Biomechanical variation of silk links spinning plasticity to spider web function

Spider silk is renowned for its high tensile strength, extensibility and toughness. However, the variability of these material properties has largely been ignored, especially at the intra-specific level. Yet, this variation could help us understand the function of spider webs. It may also point to the mechanisms used by spiders to control their silk production, which could be exploited to expand the potential range of applications for silk. In this study, we focus on variation of silk properties within different regions of cobwebs spun by the common house spider, Achaearanea tepidariorum. The cobweb is composed of supporting threads that function to maintain the web shape and hold spiders and prey, and of sticky gumfooted threads that adhere to insects during prey capture. Overall, structural properties, especially thread diameter, are more variable than intrinsic material properties, which may reflect past directional selection on certain silk performance. Supporting threads are thicker and able to bear higher loads, both before deforming permanently and before breaking, compared with sticky gumfooted threads. This may facilitate the function of supporting threads through sustained periods of time. In contrast, sticky gumfooted threads are more elastic, which may reduce the forces that prey apply to webs and allow them to contact multiple sticky capture threads. Therefore, our study suggests that spiders actively modify silk material properties during spinning in ways that enhance web function.     

Egg sac silk of Theridiosoma gemmosum (Araneae: Theridiosomatidae)

Cocoons of Theridiosoma gemmosum consist of two main parts, the egg sac case and the stalk. The inner space of the egg sac case is filled with nonsticky flocculent silk. Measuring 600–800 nm in diameter, the flocculent threads are never made up of bundles of longitudinally oriented nanofibrils. The egg case wall consists of a lower layer of highly ordered threads and an upper layer of cover silk. The lower, permanently white layer consists of threads in a mesh‐like arrangement, the thicker threads being 4–6 μm and the thinner threads being 2–3 μm in diameter. Each thread is a bundle of parallel nanofibrils, with a diameter between 150 and 300 nm. The silk secretions of these fibers, emitted from spigots, are processed by legs. The upper layer of the egg case is applied to the threads of the lower layer by direct rubbing against its surface, i.e. without the use of legs. In the lower and middle part of the egg case, the accumulated secretion forms a virtually compact encrustation, whereas in the upper, conically shaped, part of the egg case where it becomes the stalk, this secretion becomes substantially scarcer. The stalk is a continuation of the egg case, its proximal part made of fibers similar to those forming the inner layer of the egg case wall. The distal part of the stalk continues towards the suspension area either as a compact bundle of parallel fibers, or the stalk forks into two bundles of roughly the same thickness, which continue towards the suspension area separately. On the surface of objects onto which cocoons are attached, the secretion of the piriform glands acts as an adhesive sheet. J. Morphol. 2009. © 2009 Wiley‐Liss, Inc.     

棒络新妇和悦目金蛛拖丝超微结构与力学行为

采用SEM对棒络新妇Nephila clavata腹部向上和向下在水平纱窗上爬行时纺出的拖丝、悦目金蛛Argiope amoena捕食拖丝与垂直向下缓慢纺出的拖丝及其圆网的铆钉丝进行了超微结构观察,采用电子单纤强力仪对棒络新妇拖丝与悦目金蛛圆网铆钉丝进行了力学拉伸试验.结果 表明棒络新妇和悦目金蛛拖丝均呈现出一至多根细丝纤维的多样化超微结构特征,其中悦目金蛛圆网铆钉丝还呈现出"S"形似弹簧的结构.两种蜘蛛丝的力学行为和性能与各自的功能要求相一致.蜘蛛能调节拖丝的超微结构、纤维组成和直径大小以适应其在不同环境条件下对力学性能和功能的瞬时需要.研究结果有助于拓宽和加深人们对蜘蛛丝超微结构、力学性能与生物学功能之间关系的认识和理解.     

