Methods for Silk Property Analyses across Structural Hierarchies and Scales.

Silk from silkworms and spiders is an exceptionally important natural material, inspiring a range of new products and applications due to its high strength, elasticity, and toughness at low density, as well as its unique conductive and optical properties. Transgenic and recombinant technologies offer great promise for the scaled-up production of new silkworm- and spider-silk-inspired fibres. However, despite considerable effort, producing an artificial silk that recaptures the physico-chemical properties of naturally spun silk has thus far proven elusive. The mechanical, biochemical, and other properties of pre-and post-development fibres accordingly should be determined across scales and structural hierarchies whenever feasible. We have herein reviewed and made recommendations on some of those practices for measuring the bulk fibre properties; skin-core structures; and the primary, secondary, and tertiary structures of silk proteins and the properties of dopes and their proteins. We thereupon examine emerging methodologies and make assessments on how they might be utilized to realize the goal of developing high quality bio-inspired fibres.

Physico-chemical properties of functionally adhesive spider silk nanofibres.

Currently, synthetic fibre production focuses primarily on high performance materials. For high performance fibrous materials, such as silks, this involves interpreting the structure-function relationship and downsizing to a smaller scale to then harness those properties within synthetic products. Spiders create an array of fibres that range in size from the micrometre to nanometre scale. At about 20 nm diameter spider cribellate silk, the smallest of these silks, is too small to contain any of the typical secondary protein structures of other spider silks, let alone a hierarchical skin-core-type structure. Here, we performed a multitude of investigations to elucidate the structure of cribellate spider silk. These confirmed our hypothesis that, unlike all other types of spider silk, it has a disordered molecular structure. Alanine and glycine, the two amino acids predominantly found in other spider silks, were much less abundant and did not form the usual α-helices and ß-sheet secondary structural arrangements. Correspondingly, we characterized the cribellate silk nanofibre to be very compliant. This characterization matches its function as a dry adhesive within the capture threads of cribellate spiders. Our results imply that at extremely small scales there may be a limit reached below which a silk will lose its structural, but not functional, integrity. Nano-sized fibres, such as cribellate silk, thus offer a new opportunity for inspiring the creation of novel scaled-down functional adhesives and nano meta-materials.

1000 spider silkomes: Linking sequences to silk physical properties.

Spider silks are among the toughest known materials and thus provide models for renewable, biodegradable, and sustainable biopolymers. However, the entirety of their diversity still remains elusive, and silks that exceed the performance limits of industrial fibers are constantly being found. We obtained transcriptome assemblies from 1098 species of spiders to comprehensively catalog silk gene sequences and measured the mechanical, thermal, structural, and hydration properties of the dragline silks of 446 species. The combination of these silk protein genotype-phenotype data revealed essential contributions of multicomponent structures with major ampullate spidroin 1 to 3 paralogs in high-performance dragline silks and numerous amino acid motifs contributing to each of the measured properties. We hope that our global sampling, comprehensive testing, integrated analysis, and open data will provide a solid starting point for future biomaterial designs.

Nanovoid formation induces property variation within and across individual silkworm silk threads.

Silk is a unique fiber, having a strength and toughness that exceeds other natural fibers. While inroads have been made in our understanding of silkworm silk structure and function, few studies have measured structure and function at nanoscales. As a consequence, the sources of variation in mechanical properties along single silk fibers remain unresolved at multiple scales. Here we utilized state of the art spectroscopic and microscopic methodologies to show that the silks of species of wild and domesticated silkworms vary in mechanical properties along a single fiber and, what is more, this variation correlates with nanoscale void formations. These results can also explain the strain hardening behaviours observed in the silks where structural features of the proteins could not. We thereupon devised a predictive thermal model and showed that the voids contribute to temperature regulation within the silkworm cocoons.

Nutritionally induced nanoscale variations in spider silk structural and mechanical properties.

