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.     

Interspecific comparison of the mechanical properties of mussel byssus

Byssally tethered mussels are found in a variety of habitats, including rocky intertidal, salt marsh, subtidal, and hydrothermal vents. One key to the survival of mussels in these communities is a secure attachment, achieved by the production of byssal threads. Although many studies have detailed the unique biomechanical properties of byssal threads, only a few prevalent species have been examined. This study assesses the variation in the mechanical properties of byssus in a broad range of mussel species from diverse environments, including intertidal and subtidal Mytilus edulis, Modiolus modiolus, Geukensia demissa, Bathymodiolus thermophilus, and Dreissena polymorpha. A tensometer was used to measure quasi-static and dynamic mechanical properties of individual threads, and several aspects of morphology were quantified. The results indicate that thread mechanical properties vary among mussel species, and several novel properties were observed. For example, of the species examined, D. polymorpha threads were the strongest, stiffest, least resilient, and fastest to recover after partial deformation. Threads of M. modiolus were characterized by the presence of two distinct yield regions prior to tensile failure. This comparative study not only provides insight into the ecological limitations and evolution of mussels, but also suggests new models for the design of novel biomimetic polymers.     

Mussel collagen molecules with silk-like domains as load-bearing elements in distal byssal threads

Mechanically stressed biological materials like tendon, spider silk or mussel byssal threads are typically composite materials comprising multi-domain proteins, in which molecular building blocks contribute to overall material function. Mussel byssal threads are the anchorage of sessile mytilid mussels, which withstand recurring external loads from waves and tides. A single thread is elastic and ductile proximally, while the distal portion exhibits an extraordinary stiffness and toughness with a transient gradient of both mechanical features along the thread. The main components of byssal threads include a set of various collagen-like structural proteins (preCols) consisting of a collagenous core sequence flanked by globular domains. Here, structural analysis using polarized Fourier-transform infrared spectroscopy (FTIR) on stretched distal portions of mussel byssal threads determines the impact of external linear load on various molecular moieties. It is concluded that the preCol collagenous core domain is the main load-bearing element in distal byssal threads, while polyalanine beta-sheets in the flanking domains, similar to those found in spider silk proteins, provide high stiffness at low strains. Load dissipation is mediated by domain stretching of amorphous glycine-rich helical moieties followed by complete unfolding of the preCol flanking domains.     

Oxidative stress and the mechanical properties of naturally occurring chimeric collagen-containing fibers.

The byssal threads of marine mussels are a fiber-reinforced composite material. Fibers are continuous, separated by matrix, and consist of chimeric collagens that encompass within the same primary protein structure domains corresponding to collagen, polyhistidine, and either elastin or dragline spider silk. The elastic modulus (stiffness) of the proximal portion of byssal threads was measured by cyclic stress-strain analysis at 50% extension. Before measurement, the threads were conditioned by various treatments, particularly agitation in aerated or nitrogen-sparged seawater. Stiffness can be permanently increased by more than two times, e.g., from 25 MPa to a maximum of 65 MPa, by simple agitation in aerated seawater. Much but not all of this stiffening can be prevented by agitation under nitrogen. Reversible strain stiffening would seem to be a useful adaptation to lower residual stresses arising from the deformation of two joined materials, i.e., distal and proximal portions with rather different elastic moduli. The permanent strain stiffening that characterizes proximal byssal threads subjected to oxidative stress is probably due to protein cross-linking. In the short term, this results in a stronger thread but at the expense of dynamic interactions between the molecules in the structure.     

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.     

Protein gradients in byssal threads of some marine bivalve molluscs

Many marine bivalve molluscs produce byssal threads for attachment to solid substrata. Small (less than 10 mm) consecutive sections of the byssal threads of Mytilus edulis, M. californianus, Geukensia demissa, Atrina vexillum, and A. rigida were analyzed by amino acid analysis to determine if chemical composition remains constant as a function of location in thread segments. Nonlinear longitudinal protein gradients, probably involving collagen and an elastic protein, were found in the Mytilus species. In these, collagen peaks in the distal third of the thread. In Geukensia and the Atrina species, although the two differed greatly in composition, there is a clear nonvariability in composition of the thread within each species as a function of location in the thread. The adhesive plaque at the tip of the thread of all species examined differs substantially in composition from the remainder of the thread. Protein gradients in the threads of some bivalves may reflect specific adaptations evolved to respond to exposed habitats in high-energy environments.     

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.     

