Bacteria inhabiting spider webs enhance host silk extensibility.

Spider silk is a promising material with great potential in biomedical applications due to its incredible mechanical properties and resistance to degradation of commercially available bacterial strains. However, little is known about the bacterial communities that may inhabit spider webs and how these microorganisms interact with spider silk. In this study, we exposed two exopolysaccharide-secreting bacteria, isolated from webs of an orb spider, to major ampullate (MA) silk from host spiders. The naturally occurring lipid and glycoprotein surface layers of MA silk were experimentally removed to further probe the interaction between bacteria and silk. Extensibility of major ampullate silk produced by Triconephila clavata that was exposed to either Microbacterium sp. or Novosphigobium sp. was significantly higher than that of silk that was not exposed to bacteria (differed by 58.7%). This strain-enhancing effect was not observed when the lipid and glycoprotein surface layers of MA silks were removed. The presence of exopolysaccharides was detected through NMR from MA silks exposed to these two bacteria but not from those without exposure. Here we report for the first time that exopolysaccharide-secreting bacteria inhabiting spider webs can enhance extensibility of host MA silks and silk surface layers play a vital role in mediating such effects.

Alkaline water as a potential agent for biting midge control: Managing effectiveness and non-target organism impact evaluation.

Biting midge Forcipomyia taiwana is one of the common pests in East Asia. Their nuisance and blood-sucking behavior causes problems not only for human health but also for some industries. This study aims to evaluate the effectiveness of spraying alkaline water on controlling biting midge population and potential side effects of such approach on non-target organisms. Laboratory experiments were conducted to evaluate the effect of alkaline water on oviposition site preference of female biting midges as well as crickets. Effect of alkaline water on distribution pattern of earthworms was also examined. Besides, we also performed field manipulative studies by long term spraying of alkaline water to evaluate the effects on biting midge density, microalgae abundance and ground arthropod communities. The results of laboratory experiments showed that female biting midges laid significantly fewer eggs in surface treated with alkaline water. However, alkaline water treatment did not significantly affect the oviposition site choice of crickets and distribution pattern of earthworms. Result of field manipulations showed that long-term spraying of alkaline water could significantly reduce the abundance of soil microalgae and density of biting midges, but did not affect the diversity of non-target ground arthropods. These results demonstrate that long-term spraying of alkaline water could decrease biting midge density without harming co-existing non-target organisms and therefore is a potentially eco-friendly approach to control such pest.

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.

Size-related increase in inducible mechanical variability of major ampullate silk in a huntsman spider (Araneae: Sparassidae).

Most spiders use major ampullate silk (MAS) to perform many functions across their lifetimes, including prey capture, vibratory signal detection, and safety/dragline. To accommodate their various needs, adult spiders can use inducible variability to tailor MAS with specific mechanical properties. However, it is currently unknown whether this inducible mechanical variability develops gradually or remains consistent across spider size. Supercontraction -a process by which "native-state" silk fibers axially shrink when exposed to water or high humidity-can be used to reveal the extent of inducible variability in MAS. Supercontraction removes some processing effects that occur during the spinning of the solid fiber from its liquid precursor by allowing a native-state MAS fiber to return to a low energy "ground-state". Here, we examined the relative extent of inducible variability of MAS across spider size by assessing supercontraction properties and the difference between ground- and native-state MAS tensile properties using silk from the huntsman spider Heteropoda venatoria (Sparassidae). Stiffness of forcibly pulled native-state silk increased by 200% with spider size. After exposure to 90% RH and subsequent supercontraction, axial shrinkage of native-state silk fibers increased by 15% in larger spiders. Supercontracted, ground-state fibers demonstrated a 200% increase in extensibility across spider size. Our results indicate a gradual increase in inducible variability of MAS mechanical properties across spider size potentially caused by shifts in internal processing or chemical composition. These findings imply both development of inducible variability and changes in use of MAS as a safety line or aiding jumps across a spider's lifetime.

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.

Mechanical and structural properties of major ampullate silk from spiders fed carbon nanomaterials.