Spider major ampullate (MA) silk is characterized by high strength and toughness and is adaptable across environments. Experiments depriving spiders of protein have enabled researchers to examine nutritionally induced changes in gene expression, protein structures, and bulk properties of MA silk. However, it has not been elucidated if it varies in a similar way at a nanoscale. Here we used Atomic Force Microscopy (AFM) to simultaneously examine the topographic, structural, and mechanical properties of silks spun by two species of spider, Argiope keyserlingi and Latrodectus hasselti, at a nanoscale when protein fed or deprived. We found height, a measure of localized width, to substantially vary across species and treatments. We also found that Young's modulus, which may be used as an estimate of localized stiffness, decreased with protein deprivation in both species' silk. Our results suggest that nanoscale skin-core structures of A. keyserlingi's MA silk varied significantly across treatments, whereas only slight structural and functional variability was found for L. hasselti's silk. These results largely agreed with examinations of the bulk properties of each species' silk. However, we could not directly attribute the decoupling between protein structures and bulk mechanics in L. hasselti's silk to nanoscale features. Our results advance the understanding of processes inducing skin and core structural variations in spider silks at a nanoscale, which serves to enhance the prospect of developing biomimetic engineering programs.

Nanoscale Material Heterogeneity of Glowworm Capture Threads Revealed by AFM.

Adhesive materials used by many arthropods for biological functions incorporate sticky substances and a supporting material that operate synergistically by exploiting substrate attachment and energy dissipation. While there has been much focus on the composition and properties of the sticky glues of these bio-composites, less attention has been given to the materials that support them. In particular, as these materials are primarily responsible for dissipation during adhesive pull-off, little is known of the structures that give rise to functionality, especially at the nano-scale. In this study we used tapping mode atomic force microscopy (TM-AFM) to analyze unstretched and stretched glowworm (Arachnocampa tasmaniensis) capture threads and revealed nano-scale features corresponding to variation in surface structure and elastic modulus near the surface of the silk. Phase images demonstrated a high resolution of viscoelastic variation and revealed mostly globular and elongated features in the material. Increased vertical orientation of 11-15 nm wide fibrillar features was observed in stretched threads. Fast Fourier transform analysis of phase images confirmed these results. Relative viscoelastic properties were also highly variable at inter- and intra-individual levels. Results of this study demonstrate the practical usefulness of TM-AFM, especially phase angle imaging, in investigating the nano-scale structures that give rise to macro-scale function of soft and highly heterogeneous materials of both natural and synthetic origins.

Adhesion of spider cribellate silk enhanced in high humidity by mechanical plasticization of the underlying fiber.

The disruptive nature of water presents a significant challenge when designing synthetic adhesives that maintain functionality in wet conditions. However, many animal adhesives can withstand high humidity or underwater conditions, and some are even enhanced by them. An understudied mechanism in such systems is the influence of material plasticization by water to induce adhesive work through deformation. Cribellate silk is a dry adhesive used by particular spiders to capture moving prey. It presents as a candidate for testing the water plasticization model as it can remain functional at high humidity despite lacking an aqueous component. We performed herein tensile and adhesion tests on cribellate threads from the spider, Hickmania troglodytes; a spider that lives within wet cave environments. We found that the work of adhesion of its cribellate threads increased as the axial fibre deformed during pull-off experiments. This effect was enhanced when the silk was wetted and as spider body size increased. Dry threads on the other hand were stiff with low adhesion. We rationalized our experiments by a series of scaling law models. We concluded that these cribellate threads operate best when the nanofibrils and axial fibers both contribute to adhesion. Design of future synthetic materials could draw inspiration from how water facilitates, rather than diminishes, cribellate silk adhesion.

Meta-analysis reveals materiomic relationships in major ampullate silk across the spider phylogeny.

Spider major ampullate (MA) silk, with its combination of strength and extensibility, outperforms any synthetic equivalents. There is thus much interest in understanding its underlying materiome. While the expression of the different silk proteins (spidroins) appears an integral component of silk performance, our understanding of the nature of the relationship between the spidroins, their constituent amino acids and MA silk mechanics is ambiguous. To provide clarity on these relationships across spider species, we performed a meta-analysis using phylogenetic comparative methods. These showed that glycine and proline, both of which are indicators of differential spidroin expression, had effects on MA silk mechanics across the phylogeny. We also found serine to correlate with silk mechanics, probably via its presence within the carboxyl and amino-terminal domains of the spidroins. From our analyses, we concluded that the spidroin expression shifts across the phylogeny from predominantly MaSp1 in the MA silks of ancestral spiders to predominantly MaSp2 in the more derived spiders' silks. This trend was accompanied by an enhanced ultimate strain and decreased Young's modulus in the silks. Our meta-analysis enabled us to decipher between real and apparent influences on MA silk properties, providing significant insights into spider silk and web coevolution and enhancing our capacity to create spider silk-like materials.