Adhesive efficiency of spider prey capture threads

Cribellar capture threads are comprised of thousands of fine silk fibrils that are produced by the spigots of a spider's cribellum spinning plate and are supported by larger interior axial fibers. This study examined factors that constrain the stickiness of cribellar threads spun by members of the orb-weaving family Uloboridae in the Deinopoidea clade and compared the material efficiency of these threads with that of viscous capture threads produced by members of their sister clade, the Araneoidea. An independent contrast analysis confirmed the direct relationship between cribellar spigot number and cribellar thread stickiness. A model based on this relationship showed that cribellar thread stickiness is achieved at a rapidly decreasing material efficiency, as measured in terms of stickiness per spigot. Another limitation of cribellar thread was documented when the threads of two uloborid species were measured with contact plates of four widths. Unlike that of viscous threads, the stickiness of cribellar threads did not increase as plate width increased, indicating that only narrow bands along the edges of thread contact contributed to their stickiness. As thread volume increased, the gross material efficiency of cribellar threads decreased much more rapidly than that of viscous threads. However, cribellar threads achieved their stickiness at a much greater gross material efficiency than did viscous threads, making it more challenging to explain the transition from deinopoid to araneoid orb-webs.     

Investigating the ultrastructure of fibrous long spacing collagen by parallel atomic force and transmission electron microscopy

The ultrastructure of fibrous long spacing (FLS) collagen fibrils has been investigated by performing both atomic force microscopy (AFM) and transmission electron microscopy (TEM) on exactly the same area of FLS collagen fibril samples. These FLS collagen fibrils were formed in vitro from type I collagen and alpha1-acid glycoprotein (AAG) solutions. On the basis of the correlated AFM and TEM images obtained before and after negative staining, the periodic dark bands observed in TEM images along the longitudinal axis of the FLS collagen fibril correspond directly to periodic protrusions seen by AFM. This observation is in agreement with the original surmise made by Gross, Highberger, and Schmitt (Gross J, Highberger JH, Schmitt FO, Proc Natl Acad Sci USA 1954;40:679-688) that the major repeating dark bands of FLS collagen fibrils observed under TEM are thick relative to the interband region. Although these results do not refute the idea of negative stain penetration into gap regions proposed by Hodge and Petruska (Petruska JA, Hodge AJ. Aspects of protein structure. Ramachandran GN, editor. New York: Academic Press; 1963. p. 289-300), there is no need to invoke the presence of gap regions to explain the periodic dark bands observed in TEM images of FLS collagen fibrils.     

Fibrous long spacing type collagen fibrils have a hierarchical internal structure

Nanodissection of single fibrous long spacing (FLS) type collagen fibrils by atomic force microscopy (AFM) reveals hierarchical internal structure: Fibrillar subcomponents with diameters of approximately 10 to 20 nm were observed to be running parallel to the long axis of the fibril in which they are found. The fibrillar subcomponent displayed protrusions with characteristic approximately 270 nm periodicity, such that protrusions on neighboring subfibrils were aligned in register. Hence, the banding pattern of mature FLS-type collagen fibrils arises from the in-register alignment of these fibrillar subcomponents. This hierarchical organization observed in FLS-type collagen fibrils is different from that previously reported for native-type collagen fibrils, displaying no supercoiling at the level of organization observed.     

