Major Ampullate Gland Research Articles

Nearly complete 1H, 13C and 15N chemical shift assignment of monomeric form of N-terminal domain of Nephila clavipes major ampullate spidroin 2.

Spider dragline silk is well recognized due to its excellent mechanical properties. Dragline silk protein mainly consists of two proteins, namely, major ampullate spidroin 1 (MaSp1) and major ampullate spidroin 2 (MaSp2). The MaSp N-terminal domain (NTD) conformation displays a strong dependence on ion and pH gradients, which is crucial for the self-assembly behavior of spider silk. In the spider major ampullate gland, where the pH is neutral and concentration of NaCl is high, the NTD forms a monomer. In contrast, within the spinning duct, where pH becomes more acidic (to pH ~ 5) and the concentration of salt is low, NTD forms a dimer in antiparallel orientation. In this study, we report near-complete backbone and side chain chemical shift assignment of the monomeric form of NTD of MaSp2 from Nephila clavipes at pH 7 in the presence of 300mM NaCl. Our NMR data demonstrate that secondary structure of monomeric form of NTD MaSp2 consists of five helix regions.

Application of the Spider Silk Standardization Initiative (S3I) methodology to the characterization of major ampullate gland silk fibers spun by spiders from Pantanos de Villa wetlands (Lima, Peru)

Spider silk is a natural material with unique properties and a great potential for engineering and biomedical applications. In spite of its simple composition and highly conserved and stereotypical production, spider silks show a wide range of variability in their mechanical properties which, for long, have defied their classification and standardization. Here we propose to launch the Spider Silk Standardization Initiative (S3I), a methodology based on the definition of the α* parameter, in an attempt to define a systematic procedure to classify the tensile properties exhibited by major ampullate gland silk (MAS) spun by Entelegynae spiders. The α* parameter is calculated from the comparison of the true stress-true strain curve of any MAS fiber after being subjected to maximum supercontraction, with the true stress-true strain curve of the species Argiope aurantia, which is set as a reference curve. This work presents the details of the S3I methodology and, as an example, shows its application to an assemblage of Entelegynae spiders from different families collected at the Pantanos de Villa wetlands (Lima, Peru). The systematic and objective classification of the tensile properties of MAS fibers allowed by the S3I will offer insights into key aspects of the biological evolution of the material, and address questions such as how history and adaptation contributed to shape those properties. In addition, it will surely have far reaching consequences in fields such as Materials Science, and Molecular and Evolutionary Biology, by organizing the range of tensile properties exhibited by spider silk fibers.

Properties of two spliceoforms of major ampullate spidroin 1 reveal unique functions of N-linker region

Spiders produce diverse silk fibers with distinct properties for daily survival. Among these silk fibers, dragline silk spun by major ampullate gland is used for bridgelines and web radii, exhibiting both outstanding tensile strength and extensibility. Although more and more full-length major ampullate spidroin gene sequences have been reported, the research regarding alternative splicing events of spidroins are rare. Here we describe two spliceoforms of major ampullate spidroin 1 (MaSp1) from Araneus ventricosus, and both of them are lack of central repetitive region. The minor isoform only has terminal regions. For the major isoform, however, the N-linker and terminal regions are all retained. Furthermore, we investigated the functions of N-linker structure of A. ventricosus MaSp1, based on the properties of the two spliceoforms. The dimer level of major isoform (MaSp1-2) is higher than that of the minor isoform (MaSp1-1). Moreover, the MaSp1-2 protein display higher melting temperature (Tm) than MaSp1-1, and the MaSp1-2 fibers exhibit higher tensile strength than MaSp1-1 fibers. These studies demonstrate that the N-linker region promotes the formation of intermolecular disulphide bond, suggesting a strategy to enhance the thermostabilization and mechanical properties of spidroins.

Surface Analysis of Native Spider Draglines by FE-SEM and XPS.

Although the physical and biological functions of the skin layer of spider dragline have been studied and partially clarified, the morphology and elemental contents of the skin layer of silk fibers have not been investigated in detail to date. Here, the surface of Nephila clavata spider dragline was evaluated by field emission scanning electron microscopy (FE-SEM) and X-ray photoelectron spectroscopy (XPS) to obtain clear surface morphological and molecular information. The FE-SEM images of the spider dragline indicate that the spider dragline forms a bundle of microfibrils. This hierarchical structure might induce faint fibrilar and network-like patterns on the surface of the dragline. XPS analysis revealed the presence of Na, P, and S, which are reasonably explained by considering the biological components of the major ampullate gland of spiders. The results obtained here are preliminary but will be important to consider the molecular transition of silk proteins to form excellent hierarchical structures during the spider dragline spinning process.