Photoreflectance/scattering measurements of spider silks informed by standard optics.

The silks of certain orb weaving spiders are emerging as high-quality optical materials. This motivates study of the optical properties of such silk and particularly the comparative optical properties of the silks of different species. Any differences in optical properties may impart biological advantage for a spider species and make the silks interesting for biomimetic prospecting as optical materials. A prior study of the reflectance of spider silks from 18 species reported results for three species of modern orb weaving spiders (Nephila clavipes, Argiope argentata and Micrathena Schreibersi) as having reduced reflectance in the UV range. (Modern in the context used here means more recently derived.) The reduced UV reflectance was interpreted as an adaptive advantage in making the silks less visible to insects. Herein, a standard, experimental technique for measuring the reflectance spectrum of diffuse surfaces, using commercially available equipment, has been applied to samples of the silks of four modern species of orb weaving spiders: Phonognatha graeffei, Eriophora transmarina, Nephila plumipes and Argiope keyserlingi. This is a different technique than used in the previous study. Three of the four silks measured have a reduced signal in the UV. By taking the form of the silks as optical elements into account, it is shown that this is attributable to a combination of wavelength-dependent absorption and scattering by the silks rather than differences in reflectance for the different silks. Phonognatha graeffei dragline silk emerges as a very interesting spider silk with a flat 'reflectance'/scattering spectrum which may indicate it is a low UV absorbing dielectric micro-fibre. Overall the measurement emerges as having the potential to compare the large numbers of silks from different species to prospect for those which have desirable optical properties.

Author Correction: Silk physico-chemical variability and mechanical robustness facilitates intercontinental invasibility of a spider.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Silk physico-chemical variability and mechanical robustness facilitates intercontinental invasibility of a spider.

There are substantive problems associated with invasive species, including threats to endemic organisms and biodiversity. Understanding the mechanisms driving invasions is thus critical. Variable extended phenotypes may enable animals to invade into novel environments. We explored here the proposition that silk variability is a facilitator of invasive success for the highly invasive Australian house spider, Badumna longinqua. We compared the physico-chemical and mechanical properties and underlying gene expressions of its major ampullate (MA) silk between a native Sydney population and an invasive counterpart from Montevideo, Uruguay. We found that while differential gene expressions might explain the differences in silk amino acid compositions and protein nanostructures, we did not find any significant differences in silk mechanical properties across the populations. Our results accordingly suggest that B. longinqua's silk remains functionally robust despite underlying physico-chemical and genetic variability as the spider expands its range across continents. They also imply that a combination of silk physico-chemical plasticity combined with mechanical robustness might contribute more broadly to spider invasibilities.

Spider silk colour covaries with thermal properties but not protein structure.

Understanding how and why animal secretions vary in property has important biomimetic implications as desirable properties might covary. Spider major ampullate (MA) silk, for instance, is a secretion earmarked for biomimetic applications, but many of its properties vary among and between species across environments. Here, we tested the hypothesis that MA silk colour, protein structure and thermal properties covary when protein uptake is manipulated in the spider Trichonephila plumipes. We collected silk from adult female spiders maintained on a protein-fed or protein-deprived diet. Based on spectrophotometric quantifications, we classified half the silks as 'bee visible' and the other half 'bee invisible'. Wide angle X-ray diffraction and differential scanning calorimetry were then used to assess the silk's protein structure and thermal properties, respectively. We found that although protein structures and thermal properties varied across our treatments only the thermal properties covaried with colour. This ultimately suggests that protein structure alone is not responsible for MA silk thermal properties, nor does it affect silk colours. We speculate that similar ecological factors act on silk colour and thermal properties, which should be uncovered to inform biomimetic programmes.

DNP NMR spectroscopy reveals new structures, residues and interactions in wild spider silks.

DNP solid state NMR spectroscopy allows non-targeted analysis of wild spider silk in unprecedented detail at natural abundance, revealing hitherto unreported features across several species. A >50-fold signal enhancement for each silk, enables the detection of novel H-bonding networks and arginine conformations, and the post-translational modified amino acid, hydroxyproline.

Web building and silk properties functionally covary among species of wolf spider.