Aspects of Mineral Structure in Normally Calcifying Avian Tendon

Structural characteristics of normally calcifying leg tendons of the domestic turkey Meleagris gallopavo have been observed for the first time by tapping mode atomic force microscopy (TMAFM), and phase as well as corresponding topographic images were acquired to gain insight into the features of mineralizing collagen fibrils and fibers. Analysis of different regions of the tendon has yielded new information concerning the structural interrelationships in vivo between collagen fibrils and fibers and mineral crystals appearing in the form of plates and plate aggregates. TMAFM images show numerous mineralized collagen structures exhibiting characteristic periodicity (54-70 nm), organized with their respective long axes parallel to each other. In some instances, mineral plates (30-40 nm thick) are found interspersed between and in intimate contact with the mineralized collagen. The edges of such plates lie parallel to the neighboring collagen. Many of these plates appear to be aligned to form larger aggregates (475-600 nm long x 75-90 nm thick) that also retain collagen periodicity along their exposed edges. Intrinsic structural properties of the mineralizing avian tendon have not previously been described on the scale reported in this study. These data provide the first visual evidence supporting the concept that larger plates form from parallel association of smaller ones, and the data fill a gap in knowledge between macromolecular- and anatomic-scale studies of the mineralization of avian tendon and connective tissues in general. The observed organization of mineralized collagen, plates, and plate aggregates maintaining a consistently parallel nature demonstrates the means by which increasing structural complexity may be achieved in a calcified tissue over greater levels of hierarchical order.     

Polarized light microscopy, variability in spider silk diameters, and the mechanical characterization of spider silk

Abstract. Spider silks possess a remarkable combination of high tensile strength and extensibility that makes them among the toughest materials known. Despite the potential exploitation of these properties in biotechnology, very few silks have ever been characterized mechanically. This is due in part to the difficulty of measuring the thin diameters of silk fibers. The largest silk fibers are only 5–10 μm in diameter and some can be as fine as 50 nm in diameter. Such narrow diameters, coupled with the refraction of light due to the anisotropic nature of crystalline regions within silk fibers, make it difficult to determine the size of silk fibers. Here, we report upon a technique that uses polarized light microscopy (PLM) to accurately and precisely characterize the diameters of spider silk fibers. We found that polarized light microscopy is as precise as scanning electron microscopy (SEM) across repeated measurements of individual samples of silk and resulted in mean diameters that were ～0.10 μm larger than those from SEM. Furthermore, we demonstrate that thread diameters within webs of individual spiders can vary by as much as 600%. Therefore, the ability of PLM to non‐invasively characterize the diameters of each individual silk fiber used in mechanical tests can provide a crucial control for natural variation in silk diameters, both within webs and among spiders.     

Changes in spinning anatomy and thread stickiness associated with the origin of orb-weaving spiders

The cribellum is an oval spinning field whose spigots produce silk fibrils that form the outer surfaces of the primitive prey capture threads found in aerial spider webs. A comparison of the cribella and cribellar capture threads of 13 species of spiders representing seven families (Amaurobiidae, Desidae, Dictynidae, Filistatidae, Neolanidae, Oecobiidae, and Uloboridae) confirms that the stickness of a cribellar thread is directly related to the number of spigots on a spider's cribellum. This comparison also demonstrates that the origin of orb-weaving spiders from ancestors that constructed less highly organized webs was associated with increases in both the weight-specific number of cribellum spigots and the weight-specific stickiness of cribellar prey capture threads. In contrast to other cribellate spiders, the number of cribellum spigots of orb-weaving species of the family Uloboridae scales to spider mass. Thus, the origin of orb-weaving spiders involved not only behavioural changes that stylized and restricted the placement of cribellar threads, but also included morphological changes that increased the stickiness of these capture threads by endowing them with more cribellar fibrils.     

