Byssus Research Articles

Byssus production ability and degree of byssal‐gland development in the infaunal clam Ruditapes philippinarum

AbstractByssus production of Ruditapes philippinarum clams becomes reduced with growth. This tendency is well recognized but has not been analysed in detail. Additionally, it remains uninvestigated whether the lack of competence to produce byssus threads in the adult stage is caused by atrophy of the byssal glands or not. The objective of this study was to evaluate the byssus production ability of clams through the juvenile to adult stages and to examine the importance of two endogenous factors (i.e. shell size, somatic condition) in determining the byssus production probability (proportion of clams with byssus production in a population). This study also histologically confirmed the presence of byssal glands in juvenile to adult clams. For these purposes, field surveys to investigate the relationship among byssus production, shell size and somatic condition of clams collected from four intertidal sites and a histological study for byssal glands of the clams was conducted. This study revealed that byssus production probability decreases with increasing shell size and declining somatic condition and that the lack of byssus production is not caused by the loss of the byssal glands.

The Thiol-Rich Interlayer in the Shell/Core Architecture of Mussel Byssal Threads.

The mussel byssus thread is an extremely tough core-shelled fiber that dissipates substantial amounts of energy during tensile loading. The mechanical performance of the shell is critically reliant on 3,4-dihydroxyphenylalanine's (Dopa) ability to form reversible iron-catecholate complexes at pH 8. However, the formation of these coordinate cross-links is undercut by Dopa's oxidation to Dopa-quinone, a spontaneous process at seawater conditions. The large mechanical mismatch between the cuticle and the core lends itself to further complications. Despite these challenges, the mussel byssus thread performs its tethering function over long periods of time. Here, we address these two major questions: (1) how does the mussel slow/prevent oxidation in the cuticle, and (2) how is the mechanical mismatch at the core/shell interface mitigated? By combining a number of microscopy and spectroscopy techniques we have discerned a previously undescribed layer. Our results indicate this interlayer is thiol rich and thus will be called the thiol-rich interlayer (TRL). We propose the TRL serves as a long-lasting redox reservoir as well as a mechanical barrier.

Structural characterization of the buccal mass of Ariolimax californicus (Gastropoda; Stylommatophora).

Biological materials such as chiton tooth, squid beak, and byssal threads of bivalves have inspired the development of new technologies. To this end, we have characterized the acellular components in the buccal mass of the terrestrial slug Ariolimax californicus (banana slug). These components are the radula, the jaw, and the odontophore. In the radula, calcium-rich denticles are tightly interlocked one to the other on top of a nanofibrous chitin membrane. The jaw has a nanostructured morphology made of chitin to achieve compression resistance and is directly linked to the foregut cuticle, which has a protective nanofibrous structure. Finally, in the odontophore, we observed a structurally elastic microstructure that interfaces soft tissues with a highly stressed radula membrane. Based on those observations, we discuss the interaction between these components and highlight how the materials in these task-specific components have evolved. This structure-properties-function study of the A. californicus’ buccal mass may aid in the design and fabrication of novel bioinspired materials.

Bioinspired nanoplatform for enhanced delivery efficiency of doxorubicin into nucleus with fast endocytosis, lysosomal pH-triggered drug release, and reduced efflux