The dragline silk of spiders is of particular interest to science due to its unique properties that make it an exceptional biomaterial that has both high tensile strength and elasticity. To improve these natural fibers, researchers have begun to try infusing metals and carbon nanomaterials to improve mechanical properties of spider silk. The objective of this study was to incorporate carbon nanomaterials into the silk of an orb-weaving spider, Nephila pilipes, by feeding them solutions containing graphene and carbon nanotubes. Spiders were collected from the field and in the lab were fed solutions by pipette containing either graphene sheets or nanotubes. Major ampullate silk was collected and a tensile tester was used to determine mechanical properties for pre- and post-treatment samples. Raman spectroscopy was then used to test for the presence of nanomaterials in silk samples. There was no apparent incorporation of carbon nanomaterials in the silk fibers that could be detected with Raman spectroscopy and there were no significant improvements in mechanical properties. This study represents an example for the importance of attempting to replicate previously published research. Researchers should be encouraged to continue to do these types of investigations in order to build a strong consensus and solid foundation for how to go forward with these new methods for creating novel biomaterials.

Hydrothermal Effect on Mechanical Properties of Nephila pilipes Spidroin.

The superlative mechanical properties of spider silk and its conspicuous variations have instigated significant interest over the past few years. However, current attempts to synthetically spin spider silk fibers often yield an inferior physical performance, owing to the improper molecular interactions of silk proteins. Considering this, herein, a post-treatment process to reorganize molecular structures and improve the physical strength of spider silk is reported. The major ampullate dragline silk from Nephila pilipes with a high ß-sheet content and an adequate tensile strength was utilized as the study material, while that from Cyrtophora moluccensis was regarded as a reference. Our results indicated that the hydrothermal post-treatment (50-70 °C) of natural spider silk could effectively induce the alternation of secondary structures (random coil to ß-sheet) and increase the overall tensile strength of the silk. Such advantageous post-treatment strategy when applied to regenerated spider silk also leads to an increment in the strength by ~2.5-3.0 folds, recapitulating ~90% of the strength of native spider silk. Overall, this study provides a facile and effective post-spinning means for enhancing the molecular structures and mechanical properties of as-spun silk threads, both natural and regenerated.

Uncoiling springs promote mechanical functionality of spider cribellate silk.

Composites, both natural and synthetic, achieve novel functionality by combining two or more constituent materials. For example, the earliest adhesive silk in spider webs - cribellate silk - is composed of stiff axial fibers and coiled fibers surrounded by hundreds of sticky cribellate nanofibrils. Yet, little is known of how fiber types interact to enable capture of insect prey with cribellate silk. To understand the roles of each constituent fiber during prey capture, we compared the tensile performance of native-state and manipulated threads produced by the cribellate spider Psechrus clavis, and the adhesion of native threads along a smooth surface and hairy bee thorax. We found that the coiled fiber increases the work to fracture of the entire cribellate thread by up to 20-fold. We also found that the axial fiber breaks multiple times during deformation, an unexpected observation that indicates: (i) the axial fiber continues to contribute work even after breakage, and (ii) the cribellate nanofibrils may perform a previously unidentified role as a binder material that distributes forces throughout the thread. Work of adhesion increased on surfaces with more surface structures (hairy bee thorax) corresponding to increased deformation of the coiled fiber. Together, our observations highlight how the synergistic interactions among the constituents of this natural composite adhesive enhance functionality. These highly extensible threads may serve to expose additional cribellate nanofibrils to form attachment points with prey substrata while also immobilizing prey as they sink into the web due to gravity.

Nitrogen inaccessibility protects spider silk from bacterial growth.

Spider silks are protein-based fibers that are incorporated into webs with the unique combination of high mechanical toughness and resistance to microbial degradation. While spiders are undoubtedly exposed to saprophytic microorganisms in their native habitats, such as the forest understory and bush, their silks have rarely been observed to decompose in either field or laboratory studies. We performed cross-streaking assays using silk from three spider species and four bacterial strains and found no inhibition zones, indicating the absence of antibacterial properties. We also cultured all bacteria directly upon silk in Luria-Bertani (LB) broth (full nutrients), phosphate-buffered saline (PBS; no nutrients) and nitrogen-free glucose broth (NFG; full nutrients, no nitrogen), and found that bacteria grew readily on silk in LB broth but not in PBS or NFG buffer. Our results indicate that spider silk's resistance to bacterial degradation is likely due to bacteriostatic rather than antibacterial mechanisms when nitrogen is inaccessible.

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.

Nutrient intake determines post-maturity molting in the golden orb-web spider Nephila pilipes (Araneae: Araneidae).