The nanometer-scale structure of amyloid-Β visualized by atomic force microscopy

Amyloid-Β (AΒ) is the major protein component of neuritic plaques found in Alzheimer's disease. Evidence suggests that the physical aggregation state of AΒ directly influences neurotoxicity and specific cellular biochemical events. Atomic force microscopy (AFM) is used to investigate the three-dimensional structure of aggregated AΒ and characterize aggregate/fibril size, structure, and distribution. Aggregates are characterized by fibril length and packing densities. The packing densities correspond to the differential thickness of fiber aggregates along az axis (fiber height above thex-y imaging surface). Densely packed aggregates (≥100 nm thick) were observed. At the edges of these densely packed regions and in dispersed regions, three types of AΒ fibrils were observed. These were classified by fibril thickness into three size ranges: 2–3 nm thick, 4–6 nm thick, and 8–12 nm thick. Some of the two thicker classes of fibrils exhibited pronounced axial periodicity. Substructural features observed included fibril branching or annealing and a height periodicity which varied with fibril thickness. When identical samples were visualized with AFM and electron microscopy (EM) the thicker fibrils (4–6 nm and 8–12 nm thick) had similar morphology. In comparison, the densely packed regions of ～≥100 nm thickness observed by AFM were difficult to resolve by EM. The small, 2- to 3-nm-thick, fibrils were not observed by EM even though they were routinely imaged by AFM. These studies demonstrate that AFM imaging of AΒ fibrils can, for the first time, resolve nanometer-scale,z-axis, surface-height (thickness) fibril features. Concurrentx-y surface scans of fibrils reveal the surface submicrometer structure and organization of aggregated AΒ. Thus, when AFM imaging of AΒ is combined with, and correlated to, careful studies of cellular AΒ toxicity it may be possible to relate certain AΒ structural features to cellular neurotoxicity.     

The common house spider alters the material and mechanical properties of cobweb silk in response to different prey

Many spiders depend upon webs to capture prey. Web function results from architecture and mechanical performance of the silk. We hypothesized that the common house spider, Achaearanea tepidariorum, would alter the mechanical performance of its cobweb in response to different prey by varying the structural and material properties of its silk. We fed spiders either large, high kinetic energy crickets or small, low kinetic energy pillbugs for 1 week and then examined their freshly spun silk. We separated mechanical performance into structural and material effects. We measured both types of properties for silk threads collected directly from cobwebs to test for "tuning" of silk performance to different aspects of prey capture. We compared silk from two different functional regions of the cobweb-sticky gumfooted threads that adhere directly to prey and supporting threads that maintain web integrity. Supporting threads from cricket-fed spiders were stiffer and tougher than supporting threads from pillbug-fed spiders. Both types of silk from cricket-fed spiders broke at higher loads than silk from pillbug-fed spiders. We explain this variation using a simple model of forces exerted by prey and spiders on single threads and propose potential mechanisms for this change in material properties. Two alternative, nonexclusive, hypotheses are suggested by our data. Spiders may tune silk to different types of prey by spinning threads that are able to hold prey without deforming permanently. Alternatively, as spider's body mass differed dramatically between the two feeding regimes, spiders may tune silk to their own body mass.     

Capture thread extensibility of orb-weaving spiders: testing punctuated and associative explanations of character evolution

Spider orb-webs contain sticky prey capture threads and non-sticky support threads. Primitive orb-weavers of the Deinopoidea produce dry cribellar threads made of thousands of silk fibrils that surround supporting axial fibres, whereas the viscous threads of modern Araneoidea orb-weavers produce adhesive threads with an aqueous solution that coalesces as droplets around the axial fibres. We have previously shown that the greater diversity of the Araneoidea is phylogenetically significant and attributed this disparity to a number of advantages, considered key innovations, that adhesive thread has over cribellar thread. An important putative advantage of adhesive thread demonstrated by Kohler and Vollrath in their 1995 study is its greater extensibility, a feature that better adapts it to absorb the kinetic energy of a prey strike. However, this conclusion is based on a two-species comparison that does not take advantage of the modern comparative method that requires hypotheses to be tested in a phylogenetic context. Using a transformational analysis to examine threads produced by nine species, our study finds no support for the punctuated explanation that adhesive thread has a greater extensibility than cribellar thread. Instead, it strongly supports the associative null hypothesis that capture thread extensibility is tuned to spider mass and to architectural features of the web, including its capture area, capture spiral spacing, and capture area per radius.     