The transcriptome of Darwin\u2019s bark spider silk glands predicts proteins contributing to dragline silk toughness

Darwin’s bark spider (Caerostris darwini) produces giant orb webs from dragline silk that can be twice as tough as other silks, making it the toughest biological material. This extreme toughness comes from increased extensibility relative to other draglines. We show C. darwini dragline-producing major ampullate (MA) glands highly express a novel silk gene transcript (MaSp4) encoding a protein that diverges markedly from closely related proteins and contains abundant proline, known to confer silk extensibility, in a unique GPGPQ amino acid motif. This suggests C. darwini evolved distinct proteins that may have increased its dragline’s toughness, enabling giant webs. Caerostris darwini’s MA spinning ducts also appear unusually long, potentially facilitating alignment of silk proteins into extremely tough fibers. Thus, a suite of novel traits from the level of genes to spinning physiology to silk biomechanics are associated with the unique ecology of Darwin’s bark spider, presenting innovative designs for engineering biomaterials.

Two-step self-assembly of a spider silk molecular clamp

Web spiders synthesize silk fibers of unique strength and extensibility through the controlled self-assembly of protein building blocks, so-called spidroins. The spidroin C-terminal domain is highly conserved and connects two polypeptide chains through formation of an all-helical, intertwined dimer. Here we use contact-induced fluorescence self-quenching and resonance energy transfer in combination with far-UV circular dichroism spectroscopy as three orthogonal structural probes to dissect the mechanism of folding and dimerization of a spidroin C-terminal domain from the major ampullate gland of the nursery web spider Euprosthenops australis. We show that helices forming the dimer core assemble very rapidly and fold on association. Subsequently, peripheral helices fold and dock slowly onto the preformed core. Lability of outer helices facilitates formation of a highly expanded, partially folded dimer. The high end-to-end distance of chain termini in the partially folded dimer suggests an extensibility module that contributes to elasticity of spider silk.

Major ampullate silk gland transcriptomes and fibre proteomes of the golden orb-weavers, Nephila plumipes and Nephila pilipes (Araneae: Nephilidae).

Natural spider silk is one of the world’s toughest proteinaceous materials, yet a truly biomimetic spider silk is elusive even after several decades of intense focus. In this study, Next-Generation Sequencing was utilised to produce transcriptomes of the major ampullate gland of two Australian golden orb-weavers, Nephila plumipes and Nephila pilipes, in order to identify highly expressed predicted proteins that may co-factor in the construction of the final polymer. Furthermore, proteomics was performed by liquid chromatography tandem-mass spectroscopy to analyse the natural solid silk fibre of each species to confirm highly expressed predicted proteins within the silk gland are present in the final silk product. We assembled the silk gland transcriptomes of N. plumipes and N. pilipes into 69,812 and 70,123 contigs, respectively. Gene expression analysis revealed that silk gene sequences were among the most highly expressed and we were able to procure silk sequences from both species in excess of 1,300 amino acids. However, some of the genes with the highest expression values were not able to be identified from our proteomic analysis. Proteome analysis of “reeled” silk fibres of N. plumipes and N. pilipes revealed 29 and 18 proteins, respectively, most of which were identified as silk fibre proteins. This study is the first silk gland specific transcriptome and proteome analysis for these species and will assist in the future development of a biomimetic spider silk.

A Facile Measurement for Monitoring Dragline Silk Dope Concentration in Nephila pilipes upon Spinning.

In spite of all the efforts towards deciphering the silk spinning process of spiders, the underlying mechanism is yet to be fully revealed. In this research, we designed a novel approach that allowed us to quantitatively evaluate the concentration change of silk dope during the liquid-to-solid spinning process of the orb-weaver Nephila pilipes. As a prior characterization of the optimal silking conditions, we first gauged the influence of silking-rate, ranging from 1.5 to 8.0 m/min, on dragline silk diameters and silk tensile strengths obtained from the spiders. Next, to evaluate the liquid content of the silk dope, the major ampullate gland was dissected and the concentration of the sac portion was measured by thermogravimetric analysis (TGA). The solid content of the dragline fibers leaving the spinneret was investigated by calculating the ratio of collected dried silk to the weight loss of the spider recorded in situ upon spinning. As the results indicate, the tensile strength and diameter of the spun dragline fibers were 800–1100 MPa and 8–11 μm, respectively. The liquid content of silk stored in the major ampullate sac (50.0 wt%) was significantly lower than that of silk leaving the spinnerets (80.9–96.1 wt%), indicating that a liquid supplying mechanism might be involved during the spinning process. This reveals, for the first time, quantitative evidence in support of the lubricative hypothesis proposed formerly, namely that a liquid coating layer is supplemented to compensate for silking resistance during the spinning process of a spider. The spigot, at the exit of the spinneret, is speculated to serve as a valve-like controller that regulates the lubrication process along with fiber formation. Taken together, these findings provide understanding of the physiological functions in the spider spinning process and could further shed some light on the future biomimetic development of silk material fabrication.