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     

基于illumina测序的厚壳贻贝足组织转录组研究

贻贝足丝是贻贝足组织分泌的足丝蛋白形成的非细胞组织,具有在水环境下的极强粘附性能,是当前生物粘附剂及抗腐蚀材料的研发热点．为进一步了解贻贝足丝蛋白的分子多样性特征,采用新一代Illumina高通量测序平台对厚壳贻贝(Mytilus coruscus)足组织进行转录组测序,首次构建了厚壳贻贝足组织的转录组数据库．共计获得7 199 799 840 nt的碱基数据经过序列拼接和组装,获得88 825条unigene．对上述unigene开展了序列注释,共计37 007条unigene获得注释．在此基础上,经序列检索和比对,从中筛选出与目前已知的11种足丝蛋白同源的56条unigene序列并进行分析．结果表明,厚壳贻贝足丝蛋白具有明显的氨基酸偏好性,部分足丝蛋白具有重复序列,且厚壳贻贝足丝蛋白与其他种类的贻贝足丝蛋白具有较高的序列相似性．上述结果为后续贻贝足丝蛋白的批量鉴定以及在此基础上的贻贝足丝形成、固化以及粘附机制相关研究奠定了基础．     

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.     

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.     

Collagen-binding matrix proteins from elastomeric extraorganismic byssal fibers

The byssal threads of marine mussels represent a peculiar case of extraorganismic extracellular material. The threads consist of fibrous chimeric collagens such as preCol-P (with collagenous, elastin-like and histidine-rich domains) embedded in a microfibrillar matrix. We report here on the extraction, purification, and characterization of water-soluble proximal thread matrix protein 1 (PTMP1), which is preferentially located in the proximal portion of each byssal thread and decreases in a proximal to distal direction. PTMP1 has a mass of about 50 kDa as determined by matrix-assisted laser desorption-ionization with time-of-flight (MALDI-TOF) mass spectrometry. Glycine is the most common residue at 12.2 mol %, followed by asparagine/aspartic acid and glutamine/glutamic acid at 11.4 and 9.9 mol %, respectively. Glycosylation has been detected by Western blotting with biotinylated concanavalin A and neutral sugar analysis. With degenerate primers designed from the N-terminal sequence and an additional internal peptide derived by Lys-C endopeptidase digestion, a complete cDNA sequence for this protein was obtained by polymerase chain reaction (PCR) amplification of a Mytilus edulis foot cDNA library. Two variants with minor sequence differences limited to the N-terminus were found. The cDNA-deduced protein sequence reveals two symmetric internal repeats that together account for >85% of the protein. Sequence and epitope similarity of PTMP1 to the A domains of von Willebrand factor and integrin alpha(1)I suggest a capacity for collagen binding. Enzyme-linked immunosorbent assay (ELISA)-based measurement of PTMP1 binding to immobilized type I collagen shows high affinity (apparent K(D) = 0.25 microM), but the binding exhibits no dependence on metals. Using primers designed from M. edulis, we also found a PTMP1-like cDNA in a related species, M. galloprovincialis, with a deduced protein sequence having 97% identity with one M. edulis variant and 99% identity with the other. The corresponding cDNA sequences have 94% and 96% identity, respectively.     

Attachment strength of an adhesive nuisance mussel,limnoperna fortunei,against water flow

The attachment strength of the freshwater mussel Limnoperna fortunei against water flow was studied. Newton's expression successfully described the hydrodynamic drag force acting on the mussel with a drag coefficient value of 1.03. The drag‐resistant force (defined as hydrodynamic drag force at mussel detachment) was smaller than the detachment force measured using a tensile load test. A fairly good correlation was obtained between the drag‐resistant force and the number of secreted threads. The drag‐resistant force divided by the number of threads increased with shell size, suggesting that byssal thread strength increased with mussel growth. For the mussel specimens obtained from a water transmission pipe, thread width increased with shell size. However, thread width was not dependent on current velocity. There was no correlation between the number of secreted threads and shell length, which indicated that the number of secreted threads did not change with mussel size. Therefore, the water velocity needed to detach mussels increases with shell size of the mussel when the number of secreted threads is constant. The increases in the water velocity to detach mussels with larger shells suggests that the mussel becomes more resistant to water flow as it grows. It is estimated that a flow velocity of around lms^‐1 is critical for attachment/detachment of a juvenile mussel with a shell length of a few millimeters and one hundred byssal threads.     

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)     

Effect of the excretory-secretory products of some marine invertebrates on byssus production of the blue mussel <Emphasis Type="Italic">Mytilus edulis</Emphasis> (Bivalvia: Mytilidae)

Under laboratory conditions, we investigated byssus production in the blue mussel Mytilus edulis, as affected by the excretory-secretory products (ESPs) of the mussel itself and some marine invertebrates: the predatory starfish Asterias rubens, and organisms competing with mussels in White Sea fouling communities—a bivalve Hiatella arctica, the solitary ascidians Styela rustica and Molgula citrine, and a sponge Halichondria panicea. The number of attachment disks produced by a mussel per day and the thickness of byssal threads were estimated. Excretory-secretory products of H. arctica and M. citrine had no effect on the number of attachment disks, while ESPs of S. rustica, H. panacea and A. rubens stimulated mussels to produce attachment plaques. The activity of the mussel was slightly increased at low levels of its own ESPs in seawater. The thickness of byssal threads decreased with an increase in the ESPs of mussels in seawater, but it increased in experiments with the ESPs of any other species tested.     