Ultrastructure of spider thread anchorages

Spiders attach silken threads to substrates by means of glue-coated nanofibers (piriform silk), spun into disc-like structures. The organization and ultrastructure of this nano-composite silk are largely unknown, despite their implications for the biomechanical function and material properties of thread anchorages. In this work, the ultrastructure of silken attachment discs was studied in representatives of four spider families with Transmission Electron Microscopy to facilitate a mechanistic understanding of piriform silk function across spiders. Based on previous findings from comparative studies of piriform silk gland morphology, we hypothesized that the fibre-glue proportion of piriform silk differs in different spiders, while the composition of fibre and glue fractions is consistent. Results confirmed large differences in the relative proportion of glue with low amounts in the orb weaver Nephila senegalensis (Araneidae) and the hunting spider Cupiennius salei (Ctenidae), larger amounts in the cobweb spider Parasteatoda tepidariorum (Theridiidae) and a complete reduction of the fibrous component in the haplogyne spider Pholcus phalangioides (Pholcidae). We rejected our hypothesis that glue ultrastructure is consistent. The glue is a colloid with polymeric and fluid fractions that strongly differ in proportions and assembly. We further confirmed that in all species studied both dragline and piriform silk fibres do not make contact with the environmental substrate. Instead, adhesion is established by a thin dense skin layer of the piriform glue. These results advance our understanding of piriform silk function and the interspecific variation of its properties, which is significant for spider biology, web function and the bioengineering of silk.     

Corytibacladius Oliveira,Messias & Santos, 1995, a junior synonym of Limnophyes Eaton, 1875 (Diptera: Chironomidae: Orthocladiinae)

Larvae of the blackfly Simulium noelleri Friederichs aggregate at high populatio n densities on dams and spillways at the outlet of ponds. When displaced into the water column from their point of attachment, larvae can secrete silk threads as "life-lines" which enable them to maintain a link to the substratum an d up which they can climb to regain their original position. Experiments were conducted in a laboratory pond outlet to investigate this use of silk threads, larvae being displaced by means of forceps. It was demonstrated that: (i) the length of thread produced , and the speed of climbing the thread are independent of larval size; (ii) within limits, the speed of climbing was independent of both the length of the thread and the time already spent climbing, and (iii) speed of climb became more rapid as larvae neared the point of attachment. The range of locomotion in blackfly larvae is then discussed.     

The hagfish slime gland thread cell. I. A unique cellular system for the study of intermediate filaments and intermediate filament-microtubule interactions

Thread cell differentiation in the slime gland of the Pacific hagfish Eptatretus stouti has been studied using light microscopy and scanning and transmission electron microscopy. Thread cell differentiation is remarkable in that the life history of the cell is largely dedicated to the production of a single, tapered, cylindrical, highly coiled, and precisely packaged cytoplasmic thread that may attain lengths of 60 cm and diameters approaching 1.5 micron. Each tapered thread, in turn, is comprised almost entirely of large numbers of intermediate filaments (IFs) bundled in parallel. During differentiation of the thread, the IFs become progressively more tightly packed. Various numbers of microtubules (MTs) are found among the bundled IFs during differentiation of the thread but disappear during the latter stages of thread differentiation. Observations of regularly spaced dots in longitudinal bisections of developing threads, diagonal striations in tangential sections of developing threads, and circumferentially oriented, filament-like structures observed at the periphery of developing threads cut in cross section have led us to postulate a helically oriented component(s) wrapped around the periphery of the developing thread. The enormous size of the fully differentiated thread cell, its apparent singular dedication to the production of IFs, the ease of isolating and purifying the threads and IF subunits (see accompanying paper), and the unique position of the hagfish in the phylogenetic scheme of vertebrate evolution all contribute to the attractiveness of the hagfish slime gland thread cell as a potential model system for studying IF subunit synthesis, IF formation from IF subunits, aggregation of IFs into IF bundles and the interaction(s) of IFs and MTs.     

Microvoids in Bombyx mori silk. An electron microscope study.