A novel bioinspired nanoplatform capable of fast endocytosis, lysosomal pH-triggered drug release, and reduced drug efflux based on PBA-PEG-b-P(Glu-co-GluDA) copolymer was developed in this study. The synthesized copolymer could facilitate doxorubicin encapsulation with relatively high drug-loading content and efficiency. Inspired by mussel byssal threads, a core crosslinking strategy based on the coordination between catechol and ferric ions was introduced to improve the stability of nanomicelles and realize lysosomal pH-controlled drug release. This nanoplatform could maintain integrity even after being dissolved in a good solvent, demonstrating its the potential to withstand infinite dilution of plasma after intravenous injection. Moreover, this nanoplatform demonstrated lysosomal pH-triggered drug release, and the cumulative release amount of doxorubicin under a simulated lysosomal condition was 13 times higher than that under a simulated plasma condition. Moreover, as a result of the high binding capacity between phenylboronic acid (PBA) and sialic acid on the surface of human hepatoma cell line (HepG2), the fast and enhanced endocytosis in addition to lysosomal pH-triggered release property and significantly low efflux, this nanoplatform exhibits improved delivery efficiency of doxorubicin into the nucleus and notably outstanding antiproliferative effects compared with doxorubicin. Furthermore, the PBA modification remarkably increased the mean fluorescence intensity of this nanoplatform endocytosed by HepG2 cells to twice that of doxorubicin after one hour of incubation. The nanoplatform exhibited an inhibition rate of 70% against tumor growth. Thus, this novel nanoplatform based on PBA-PEG-b-P(Glu-co-GluDA) copolymer displayed multifunctionality and exhibited great potential as an intelligent nanoplatform for antitumor drug delivery.

Seawater acidification and temperature modulate anti-predator defenses in two co-existing Mytilus species

The effects of short-term (7 days) experimental ocean acidification (−0.4 pH units) and warming (+5 °C) on anti-predator defenses of two sympatric Mytilus species from China, M. coruscus and M. edulis, in the presence and absence of predator cues were investigated. Results suggested species-specific independent negative effects of acidification and warming on the number and weight of byssal threads, the force of thread attachment, and total thread plaque area. Similar negative effects were observed for clustering behaviour, with acidification and warming independently increasing the number of solitary individuals and decreasing the percentage of mussels in clusters. Acidification effects on byssus were strongly exacerbated when predators were present. Ultimately, this study suggests that short-term exposure to experimental warming and acidification can negatively impact anti-predator defense strategies in mussels with potential ramifications for predator-prey interactions and ecological functioning in systems where mussel beds play a key ecological role.

The benthic sea-silk-thread displacement of a sessile bivalve, Pinctada imbricata radiata (Leach, 1819) in the Arabian-Persian Gulf.

A number of molluscs within the Class Bivalvia are defined by their ability to secrete fine silk like threads known as byssus which are used to anchor themselves to solid substrates. With relatively few exceptions the majority of these species remain in a sedentary state throughout their life attached via their byssal threads. However, observations of adult Pinctada imbricata radiata pearl oysters made during this study revealed this species’ ability to implement active movement. Byssal threads were secreted in a sequence of attachment and detachment phases, which resulted in the active displacement of the oyster. The oyster was observed, in the laboratory over a 9 day period, travelling a distance of 28cm in a horizontal path. After horizontal displacement, a vertical climbing phase was observed until the oyster reached the water surface at which point the byssus was discarded and the animal dropped, drifting in accordance with water current intensity. It is possible that these adaptations of byssal use are a result of environmentally induced evolutionary change within P. i. radiata.

Bioinspired Metal⁻Polyphenol Materials: Self-Healing and Beyond.

The blue mussel incorporates the polyphenolic amino acid l-3,4-dihydroxyphenylalanine (DOPA) to achieve self-healing, pH-responsiveness, and impressive underwater adhesion in the byssus threads that ensure the survival of the animal. This is achieved by a pH-dependent and versatile reaction chemistry of polyphenols, including both physical interactions as well as reversible and irreversible chemical bonding. With a short introduction to the biological background, we here review the latest advances in the development of smart materials based on the metal-chelating capabilities of polyphenols. We focus on new ways of utilizing the polyphenolic properties, including studies on the modifications of the nearby chemical environment (on and near the polyphenolic moiety) and on the incorporation of polyphenols into untraditional materials.

Bioinspired Histidine⁻Zn2+ Coordination for Tuning the Mechanical Properties of Self-Healing Coiled Coil Cross-Linked Hydrogels.

