels (p < or = 0.05, ANOVA). There was no difference in mussel of reattachment between the dish surface and the shells of other mussels in most control polystyrene dishes (p > 0.05, ANOVA), whereas in filmed polystyrene the percentage of reattachment to the dish surface was greater than to the shells of other mussels (p < or = 0.05, ANOVA). These results indicate that substratum wettability and the presence of biofilms on some types of substrata can be factors in the reattachment of young adult zebra mussels.     

Predatory blue crabs induce byssal thread production in hooked mussels

Abstract. Blue crabs (Callinectes sapidus) prey on hooked mussels (Ischadium recurvum) growing epizoically on oyster clumps in estuaries along the Louisiana coast. In prey size‐selection experiments, blue crabs preferred small mussels (<30‐mm shell length) to larger mussels, possibly because handling time increased with mussel size. When crabs were given a choice of solitary mussels versus mussels in clumps on oysters in the laboratory, mortality was lower by 86% in clumped mussels. However, no size selection by crabs occurred with mussels in clumps, likely because smaller mussels escaped predation in crevices between larger mussels or oysters. When individuals of two size classes of mussels were exposed to water containing the scent of crabs and of mussels consumed by blue crabs, an increase in byssal thread production was induced in all mussels, but byssal thread production rate was higher for small mussels than for large mussels. We conclude that increased predation risk for small mussels has resulted in higher size‐specific production of byssal threads, and that predator‐induced production of byssal threads, which may increase clumping behavior, may reduce their risk of mortality to predatory blue crabs.     

Linking adhesive and structural proteins in the attachment plaque of Mytilus californianus

The byssal attachment of California mussels Mytilus californianus provides secure adhesion in the presence of moisture, a feat that still eludes most synthetic polymers. Matrix-assisted laser desorption ionization mass spectrometry was used to probe the footprints of byssal attachment plaques on glass cover slips for adhesive proteins. Besides the abundant mcfp-3 protein family (Zhao, H., Robertson, N. B., Jewhurst, S. A., and Waite, J. H. (2006) J. Biol. Chem. 281, 11090-11096), two new proteins, mcfp-5 and mcfp-6, with masses of 8.9 kDa and 11.6 kDa, respectively, were identified in footprints, partially characterized and completely sequenced from a cDNA library. mcfp-5 resembles mcfp-3 in its basic pI and abundant 3,4-dihydroxyphenyl-L-alanine (Dopa; 30 mol %), but is distinct in two respects: it is more homogeneous in primary sequence and is polyphosphorylated. mcfp-6 is basic and contains a small amount of Dopa (<5 mol %). In contrast to mcfp-3 and -5, tyrosine prevails at 20 mol %, and cysteine is present at 11 mol %, one-third of which remains thiolate. Given the oxidative instability of Dopa and cysteine at pH 8.2 (seawater), we tested the hypothesis that thiols serve to scavenge dopaquinones by adduct formation. Plaque footprints were hydrolyzed and screened for cysteine dopaquinone adducts using phenylboronate affinity chromatography. 5-S-Cysteinyldopa was detected at nearly 1 mol %. The results suggest that mcfp-6 may provide a cohesive link between the surface-coupling Dopa-rich proteins and the bulk of the plaque proteins.     

Inhibition of the reattachment of young adult zebra mussels by single-species biofilms and associated exopolymers

AIMS: To determine the effects of single-species bacterial films and their associated extracellular products on the reattachment of young adult zebra mussels. MATERIALS AND RESULTS: Ten strains of bacteria were isolated from surfaces where adult zebra mussels can be found attached in nature. Single-species biofilms were developed on both glass and polystyrene using these bacteria. The reattachment of zebra mussels (i.e. with byssal threads) was compared between surfaces with and without films. Although no differences were observed in mussel reattachment between glass surfaces with and without films (P > 0.05, anova), a reduction in mussel reattachment between polystyrene surfaces with and without films was observed for seven of the 10 strains (P < or = 0.05 to <0.001, anova). Bacterial extracellular products (BEP) were isolated from five bacterial films and tested for their effects on mussel reattachment. Four of the five sets of isolated extracellular products evoked the same effects as their respective intact biofilms. CONCLUSIONS: We conclude that depending on the substratum, individual strains of bacteria in biofilms can inhibit the reattachment of adult zebra mussels. In some cases, BEP were the source of the inhibitory effects. SIGNIFICANCE AND IMPACT OF THE STUDY: The nature of the substratum on which the biofilms develop affects properties of the biofilm and its extracellular components, which subsequently influences zebra mussel reattachment.     