While molting occurs in the development of many animals, especially arthropods, post-maturity molting (PMM, organisms continue to molt after sexual maturity) has received little attention. The mechanism of molting has been studied intensively; however, the mechanism of PMM remains unknown although it is suggested to be crucial for the development of body size. In this study, we investigated factors that potentially induce PMM in the golden orb-web spider Nephila pilipes, which has the greatest degree of sexual dimorphism among terrestrial animals. We manipulated the mating history and the nutrient consumption of the females to examine whether they affect PMM. The results showed that female spiders under low nutrition were more likely to molt as adults, and mating had no significant influence on the occurrence of PMM. Moreover, spiders that underwent PMM lived longer than those that did not and their body sizes were significantly increased. Therefore, we concluded that nutritional condition rather than mating history affect PMM.

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.

Evidence of Decoupling Protein Structure from Spidroin Expression in Spider Dragline Silks.

The exceptional strength and extensibility of spider dragline silk have been thought to be facilitated by two spidroins, major ampullate spidroin 1 (MaSp1) and major ampullate spidroin 2 (MaSp2), under the assumption that protein secondary structures are coupled with the expressed spidroins. We tested this assumption for the dragline silk of three co-existing Australian spiders, Argiope keyserlingi, Latrodectus hasselti and Nephila plumipes. We found that silk amino acid compositions did not differ among spiders collected in May. We extended these analyses temporally and found the amino acid compositions of A. keyserlingi silks to differ when collected in May compared to November, while those of L. hasselti did not. To ascertain whether their secondary structures were decoupled from spidroin expression, we performed solid-state nuclear magnetic resonance spectroscopy (NMR) analysis on the silks of all spiders collected in May. We found the distribution of alanine toward ß-sheet and 3,10helix/random coil conformations differed between species, as did their relative crystallinities, with A. keyserlingi having the greatest 3,10helix/random coil composition and N. plumipes the greatest crystallinity. The protein secondary structures correlated with the mechanical properties for each of the silks better than the amino acid compositions. Our findings suggested that a differential distribution of alanine during spinning could decouple secondary structures from spidroin expression ensuring that silks of desirable mechanical properties are consistently produced. Alternative explanations include the possibility that other spidroins were incorporated into some silks.

Spider web and silk performance landscapes across nutrient space.

Predators have been shown to alter their foraging as a regulatory response to recent feeding history, but it remains unknown whether trap building predators modulate their traps similarly as a regulatory strategy. Here we fed the orb web spider Nephila pilipes either live crickets, dead crickets with webs stimulated by flies, or dead crickets without web stimulation, over 21 days to enforce spiders to differentially extract nutrients from a single prey source. In addition to the nutrients extracted we measured web architectures, silk tensile properties, silk amino acid compositions, and web tension after each feeding round. We then plotted web and silk "performance landscapes" across nutrient space. The landscapes had multiple peaks and troughs for each web and silk performance parameter. The findings suggest that N. pilipes plastically adjusts the chemical and physical properties of their web and silk in accordance with its nutritional history. Our study expands the application of the geometric framework foraging model to include a type of predatory trap. Whether it can be applied to other predatory traps requires further testing.

Can differential nutrient extraction explain property variations in a predatory trap?

Predators exhibit flexible foraging to facilitate taking prey that offer important nutrients. Because trap-building predators have limited control over the prey they encounter, differential nutrient extraction and trap architectural flexibility may be used as a means of prey selection. Here, we tested whether differential nutrient extraction induces flexibility in architecture and stickiness of a spider's web by feeding Nephila pilipes live crickets (CC), live flies (FF), dead crickets with the web stimulated by flies (CD) or dead flies with the web stimulated by crickets (FD). Spiders in the CD group consumed less protein per mass of lipid or carbohydrate, and spiders in the FF group consumed less carbohydrates per mass of protein. Spiders from the CD group built stickier webs that used less silk, whereas spiders in the FF group built webs with more radii, greater catching areas and more silk, compared with other treatments. Our results suggest that differential nutrient extraction is a likely explanation for prey-induced spider web architecture and stickiness variations.

Crypsis via leg clustering: twig masquerading in a spider.

The role of background matching in camouflage has been extensively studied. However, contour modification has received far less attention, especially in twig-mimicking species. Here, we studied this deceptive strategy by revealing a special masquerade tactic, in which the animals protract and cluster their legs linearly in the same axis with their bodies when resting, using the spider Ariamnes cylindrogaster as a model. We used cardboard papers to construct dummies resembling spiders in appearance and colour. To differentiate the most important factors in the concealment effect, we manipulated body size (long or short abdomen) and resting postures (leg clustered or spread) of the dummies and recorded the responses of predators to different dummy types in the field. The results showed that dummies with clustered legs received significantly less attention from predators, regardless of the body length. Thus, we conclude that A. cylindrogaster relies on the resting posture rather than body size for predator avoidance. This study provides, to the best of our knowledge, empirical evidence for the first time that twig-mimicking species can achieve effective camouflage by contour modification.