The size and distribution of microvoids in Bombyx mori silk were examined by transmission electron microscopy of silver sulphide 'stained' filaments. Silver sulphide deposited in voids and accessible regions of molecular structure appears as dense particles in thin transverse and longitudinal sections of silk filaments. Small particles (about 8 nm or less in diameter) occur around or adjacent to the periphery of the filaments. Larger particles (around 10-15 nm in diameter) occur in the form of dendritic arrays in the core region of the filaments. The leading edges of the dendritic arrays are oriented towards the fibre periphery. The particles (microvoids) appear to be either spherical or rod-like in shape and are aligned parallel to the long axis of the filament. A skin/core structure is proposed.     

The silk road of <Emphasis Type="Italic">Tetranychus urticae</Emphasis>: is it a single or a double lane?

Tetranychus urticae (Acari: Tetranychidae) is a phytophagous mite that forms huge colonies. All active members of a colony (immatures and matures, females and males) spin silken threads. These mites construct a common web that protects the colony from external aggression. The silk coverage is well-known to provide advantages to the colony but very little is known about the characteristics of the threads themselves. Here is the first quantification of the diameter of silken threads spun by two different stages (adult females and larvae) and its relationship with body size of the spinning individuals. Moreover, we observed how silk was deposited on the substrate through their two pedipalps. Threads were observed by means of transmission electron and fluorescence microscopy. Silken threads spun by larvae (0.055 ± 0.018 μm) were significantly thinner than threads spun by adult females (0.111 ± 0.038 μm). In the first step of the silk depositing behaviour, the mite attached the thread to the substrate by putting its pedipalps in contact with the surface (adhesion, double silken threads). When walking, silken threads became detached from the substrate and spitted up (silken threads were free). Finally, silken threads adhered to the surface. The presence of single and double threads makes thread diameter highly variable.     

Nanoscale visualization of a fibrillar array in the cell wall of filamentous cyanobacteria and its implications for gliding motility

Many filamentous cyanobacteria are motile by gliding, which requires attachment to a surface. There are two main theories to explain the mechanism of gliding. According to the first, the filament is pushed forward by small waves that pass along the cell surface. In the second, gliding is powered by the extrusion of slime through pores surrounding each cell septum. We have previously shown that the cell walls of several motile cyanobacteria possess an array of parallel fibrils between the peptidoglycan and the outer membrane and have speculated that the function of this array may be to generate surface waves to power gliding. Here, we report on a study of the cell surface topography of two morphologically different filamentous cyanobacteria, using field emission gun scanning electron microscopy (FEGSEM) and atomic force microscopy (AFM). FEGSEM and AFM images of Oscillatoria sp. strain A2 confirmed the presence of an array of fibrils, visible as parallel corrugations on the cell surface. These corrugations were also visualized by AFM scanning of fully hydrated filaments under liquid; this has not been achieved before for filamentous bacteria. FEGSEM images of Nostoc punctiforme revealed a highly convoluted, not parallel, fibrillar array. We conclude that an array of parallel fibrils, beneath the outer membrane of Oscillatoria, may function in the generation of thrust in gliding motility. The array of convoluted fibrils in N. punctiforme may have an alternative function, perhaps connected with the increase in outer membrane surface area resulting from the presence of the fibrils.     

Economics of spider orb-webs: the benefits of producing adhesive capture thread and of recycling silk

1. The replacement of dry, fuzzy cribellar prey capture thread by viscous, adhesive capture thread was a major event in the evolution of orb-weaving spiders. Over 95% of all orb-weaving species now produce adhesive threads.
2. Adhesive thread achieves its stickiness with a much greater material economy than does cribellar thread.
3. Transformational analyses show that, relative to spider mass, adhesive orb-weavers invest less material per mm of capture thread and produce stickier capture threads than do cribellate orb-weavers.
4. The total cost of producing an orb-web that contains cribellar thread is reduced by 32% when a spider recycles its silk and another 34% when these capture threads are replaced by adhesive threads of equal stickiness.
5. The increased economy with which adhesive capture thread achieves its stickiness may have been an important factor that favoured the origin and success of modern orb-weaving spiders that produce adhesive capture threads.