Byssogenesis in the juvenile pink heelsplitter mussel,Potamilus alatus (Bivalvia: Unionidae)

The North American pink heelsplitter (Potamilus alatus) differs from most freshwater mussels in China by the ability to secrete an ephemeral byssus during its juvenile stage. In the present study, light microscopy, scanning electron microscopy (SEM), and transmission electron microscopy (TEM) were used to investigate this ephemeral byssal structure, and amino acid composition was analyzed and compared with that of other species. The results revealed that the byssus consists of a long byssal thread and a few adhesive plaques which are randomly set up along the thread and assembled by petioles. There is a thin but distinctive cuticle with a continuous homogeneous matrix surrounding the byssal thread. Structural variation occurred when the byssal thread was differentially stretched. Four‐stranded helical primary fasciculi, which form a stable rope‐like structure, become evident after removal of the cuticle. The primary fasciculi consist of bundles of hundreds of parallel secondary fasciculi, each measuring about 5 μm in diameter. All evidence indicates that the byssus of the pink heelsplitter has a significantly different macrostructure and microstructure than the permanent byssus of the marine mussel Mytilus. Byssogenesis ceases when juveniles exceed 30 mm in length, although it varies greatly even among juveniles of similar size. Byssus formation is influenced by substrate type. The unique characteristics of the byssus have important advantages for survival, transition, and aggregation during the early life history. This study not only provides first insight into the structure of the ephemeral byssus and its relationship to freshwater mussel development and growth, but also suggests possibilities for the synthesis of novel biopolymer materials particularly useful in freshwater ecosystems. J. Morphol. 276:1273–1282, 2015. © 2015 Wiley Periodicals, Inc.     

Byssal proteins of the freshwater zebra mussel,Dreissena polymorpha

The freshwater zebra mussel (Dreissena polymorpha) is a notorious biofouling organism. It adheres to a variety of substrata underwater by means of a proteinaceous structure called the byssus, which consists of a number of threads with adhesive plaques at the tips. The byssal proteins are difficult to characterize due to extensive cross-linking of 3,4-dihydroxyphenylalanine (DOPA), which renders the mature structure largely resistant to protein extraction and immunolocalization. By inducing secretion of fresh threads and plaques in which cross-linking is minimized, three novel zebra mussel byssal proteins were identified following extraction and separation by gel electrophoresis. Peptide fragment fingerprinting was used to match tryptic digests of several gel bands against a cDNA library of genes expressed uniquely in the mussel foot, the organ which secretes the byssus. This allowed identification of a more complete sequence of Dpfp2 (D. polymorpha foot protein 2), a known DOPA-containing byssal protein, and a partial sequence of Dpfp5, a novel protein with several typical characteristics of mussel adhesive proteins.     

Purification, cDNA clone and recombinant expression of foot protein-3 from Mytilus coruscus

Mussels Mytilus coruscus can adhere to various solid surface in the presence of moisture. Mussel foot protein-3 (mfp-3) has been suggested as the main adhesive protein in the plaques closest to the adhesion interface and been the focus of substantial biomaterials development research within the last decade. The byssal plaques of M. coruscus were accumulated and variants of a family known as mcofp3 (Mytilus coruscus foot protein 3) were purified from acetic acid/urea extracts of plaques, with their N-terminal sequences determined thereafter. The cDNA sequence coding for the mcofp3 precursor was obtained from M. coruscus foot cDNA library. These precursors contain a putative signal peptide of 24 residues, a mature peptide sequence of 41-56 amino acids rich in Tyr, Gly, Pro, and Asn. The recombinant mcofp3 fused with a hexa-histidine affinity ligand was successfully expressed through an Escherichia coli expression system, and the recombinant mcofp3 was purified using affinity chromatography followed by reverse phase high performance liquid chromatography (HPLC). The DOPA content and adhesive properties of purified recombinant mcofp3 with or without tyrosinase modification were compared with the native mcofp3. These assays showed that recombinant mcofp3 has significant adhesive ability and may be useful as a bioadhesive in medical or underwater environments.     

