The mussel cuticle, a thin layer that shields byssal threads from environmental exposure, is a model among high-performance coatings for being both hard and hyper-extensible. However, despite avid interest in translating its features into an engineered material, the mechanisms underlying this performance are manifold and incompletely understood. To deepen our understanding of this biomaterial, we explore here the ultrastructural, scratch-resistant, and mechanical features at the submicrometer scale and relate our observations to individual cuticular components. These investigations show that cuticle nanomechanics are governed by granular microinclusions/nanoinclusions, which, contrary to previous interpretations, are three-fold softer than the surrounding matrix. This adaptation, which is found across several related mussel species, is linked to the level of hydration and presumed to maintain bulk performance during tidal exposures. Given the interest in implementing transfer of biological principles to modern materials, these findings may have noteworthy implications for the design of durable synthetic coatings.

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.

The reversible assembly of reflectin proteins drives dynamic iridescence in cephalopods. Squid dynamically tune the intensity and colors of iridescence generated by constructive interference from intracellular Bragg reflectors in specialized skin cells called iridocytes. Analysis of the tissue specificity of reflectin subtypes reveals that tunability is correlated with the presence of one specific reflectin sequence. Differential phosphorylation and dephosphorylation of the reflectins in response to activation by acetylcholine, as well as differences in their tissue-specific and subcellular spatial distributions, further support the suggestion of different roles for the different reflectin subtypes.

Whether and how intramolecular crosslinks in polymeric materials contribute to mechanical properties is debated in both experimental and theoretical arenas. The tethering threads of Octopus bimaculoides egg cases provide a rare window to investigate this question in a biomaterial. The only detectable component of the load-bearing fibers in octopus threads is a 135 kDa protein, octovafibrin, comprising 29 tandem repeats of epidermal growth factor (EGF) each of which contains 3 intramolecular disulfide linkages. The N- and C-terminal C-type lectins mediate linear end-to-end octovafibrin self-assembly. Mechanical testing of threads shows that the regularly spaced disulfide linkages result in improved stiffness, toughness, and energy dissipation. In response to applied loads, molecular dynamics and X-ray scattering show that EGF-like domains deform by recruiting two hidden length β-sheet structures nested between the disulfides. The results of this study further the understanding of intramolecular crosslinking in polymers and provide a foundation for the mechanical contributions of EGF domains to the extracellular matrix.

The adaptive attachment of marine mussels to a wide range of substrates in a high-energy, saline environment has been explored for decades and is a significant driver of bioinspired wet adhesion research. Mussel attachment relies on a fibrous holdfast known as the byssus, which is made by a specialized appendage called the foot. Multiple adhesive and structural proteins are rapidly synthesized, secreted and moulded by the foot into holdfast threads. About 10 well-characterized proteins, namely the mussel foot proteins (Mfps), the preCols and the thread matrix proteins, are reported as representing the bulk of these structures. To explore how robust this proposition is, we sequenced the transcriptome of the glandular tissues that produce and secrete the various holdfast components using next-generation sequencing methods. Surprisingly, we found around 15 highly expressed genes that have not previously been characterized, but bear key similarities to the previously defined mussel foot proteins, suggesting additional contribution to byssal function. We verified the validity of these transcripts by polymerase chain reaction, cloning and Sanger sequencing as well as confirming their presence as proteins in the byssus. These newly identified proteins greatly expand the palette of mussel holdfast biochemistry and provide new targets for investigation into bioinspired wet adhesion.

The adaptive attachment of marine mussels to a wide range of substrates in a high-energy, saline environment has been explored for decades and is a significant driver of bioinspired wet adhesion research. Mussel attachment relies on a fibrous holdfast known as the byssus, which is made by a specialized appendage called the foot. Multiple adhesive and structural proteins are rapidly synthesized, secreted and moulded by the foot into holdfast threads. About 10 well-characterized proteins, namely the mussel foot proteins (Mfps), the preCols and the thread matrix proteins, are reported as representing the bulk of these structures. To explore how robust this proposition is, we sequenced the transcriptome of the glandular tissues that produce and secrete the various holdfast components using next-generation sequencing methods. Surprisingly, we found around 15 highly expressed genes that have not previously been characterized, but bear key similarities to the previously defined mussel foot proteins, suggesting additional contribution to byssal function. We verified the validity of these transcripts by polymerase chain reaction, cloning and Sanger sequencing as well as confirming their presence as proteins in the byssus. These newly identified proteins greatly expand the palette of mussel holdfast biochemistry and provide new targets for investigation into bioinspired wet adhesion.

Marine mussels of the genus Mytilus live in the hostile intertidal zone, attached to rocks, bio-fouled surfaces and each other via collagen-rich threads ending in adhesive pads, the plaques. Plaques adhere in salty, alkaline seawater, withstanding waves and tidal currents. Each plaque requires a force of several newtons to detach. Although the molecular composition of the plaques has been well studied, a complete understanding of supra-molecular plaque architecture and its role in maintaining adhesive strength remains elusive. Here, electron microscopy and neutron scattering studies of plaques harvested from Mytilus californianus and Mytilus galloprovincialis reveal a complex network structure reminiscent of structural foams. Two characteristic length scales are observed characterizing a dense meshwork (approx. 100 nm) with large interpenetrating pores (approx. 1 µm). The network withstands chemical denaturation, indicating significant cross-linking. Plaques formed at lower temperatures have finer network struts, from which we hypothesize a kinetically controlled formation mechanism. When mussels are induced to create plaques, the resulting structure lacks a well-defined network architecture, showcasing the importance of processing over self-assembly. Together, these new data provide essential insight into plaque structure and formation and set the foundation to understand the role of plaque structure in stress distribution and toughening in natural and biomimetic materials.