Natural biopolymeric materials often possess properties superior to their individual components. In mussel byssus, reversible histidine (His)–metal coordination is a key feature, which mediates higher-order self-assembly as well as self-healing. The byssus structure, thus, serves as an excellent natural blueprint for the development of self-healing biomimetic materials with reversibly tunable mechanical properties. Inspired by byssal threads, we bioengineered His–metal coordination sites into a heterodimeric coiled coil (CC). These CC-forming peptides serve as a noncovalent cross-link for poly(ethylene glycol)-based hydrogels and participate in the formation of higher-order assemblies via intermolecular His–metal coordination as a second cross-linking mode. Raman and circular dichroism spectroscopy revealed the presence of α-helical, Zn2+ cross-linked aggregates. Using rheology, we demonstrate that the hydrogel is self-healing and that the addition of Zn2+ reversibly switches the hydrogel properties from viscoelastic to elastic. Importantly, using different Zn2+:His ratios allows for tuning the hydrogel relaxation time over nearly three orders of magnitude. This tunability is attributed to the progressive transformation of single CC cross-links into Zn2+ cross-linked aggregates; a process that is fully reversible upon addition of the metal chelator ethylenediaminetetraacetic acid. These findings reveal that His–metal coordination can be used as a versatile cross-linking mechanism for tuning the viscoelastic properties of biomimetic hydrogels.

Healing through Histidine: Bioinspired Pathways to Self-Healing Polymers via Imidazole⁻Metal Coordination.

Biology offers a valuable inspiration toward the development of self-healing engineering composites and polymers. In particular, chemical level design principles extracted from proteinaceous biopolymers, especially the mussel byssus, provide inspiration for design of autonomous and intrinsic healing in synthetic polymers. The mussel byssus is an acellular tissue comprised of extremely tough protein-based fibers, produced by mussels to secure attachment on rocky surfaces. Threads exhibit self-healing response following an apparent plastic yield event, recovering initial material properties in a time-dependent fashion. Recent biochemical analysis of the structure–function relationships defining this response reveal a key role of sacrificial cross-links based on metal coordination bonds between Zn2+ ions and histidine amino acid residues. Inspired by this example, many research groups have developed self-healing polymeric materials based on histidine (imidazole)–metal chemistry. In this review, we provide a detailed overview of the current understanding of the self-healing mechanism in byssal threads, and an overview of the current state of the art in histidine- and imidazole-based synthetic polymers.

Nano-ZnO impairs anti-predation capacity of marine mussels under seawater acidification.

Artificial nanoparticles and ocean acidification (OA) caused by the rapid increase of CO2 absorbed by the ocean are both ecologically hazardous to marine organisms. The combined effects of the two environmental stressors on the anti-predation ability of marine mussels were studied. Mytilus coruscus was exposed to three different gradient concentrations of nano-ZnO (0, 2.5, 10 mg/L) in combination of two pH levels (7.7 and 8.1). The crab Charybdis japonica was used as its predator. During the experiment, anti-predator indexes, including number of byssus threads (NBT), shell-closing strength (SCS), diameter of byssus thread (BTD), length of byssus thread (BTL), cumulative length of byssus thread (CBTL) and cumulative volume of byssus thread (CBTV) were studied. The results showed that predator induced the anti-predation responses in M. coruscus, and NBT, SCS, BTL, CBTL and CBTV were significantly increased. Under the conditions of pH 7.7 and 10 mg/L nano-ZnO, NBT, SCS, BTD, BTL, CBTL, and CBTV were significantly reduced. What's more, significant interactions among pH, nano-ZnO and predator were observed in CBTL and CBTV. Therefore, the joint treatment of nano-ZnO and low pH reduces the adhesion strength of byssus thread and may increase the probability of mussels being preyed.

Only as strong as the weakest link: structural analysis of the combined effects of elevated temperature and pCO2 on mussel attachment.