Top down and bottom up selection drives variations in frequency and form of a visual signal.

The frequency and form of visual signals can be shaped by selection from predators, prey or both. When a signal simultaneously attracts predators and prey selection may favour a strategy that minimizes risks while attracting prey. Accordingly, varying the frequency and form of the silken decorations added to their web may be a way that Argiope spiders minimize predation while attracting prey. Nonetheless, the role of extraneous factors renders the influences of top down and bottom up selection on decoration frequency and form variation difficult to discern. Here we used dummy spiders and decorations to simulate four possible strategies that the spider Argiope aemula may choose and measured the prey and predator attraction consequences for each in the field. The strategy of decorating at a high frequency with a variable form attracted the most prey, while that of decorating at a high frequency with a fixed form attracted the most predators. These results suggest that mitigating the cost of attracting predators while maintaining prey attraction drives the use of variation in decoration form by many Argiope spp. when decorating frequently. Our study highlights the importance of considering top-down and bottom up selection pressure when devising evolutionary ecology experiments.

Mechanical performance of spider silk is robust to nutrient-mediated changes in protein composition.

Spider major ampullate (MA) silk is sought after as a biomimetic because of its high strength and extensibility. While the secondary structures of MA silk proteins (spidroins) influences silk mechanics, structural variations induced by spinning processes have additional effects. Silk properties may be induced by spiders feeding on diets that vary in certain nutrients, thus providing researchers an opportunity to assess the interplay between spidroin chemistry and spinning processes on the performance of MA silk. Here, we determined the relative influence of spidroin expression and spinning processes on MA silk mechanics when Nephila pilipes were fed solutions with or without protein. We found that spidroin expression differed across treatments but that its influence on mechanics was minimal. Mechanical tests of supercontracted fibers and X-ray diffraction analyses revealed that increased alignment in the amorphous region and to a lesser extent in the crystalline region led to increased fiber strength and extensibility in spiders on protein rich diets.

Evidence of bird dropping masquerading by a spider to avoid predators.

Masquerading comes at various costs and benefits. The principal benefit being the avoidance of predators. The orb-web spider Cyclosa ginnaga has a silver body and adds a white discoid-shaped silk decoration to its web. The size, shape and colour of C. ginnaga's body resemble, when viewed by the human eye against its decoration, a bird dropping. We therefore hypothesized that their body colouration might combine with its web decoration to form a bird dropping masquerade to protect it from predators. We measured the spectral reflectance of: (i) the spider's body, (ii) the web decoration, and (iii) bird droppings, in the field against a natural background and found that the colour of the spider bodies and decorations were indistinguishable from each other and from bird droppings when viewed by hymentopteran predators. We monitored the predatory attacks on C. ginnaga when the spider's body and/or its decorations were blackened and found that predator attack probabilities were greater when only the decorations were blackened. Accordingly, we concluded that C. ginnaga's decoration and body colouration forms a bird dropping masquerade, which reduces its probability of predation.

Nutrient deprivation induces property variations in spider gluey silk.

Understanding the mechanisms facilitating property variability in biological adhesives may promote biomimetic innovations. Spider gluey silks such as the spiral threads in orb webs and the gumfoot threads in cobwebs, both of which comprise of an axial thread coated by glue, are biological adhesives that have variable physical and chemical properties. Studies show that the physical and chemical properties of orb web gluey threads change when spiders are deprived of food. It is, however, unknown whether gumfoot threads undergo similar property variations when under nutritional stress. Here we tested whether protein deprivation induces similar variations in spiral and gumfoot thread morphology and stickiness. We manipulated protein intake for the orb web spider Nephila clavipes and the cobweb spider Latrodectus hesperus and measured the diameter, glue droplet volume, number of droplets per mm, axial thread width, thread stickiness and adhesive energy of their gluey silks. We found that the gluey silks of both species were stickier when the spiders were deprived of protein than when the spiders were fed protein. In N. clavipes a concomitant increase in glue droplet volume was found. Load-extension curves showed that protein deprivation induced glue property variations independent of the axial thread extensions in both species. We predicted that changes in salt composition of the glues were primarily responsible for the changes in stickiness of the silks, although changes in axial thread properties might also contribute. We, additionally, showed that N. clavipes' glue changes color under protein deprivation, probably as a consequence of changes to its biochemical composition.