Mini-review: The role of redox in Dopa-mediated marine adhesion

3, 4-Dihydroxyphenylanine (Dopa)-containing proteins are key to wet adhesion in mussels and possibly other sessile organisms also. However, Dopa-mediated adhesive bonding is a hard act to follow in that, at least in mussels, bonding depends on Dopa in both reduced and oxidized forms, for adhesion and cohesion, respectively. Given the vulnerability of Dopa to spontaneous oxidation, the most significant challenge to using it in practical adhesion is controlling Dopa redox in a temporally- and spatially defined manner. Mussels appear to achieve such control in their byssal attachment plaques, and factors involved in redox control can be measured with precision using redox probes such as the diphenylpicryl hydrazyl (DPPH) free radical. Understanding the specifics of natural redox control may provide fundamentally important insights for adhesive polymer engineering and antifouling strategies.     

Effects of Environmental Factors on Byssal Thread Formation in Some Members of the Family Mytilidae from the Sea of Japan

The ability to attach repeatedly to a substrate (glass, boulders, sand) in three common mussel species of the upper sublittoral zone of the Sea of Japan, Grayan's mussel Crenomytilus grayanus, the Korean mussel Mytilus coruscus, and northern horse mussel Modiolus modiolus, was studied under experimental conditions. It was found that during 120 h of the experiment C. grayanus and M. modiolus produced more byssal threads than M. coruscus. A decrease in the water temperature from 20 to 0°C slowed the rate of production of byssal threads down to full passivity in some experimental mollusks. This was more typical of M. coruscus and less typical of C. grayanus. Renewed threads differed in their length, thick, size of the adhesive plate, and strength. M. coruscus formed the shortest, thickest, and strongest threads with rather a large adhesive disk. The observed differences are discussed from the position of morphophysiological adaptations of species for colonization of different natural substrata under contrasting conditions of the upper sublittoral zone.     

Mini-review: The role of redox in Dopa-mediated marine adhesion

3, 4-Dihydroxyphenylanine (Dopa)-containing proteins are key to wet adhesion in mussels and possibly other sessile organisms also. However, Dopa-mediated adhesive bonding is a hard act to follow in that, at least in mussels, bonding depends on Dopa in both reduced and oxidized forms, for adhesion and cohesion, respectively. Given the vulnerability of Dopa to spontaneous oxidation, the most significant challenge to using it in practical adhesion is controlling Dopa redox in a temporally- and spatially defined manner. Mussels appear to achieve such control in their byssal attachment plaques, and factors involved in redox control can be measured with precision using redox probes such as the diphenylpicryl hydrazyl (DPPH) free radical. Understanding the specifics of natural redox control may provide fundamentally important insights for adhesive polymer engineering and antifouling strategies.     

Rotational echo double resonance detection of cross-links formed in mussel byssus under high-flow stress.

13C2H rotational echo double resonance NMR has been used to provide the first evidence for the formation of quinone-derived cross-links in mussel byssal plaques. Labeling of byssus was achieved by allowing mussels to filter feed from seawater containing L-[phenol-4-13C]tyrosine and L-[ring-d4]tyrosine for 2 days. Plaques and threads were harvested from two groups of mussels over a period of 28 days. One group was maintained in stationary water while the other was exposed to turbulent flow at 20 cm/s. The flow-stressed byssal plaques exhibited significantly enhanced levels of 5, 5'-di-dihydroxyphenylalanine cross-links. The average concentration of di-dihydroxyphenylalanine cross-links in byssal plaques is 1 per 1800 total protein amino acid residues.     

Effects of artificial epibionts on byssogenesis,attachment strength,and movement in two size classes of the blue mussel,Mytilus edulis

Blue mussels (Mytilus edulis) can alter the strength of byssal attachment and move between and within mussel aggregations on wave‐swept shores, but this movement ability may be limited by epibiont fouling. We quantified the effects of artificial epibiont fouling on the production of byssal threads, attachment strength, and movement in two size classes of blue mussels. In a factorial experiment, large epibiont‐covered mussels produced more functional byssal threads (i.e., those continuous from animal to substrate) after 24 h than large unfouled and small fouled mussels, but not more than small unfouled mussels. Small unfouled mussels formed and released more byssus bundles compared to any other treatment group, which indicates increased movement. Conversely, epibiont fouling resulted in decreased numbers of byssus bundles shed, and therefore reduced movement in small mussels. Epibiont‐covered mussels started producing byssal threads sooner than unfouled mussels, while small mussels began producing byssal threads earlier compared to large mussels. Mean attachment strength from both size classes increased by 9.5% when mussels were artificially fouled, and large mussels had a 34% stronger attachment compared to small mussels. On the other hand, a 2.3% decrease in attachment strength was found with increasing byssus bundles shed. Our results suggest that fouling by artificial epibionts influences byssal thread production and attachment strength in large mussels, whereas epibionts on small mussels impact their ability to move. Mussels are able to respond rapidly to fouling, which carries implications for the dynamics of mussel beds in their intertidal and subtidal habitats, especially in relation to movement of mussels within and among aggregations.     