Predicting how combinations of stressors will affect failure risk is a key challenge for the field of ecomechanics and, more generally, ecophysiology. Environmental conditions often influence the manufacture and durability of biomaterials, inducing structural failure that potentially compromises organismal reproduction, growth, and survival. Species known for tight linkages between structural integrity and survival include bivalve mussels, which produce numerous byssal threads to attach to hard substrate. Among the current environmental threats to marine organisms are ocean warming and acidification. Elevated pCO2 exposure is known to weaken byssal threads by compromising the strength of the adhesive plaque. This study uses structural analysis to evaluate how an additional stressor, elevated temperature, influences byssal thread quality and production. Mussels (Mytilus trossulus) were placed in controlled temperature and pCO2 treatments, and then, newly produced threads were counted and pulled to failure to determine byssus strength. The effects of elevated temperature on mussel attachment were dramatic; mussels produced 60% weaker and 65% fewer threads at 25°C in comparison to 10°C. These effects combine to weaken overall attachment by 64–88% at 25°C. The magnitude of the effect of pCO2 on thread strength was substantially lower than that of temperature and, contrary to our expectations, positive at high pCO2 exposure. Failure mode analysis localized the effect of temperature to the proximal region of the thread, whereas pCO2 affected only the adhesive plaques. The two stressors therefore act independently, and because their respective target regions are interconnected (resisting tension in series), their combined effects on thread strength are exactly equal to the effect of the strongest stressor. Altogether, these results show that mussels, and the coastal communities they support, may be more vulnerable to the negative effects of ocean warming than ocean acidification.

Self-healing silk from the sea: role of helical hierarchical structure in Pinna nobilis byssus mechanics.

The byssus fibers of Mytilus mussel species have become an important role model in bioinspired materials research due to their impressive properties (e.g. high toughness, self-healing); however, Mytilids represent only a small subset of all byssus-producing bivalves. Recent studies have revealed that byssus from other species possess completely different protein composition and hierarchical structure. In this regard, Pinna nobilis byssus is especially interesting due to its very different morphology, function and its historical use for weaving lightweight golden fabrics, known as sea silk. P. nobilis byssus was recently discovered to be comprised of globular proteins organized into a helical protein superstructure. In this work, we investigate the relationships between this hierarchical structure and the mechanical properties of P. nobilis byssus threads, including energy dissipation and self-healing capacity. To achieve this, we performed in-depth mechanical characterization, as well as tensile testing coupled with in situ X-ray scattering. Our findings reveal that P. nobilis byssus, like Mytilus, possesses self-healing and energy damping behavior and that the initial elastic behavior of P. nobilis byssus is due to stretching and unraveling of the previously observed helical building blocks comprising the byssus. These findings have biological relevance for understanding the convergent evolution of mussel byssus for different species, and also for the field of bio-inspired materials.

A Facile and Sensitive Colorimetric Approach to Confirming the Presence of Polydopamine Thin Films on (Bio)Material Surfaces

Mussel foot adhesive proteins are one of nature's remarkable triumphs; they guarantee a secure and robust bond between the mollusc's byssal threads and the surface to which they settle ensuring that they are securely fastened. We now know that some of these adhesive proteins are especially rich in lysine and 3,4-dihydroxy-L-phenylalanine (DOPA) residues.

Exposure to microplastics reduces attachment strength and alters the haemolymph proteome of blue mussels (Mytilus edulis)

The contamination of marine ecosystems with microplastics, such as the polymer polyethylene, a commonly used component of single-use packaging, is of global concern. Although it has been suggested that biodegradable polymers, such as polylactic acid, may be used to replace some polyethylene packaging, little is known about their effects on marine organisms. Blue mussels, Mytilus edulis, have become a “model organism” for investigating the effects of microplastics in marine ecosystems. We show here that repeated exposure, over a period of 52 days in an outdoor mesocosm setting, of M. edulis to polyethylene microplastics reduced the number of byssal threads produced and the attachment strength (tenacity) by ∼50%. Exposure to either type of microplastic altered the haemolymph proteome and, although a conserved response to microplastic exposure was observed, overall polyethylene resulted in more changes to protein abundances than polylactic acid. Many of the proteins affected are involved in vital biological processes, such as immune regulation, detoxification, metabolism and structural development. Our study highlights the utility of mass spectrometry-based proteomics to assess the health of key marine organisms and identifies the potential mechanisms by which microplastics, both conventional and biodegradable, could affect their ability to form and maintain reefs.