Although phylogenetic studies have shown covariation between the properties of spider major ampullate (MA) silk and web building, both spider webs and silks are highly plastic so we cannot be sure whether these traits functionally covary or just vary across environments that the spiders occupy. As MaSp2-like proteins provide MA silk with greater extensibility, their presence is considered necessary for spider webs to effectively capture prey. Wolf spiders (Lycosidae) are predominantly non-web building, but a select few species build webs. We accordingly collected MA silk from two web-building and six non-web-building species found in semirural ecosystems in Uruguay to test whether the presence of MaSp2-like proteins (indicated by amino acid composition, silk mechanical properties and silk nanostructures) was associated with web building across the group. The web-building and non-web-building species were from disparate subfamilies so we estimated a genetic phylogeny to perform appropriate comparisons. For all of the properties measured, we found differences between web-building and non-web-building species. A phylogenetic regression model confirmed that web building and not phylogenetic inertia influences silk properties. Our study definitively showed an ecological influence over spider silk properties. We expect that the presence of the MaSp2-like proteins and the subsequent nanostructures improves the mechanical performance of silks within the webs. Our study furthers our understanding of spider web and silk co-evolution and the ecological implications of spider silk properties.

Multiscale mechanisms of nutritionally induced property variation in spider silks.

Variability in spider major ampullate (MA) silk properties at different scales has proven difficult to determine and remains an obstacle to the development of synthetic fibers mimicking MA silk performance. A multitude of techniques may be used to measure multiscale aspects of silk properties. Here we fed five species of Araneoid spider solutions that either contained protein or were protein deprived and performed silk tensile tests, small and wide-angle X-ray scattering (SAXS/WAXS), amino acid composition analyses, and silk gene expression analyses, to resolve persistent questions about how nutrient deprivation induces variations in MA silk mechanical properties across scales. Our analyses found that the properties of each spider's silk varied differently in response to variations in their protein intake. We found changes in the crystalline and non-crystalline nanostructures to play specific roles in inducing the property variations we found. Across treatment MaSp expression patterns differed in each of the five species. We found that in most species MaSp expression and amino acid composition variations did not conform with our predictions based on a traditional MaSp expression model. In general, changes to the silk's alanine and proline compositions influenced the alignment of the proteins within the silk's amorphous region, which influenced silk extensibility and toughness. Variations in structural alignment in the crystalline and non-crystalline regions influenced ultimate strength independent of genetic expression. Our study provides the deepest insights thus far into the mechanisms of how MA silk properties vary from gene expression to nanostructure formations to fiber mechanics. Such knowledge is imperative for promoting the production of synthetic silk fibers.

Fitness consequences of plasticity in an extended phenotype.

Like regular phenotypes, extended phenotypes have demonstrable fitness advantages and their properties may vary plastically across environments. However, the fitness advantages of plasticity are only known for a select few extended phenotypes. It is known that the form and functions of spider orb webs can be manipulated by laboratory experiments. For instance, the physical and chemical properties of the spiral and gluey silks vary in property as protein intake varies. Orb web spiders thus represent good models for extended phenotypic plasticity studies. We performed experiments manipulating the protein intake of two vertically aligned orb web building spiders to determine whether variations in the chemical and physical properties of their spiral and gluey silk affect prey retention in their webs. We found in both spider species that individuals deprived of protein had a greater gluey silk glycoprotein core volume, and this correlated strongly with spiral thread stickiness and increased prey retention by the webs. Moreover, we found strong positive correlations between glue droplet volume and glycoprotein core volume for spiders in the protein-deprived treatment, but weaker correlations for protein-fed spiders. We interpreted these findings as the spiders investing more in glycoprotein when nutrient deprived. We attribute the associated increase in prey retention capacity as a fitness consequence of plasticity in the spiral properties.

Clarity of objectives and working principles enhances the success of biomimetic programs.