Clumping behavior and byssus production as strategies for substrate competition in Mytilus edulis

Laboratory experiments showed that the mussel Mytilus edulis aggregated more intensely around living organisms (the bivalve Hiatella arctica and the solitary ascidian Styela rustica, which commonly co‐occur with mussels in fouling communities) than around inanimate objects. When exposed to an inanimate object, mussels attached their byssal threads primarily to the substrate, close to the object, but when exposed to a living organism, they attached their byssal threads directly to the organism. The ascidian was more intensely covered with byssal threads than was the bivalve. Mussel attachment to the ascidians was apparently determined by the physical characteristics of the tunic and to a lesser extent by the excretion‐secretion products released by S. rustica. This study indicates that mussels can use byssus threads as a means of entrapment of potential competitors for space. It remains unclear why mussels preferentially attached to ascidians compared to the bivalve. This can be explained either by competitive interactions, or by attractiveness of the ascidian tunic as an attachment substratum.     

The effect of food availability on byssogenesis by the zebra mussel (Dreissena polymorpha Pallas)

The effect of varying algal availability on byssal thread productionby re–attaching zebra mussels (Dreissena polymorpha) wasquantified. The byssal apparatus was severed and mussels allowedto re–attach to a hard substratum for a 21 day periodduring which they were fed at algal concentrations of 0.0, 0.1,0.5, 1.0 or 2.0 mg C l^-1. Byssal thread production was quantifiedby counting the number of new attachment plaques present eachday. Results showed that starved mussels continued to partition organiccarbon towards thread production but the resultant byssal masswas compromised, containing fewer threads than those producedby fed mussels. The daily average byssal thread production bymussels fed at 2.0 mg C l^-1 was greater than that of starved musselsand the final mean dry soft tissue weight higher. At algal Cconcentrations below maintenance requirements byssal threadproduction was elevated compared to starved mussels, but therewas no concurrent increase in soft tissue. This suggests thatbelow maintenance levels assimilated carbon was solely partitionedtowards byssus production and stored reserves may have beenutilized. The ratio of organic carbon contained in the byssusto that in the soft tissues remained relatively constant acrossall feeding levels. This suggests that the carbon content ofthe byssus is a constant function of that of the soft tissuemass. These results may explain seasonal variation in attachmentstrength of numerous byssate species and seasonal vertical migrationsby D. polymorpha. Present Address-Queen Mary & Westfield College, Dept of Biology,University of London, Mile End Road, London E1 4NS, UK (Received 16 March 1998; accepted 30 September 1998)     

Reduced byssal thread production and movement by the intertidal mussel<Emphasis Type="Italic"> Hormomya mutabilis</Emphasis> in response to effluent from predators

Laboratory experiments were carried out to investigate byssal thread production by the intertidal mytilid mussel Hormomya mutabilis in response to effluent from the predatory crab Eriphia smithii and the starfish Coscinasterias acutispina. During the early period of the experiment, large H. mutabilis exposed to crab effluent produced a significantly smaller number of functional byssal threads than mussels in crab-free water. No significant difference in the diameter of threads produced in the two treatments was detected. The number of functional byssal threads produced by small H. mutabilis exposed to crab effluent did not differ significantly from that of mussels in crab-free water. However, small H. mutabilis exposed to crab effluent tended to discard fewer byssal bundles, that is, they shifted their attaching sites less frequently than similar mussels in crab-free water. In the presence of waterborne cues from the crab, H. mutabilis tended to reduce both the secretion of byssal threads and movement across the substratum. No significant differences in behaviour were observed between large mussels exposed to effluent from the starfish and those unexposed. The adaptive significance of the responses shown by H. mutabilis is discussed in terms of protection against predators differing in foraging behaviour. Electronic Publication