Microbially Synthesized Repeats of Mussel Foot Protein Display Enhanced Underwater Adhesion.

Mussels strongly adhere to a variety of surfaces by secreting byssal threads that contain mussel foot proteins (Mfps). Recombinant production of Mfps presents an attractive route for preparing advanced adhesive materials. Using synthetic biology strategies, we synthesized Mfp5 together with Mfp5 oligomers containing two or three consecutive, covalently-linked Mfp5 sequences named Mfp5(2) and Mfp5(3). The force and work of adhesion of these proteins were measured underwater with a colloidal probe mounted on an atomic force microscope and the adsorption was measured with a quartz crystal microbalance. We found positive correlations between Mfp5 molecular weight and underwater adhesive properties, including force of adhesion, work of adhesion, protein layer thickness, and recovery distance. DOPA-modified Mfp5(3) displayed a high force of adhesion (201 ± 36 nN μm-1) and a high work of adhesion (68 ± 21 fJ μm-1) for a cure time of 200 s, which are higher than those of previously reported Mfp-mimetic adhesives. Results presented in this study highlight the power of synthetic biology in producing biocompatible and highly adhesive Mfp-based materials.

The impact of ocean acidification on the byssal threads of the blue mussel (Mytilus edulis)

Blue mussel (Mytilus edulis) produce byssal threads to anchor themselves to the substrate. These threads are always exposed to the surrounding environmental conditions. Understanding how environmental pH affects these threads is crucial in understanding how climate change can affect mussels. This work examines three factors (load at failure, thread extensibility, and total thread counts) that indicate the performance of byssal threads as well as condition index to assess impacts on the physiological condition of mussels held in artificial seawater acidified by the addition of CO2. There was no significant variation between the control (~786 μatm CO2 / ~7.98 pH/ ~2805 μmol kg-1 total alkalinity) and acidified (~2555 μatm CO2 / ~7.47 pH/ ~2650 μmol kg-1 total alkalinity) treatment groups in any of these factors. The results of this study suggest that ocean acidification by CO2 addition has no significant effect on the quality and performance of threads produced by M. edulis.

Biological characteristics of the rafting bivalve Gaimardia trapesina in the Southern Ocean

Rafting invertebrate species represent an important link for the dispersal and colonisation of islands in the Southern Ocean. They are sensitive to changes in hydrodynamics through effects on transport and food availability, particularly in the case of filter-feeding species. This gives them the potential to act as sentinels of environmental change. The rafting kelp-associated Gaimardia trapesina is one such species, widely distributed in the Southern Ocean. In 2015 and 2016, the size structure, diet, and distribution of G. trapesina were examined around the Sub-Antarctic Prince Edward Islands (PEI) to establish benchmark data and explore their potential use for monitoring long-term change. Gaimardia trapesina and its potential food sources were collected and analysed for size structure, attachment strength, and diet, using stable isotope and fatty acid analyses. The populations examined were dominated by relatively small individuals. The strength of attachment to kelp blades was low and the byssal threads showed high elasticity. The PEI lie in the path of the west–east flowing Antarctic Circumpolar Current and the highest abundances of G. trapesina were found on the downstream sides of both islands, while the species was absent from the western, upstream coasts. Both diet analyses and SIAR mixing models indicated that G. trapesina feeds on suspended particulate matter, while kelp particles contribute minimally to the diet. Considering the wide distribution of G. trapesina, its importance as food for birds and fish, and its sensitivity to environmental conditions, there is good potential to use this species as an indicator of environmental change in the Southern Ocean.