Mussel attachment on rocky shores: the effect of flow on byssus production

Mussels rely on a strong byssal attachment to persist in a range of habitats with differing rates of water flow. Recent studies, however, suggest that the ability of one mussel species to sense and respond adaptively to the flow in its environment is limited under even modest flow conditions because the process of byssal thread formation is disrupted. This study extends these findings to four mussel species, Mytilus trossulus, M. galloprovincialis, M. californianus, and Modiolus modiolus. Collectively, the response of byssal thread formation decreased with rates of flow above ～25 cm/s and the critical flow threshold was estimated to be <50 cm/s. How can mussels persist on shores where flow is an order of magnitude higher? Using a combination of techniques for measuring flow, velocity profiles were obtained above and within mussel aggregations in the laboratory and in the field. Flow was greatly reduced within mussel aggregations, ranging from 0.1% to 10% of free-stream velocity. These results suggest one key to the success of mussels in habitats with high rates of flow is the ability to form aggregations that ameliorate flows to a level that is conducive to byssal thread formation.     

Giant bent-core mesogens in the thread forming process of marine mussels

In marine mussels (Mytilus), byssal threads are made in minutes from prefabricated smectic polymer liquid crystals by a process resembling reaction injection molding. The mesogens in these arrays are known to be natural block copolymers with rodlike collagen cores. Using atomic force microscopy, it was shown that these collagenous mesogens are bent-core or banana-shaped in a manner that is consistent with and predictable from their amino acid sequence. The overall bend angle in preCOL-NG in Mytilus galloprovincialis is about 130 degrees. The mesogens have a center-to-center separation of approximately 22 nm and a length of 200 nm. It is evident that the smectic structure of the prefabricated mesophases remains largely intact over 1-3 microm distances in the molded fibers and is presumably locked in place during molding by cross-linking. Like the smectic liquid crystals of many synthetic banana mesogens, the collagenous mesogens of the byssal threads exhibit SmC(2) symmetry with a characteristic tilt of 24.6 degrees. At about 100% extension, this tilt is considerably reduced and the globular end domains are no longer visible presumably because they have been unraveled.     

pH-dependent locking of giant mesogens in fibers drawn from mussel byssal collagens

Byssal threads are tough collagenous fibers that mussels use to secure themselves against dislodgement by waves in the marine intertidal zone. Here, preCol, a family of hybrid collagens comprising up to 96% of the protein content in certain regions of byssal threads, was purified in mg amounts from mussel foot tissue for the first time. Conditions for drawing preCols into quality fibers ex vivo were investigated. The most important factor affecting fiber formation was the pH of the drawing solution. The morphology and tensile properties of drawn fibers were also characterized and suggest that a liquid crystal mesophase combined with cross-linking by His-metal coordination plays a role in the assembly/mechanics of drawn fibers and likely in native byssal threads as well.     

Location and analysis of byssal structural proteins of Mytilus edulis

The acellular attachment organ (byssus) of the marine mussel Mytilus edulis L. is composed of threads that emanate from the body of the mussel to adhesive discs that anchor the threads to rocks, sand and other mussels. Three proteins have been purified by immunohistological methods and located to specific regions of the byssus. A collagenous protein with subunit molecular weights of 53,000, 55,000 and 65,000 is found in the matrix of the elastic thread region. Its 73,000-MW precursor was extracted from foot glands in the area proximal to the animal body and was identified by immune cross-reactivity. A cystine-rich, acidic protein was found in all regions of the byssus associated with a third protein, the polyphenolic protein. The L-dopa-containing polyphenolic protein appears in the cortex of the entire thread and adhesive plaque and at the substrate-plaque interface. Antiserum to this protein stains spherical vesicles in the phenol gland of the foot. Using immuno-electrophoretic methods, the polyphenolic protein and the cystine-rich protein were shown to form high molecular weight aggregates with aging of the byssus.     

Morphology of the Bivalve Mollusks Crenomytilus grayanus and Mytilus coruscus in Relation to Their Spatial Distribution in the Upper Subtidal Zone

The morphology of the shell and byssus threads was studied in two closely related mussel species Crenomytilus grayanus and Mytilus coruscus. The two species differ significantly from each other in the shell shape and in the degrees of development and deformation of byssus threads. These differences, in turn, determine (either directly or indirectly) the differences in strength of the byssal attachment and are discussed in terms of their functional morphology with respect to the spatial distribution of the mussels in marine coastal zones.