Biomimetics, the transfer of functional principles from living systems into product designs, is increasingly being utilized by engineers. Nevertheless, recurring problems must be overcome if it is to avoid becoming a short-lived fad. Here we assess the efficiency and suitability of methods typically employed by examining three flagship examples of biomimetic design approaches from different disciplines: (1) the creation of gecko-inspired adhesives; (2) the synthesis of spider silk, and (3) the derivation of computer algorithms from natural self-organizing systems. We find that identification of the elemental working principles is the most crucial step in the biomimetic design process. It bears the highest risk of failure (e.g. losing the target function) due to false assumptions about the working principle. Common problems that hamper successful implementation are: (i) a discrepancy between biological functions and the desired properties of the product, (ii) uncertainty about objectives and applications, (iii) inherent limits in methodologies, and (iv) false assumptions about the biology of the models. Projects that aim for multi-functional products are particularly challenging to accomplish. We suggest a simplification, modularisation and specification of objectives, and a critical assessment of the suitability of the model. Comparative analyses, experimental manipulation, and numerical simulations followed by tests of artificial models have led to the successful extraction of working principles. A searchable database of biological systems would optimize the choice of a model system in top-down approaches that start at an engineering problem. Only when biomimetic projects become more predictable will there be wider acceptance of biomimetics as an innovative problem-solving tool among engineers and industry.

Nanostructural and mechanical property changes to spider silk as a consequence of insecticide exposure.

Neonicotinoids are one of the world's most extensively used insecticides, but their sub-lethal influences on non-target and beneficial organisms are not well known. Here we exposed the orb web spider Parawixia audax, which is found on arable lands in Uruguay, to a sub-lethal concentration of the broad spectrum insecticide Geonex (thiamethoxam + lambda-cyhalothrin) and monitored their web building. We collected their major ampullate silk and subjected it to tensile tests, wide-angle X-ray diffraction (WAXS) analysis, and amino acid composition analysis. Around half of the exposed spiders failed to build webs. Those that built webs produced irregular webs lacking spiral threads. The mechanical properties, nanostructures, and amino acid compositions of the silk were all significantly affected when the spiders were exposed to insecticides. We found that silk proline, glutamine, alanine and glycine compositions differed between treatments, indicating that insecticide exposure induced downregulation of the silk protein MaSp2. The spiders in the control group had stronger, tougher and more extensible silks than those in the insecticide exposed group. Our WAXS analyses showed the amorphous region nanostructures became misaligned in insecticide exposed silks, explaining their greater stiffness. While the insecticide dose we subjected P. audax to was evidently sub-lethal, the changes in silk physicochemical properties and the impairment to web building will indelibly affect their ability to catch prey.

Diet-induced co-variation between architectural and physicochemical plasticity in an extended phenotype.

The adaptive benefits of extended phenotypic plasticity are imprecisely defined due to a paucity of experiments examining traits that are manipulable and measurable across environments. Spider webs are often used as models to explore the adaptive benefits of variations in extended phenotypes across environments. Nonetheless, our understanding of the adaptive nature of the plastic responses of spider webs is impeded when web architectures and silk physicochemical properties appear to co-vary. An opportunity to examine this co-variation is presented by modifying prey items while measuring web architectures and silk physiochemical properties. Here, we performed two experiments to assess the nature of the association between web architectures and gluey silk properties when the orb web spider Argiope keyserlingi was fed a diet that varied in either mass and energy or prey size and feeding frequency. We found web architectures and gluey silk physicochemical properties to co-vary across treatments in both experiments. Specifically, web capture area co-varied with gluey droplet morphometrics, thread stickiness and salt concentrations when prey mass and energy were manipulated, and spiral spacing co-varied with gluey silk salt concentrations when prey size and feeding frequency were manipulated. We explained our results as A. keyserlingi plastically shifting its foraging strategy as multiple prey parameters simultaneously varied. We confirmed and extended previous work by showing that spiders use a variety of prey cues to concurrently adjust web and silk traits across different feeding regimes.

Physicochemical Property Variation in Spider Silk: Ecology, Evolution, and Synthetic Production.

The unique combination of great stiffness, strength, and extensibility makes spider major ampullate (MA) silk desirable for various biomimetic and synthetic applications. Intensive research on the genetics, biochemistry, and biomechanics of this material has facilitated a thorough understanding of its properties at various levels. Nevertheless, methods such as cloning, recombination, and electrospinning have not successfully produced materials with properties as impressive as those of spider silk. It is nevertheless becoming clear that silk properties are a consequence of whole-organism interactions with the environment in addition to genetic expression, gland biochemistry, and spinning processes. Here we assimilate the research done and assess the techniques used to determine distinct forms of spider silk chemical and physical property variability. We suggest that more research should focus on testing hypotheses that explain spider silk property variations in ecological and evolutionary contexts.