Hypoxia weakens mussel attachment by interrupting DOPA cross-linking during adhesive plaque curing.

Marine mussels (Mytilus spp.) attach to a wide variety of surfaces underwater using a network of byssal threads, each tipped with a protein-based adhesive plaque that uses the surrounding seawater environment as a curing agent. Plaques undergo environmental post-processing, requiring a basic seawater pH be maintained for up to 8 days for the adhesive to strengthen completely. Given the sensitivity of plaques to local pH conditions long after deposition, we investigated the effect of other aspects of the seawater environment that are known to vary in nearshore habitats on plaque curing. The effect of seawater temperature, salinity and dissolved oxygen concentration were investigated using tensile testing, atomic force microscopy and amino acid compositional analysis. High temperature (30°C) and hyposalinity (1 PSU) had no effect on adhesion strength, while incubation in hypoxia (0.9 mg l-1) caused plaques to have a mottled coloration and prematurely peel from substrates, leading to a 51% decrease in adhesion strength. AFM imaging of the plaque cuticle found that plaques cured in hypoxia had regions of lower stiffness throughout, indicative of reductions in DOPA cross-linking between adhesive proteins. A better understanding of the dynamics of plaque curing could aid in the design of better synthetic adhesives, particularly in medicine where adhesion must take place within wet body cavities.

Exploring mussel byssus fabrication with peptide-polymer hybrids: Role of pH and metal coordination in self-assembly and mechanics of histidine-rich domains

Mussel byssal threads are biopolymeric fibers that possess high toughness and self-healing capacity owing to a hierarchical arrangement of collagenous protein building blocks known as preCols. To investigate the proposed role of histidine-rich domains (HRDs) at preCol termini in the rapid self-assembly and properties of mussel byssus, we functionalized 4-arm star-shaped polyethyleneglycol (star-PEG) molecules with peptides based on an evolutionarily-conserved HRD sequence motif. PEG-HRD hybrid molecules instantly and reversibly form physical hydrogels without metal ions, using a simple pH trigger mimicking the natural assembly process. Spectroscopic analysis indicates that gelation is mediated via a conformational transition into β-sheet crystalline structure, comprised of multiple peptide groups that cross-link the star-PEGs. Additionally, metal ions introduced during gelation form His-metal coordination bonds within the network and increase viscoelastic damping of the gels, as proposed for native fibers. These findings help disentangle the different roles of pH and metal coordination during natural byssus formation/function and may have important implications for generating novel metallopolymeric materials with hierarchical structure.

Native versus invasive crab effluent effects on byssal thread production in the mussel, Mytilus trossulus (Gould, 1950)

Mussels have evolved many adaptations to protect themselves, including the production of byssal threads. These are strong, proteinaceous fibres that mussels secrete to adhere themselves to rocks, preventing detachment by waves and predators. These byssal threads may be strengthened if mussels can recognize potential threats, such as native crabs, as their populations have a long history of coevolution. Unfortunately, the introduction of invasive predators poses a challenge for prey, which may not be capable of recognizing them. In this study, byssal thread production in the Pacific blue mussel (Mytilus trossulus ) was observed when exposed to effluent from the native red rock crab (Cancer productus) or the invasive European Green crab (Carcinus maenas). M. trossulus were placed in closed systems with effluent from either C. productus , C. maenas or control (no predator), over a 24-hour time period. Final measurements of number, length and diameter of byssal threads were recorded. M. trossulus exposed to effluent from C. productus produced byssal threads at a statistically significantly faster rate than in the control group over the first 7.5 hours. M. trossulus exposed to effluent from C. maenas produced byssal threads at a statistically significantly faster rate than both the C. productus and control groups. However, after 24 hours, there was no statistically significant difference between the mean number of byssal threads for any treatment. Additionally, we found no statistically significant difference between the mean diameter of byssal threads produced or length of byssal threads produced for any treatment.