Energy Harvesters Incorporating Silk from the Taiwan-Native Spider Nephila pilipes

Silks from Nephila pilipes spiders native to Taiwan, obtained from the major ampullate gland, display novel piezoelectric properties that have been exploited in the preparation of energy harvesters. An energy harvester incorporating approximately 100 pieces of spider silk, each approximately 10 mm long, produced a voltage output of 13.4 mV when vibrated at 4 Hz. A repolarization process enhanced the piezoelectric properties of the spider silks significantly, notably increasing the output voltage to 40.7 mV at 4 Hz. Electrical testing indicated that the output voltages of the energy harvesters incorporating the repolarized silks were 3- to 4-fold higher than those of the harvesters containing the nonpolarized silks. The maximum output power (59.5 pW) was generated by the energy harvester incorporating the repolarized silks when connected to an external load resistor of 8.2 MΩ. The mechanical properties of the spider silks were examined through both microtensile and nanoindenter tests, revealing elastic mod...

The ontogeny of the spinning apparatus of Tengella perfuga (Araneae: Zoropsidae)

AbstractSilk is the most recognizable trait of spiders, and silk use has changed throughout spider evolutionary history. While morphology of the adult silk spigot has been a useful character for systematics, few studies have examined the ontogeny of the spinning apparatus, and none of these included cribellate spiders. Here, we report the first published full ontogeny of the spinning apparatus of a cribellate spider, Tengella perfuga. We found the presence of expected spigots: major ampullate gland and piriform gland spigots on the anterior lateral spinneret, minor ampullate gland and aciniform gland spigots on the posterior median spinneret, and aciniform gland spigots on the posterior lateral spinneret. Females, but not males, possessed cylindrical gland spigots on both the posterior median and lateral spinnerets. Spiderlings did not possess a functioning cribellum until the third instar. The cribellum grew with increasing numbers of spigots, but functionality was lost in adult males. Most intriguingly, second instars possessed a distinct triad of pre‐spigots on the posterior lateral spinneret. From the third instar onward, these structures formed the modified spigot along with two flanking spigots (in females) or formed nubbins (in males). We suggest that the modified spigot serves as the source of axial lines in the cribellate silk produced in T. perfuga. We also compare spigot ontogeny from previous studies of ecribellate spiders. These comparisons warrant further exploration using the recent spider tree of life in a phylogenetic comparative analysis of spigot ontogeny datasets, which could yield evidence for homologous spigots across the Araneomorphae, notably the Araneoidea and the Retrolateral Tibial Apophysis (RTA) clades.

Systematics and evolution of ground spiders revisited (Araneae, Dionycha, Gnaphosidae).

Gnaphosidae Pocock are a very diverse spider family with remarkable spinning organ morphology. Although the family has received intense taxonomic attention in recent years, its intergeneric relationships remain obscure. A phylogenetic analysis of Gnaphosidae genera was performed to untangle the evolutionary history of the family. A matrix of 324 morphological characters, scored for 71 gnaphosid genera and 29 outgroup taxa, was analysed through parsimony and Bayesian phylogenetic inference. Gnaphosidae are not recovered as a monophyletic group, neither were most of the previously proposed intrafamiliar groupings. In accordance with the phylogenetic results obtained, Vectius Simon and Hemicloea Thorell are transferred to Trochanteriidae, and Xenoplectus Schiapelli & Gerschman de Pikelin to Liocranidae. Micaria Westring, Nauhea Forster and Verita Ramírez & Grismado (and some related genera) are probably not gnaphosids, although their phylogenetic placement is uncertain. Gnaphosidae s.s. are defined as spiders with enlarged piriform gland spigots, longer and wider than the major ampullate gland spigots. Within Gnaphosidae s.s., well-supported clades allow the redefinition, on the basis of quantitative phylogenetic evidence, of Gnaphosinae Pocock, Zelotinae Platnick, Herpyllinae Platnick, Drassodinae Simon, Prodidominae Simon rankres. and the newly proposed Leptodrassinae subfam. nov. Many genera are not assigned to subfamily given their poorly supported and unstable relationships. The homology and evolution of structures such as the claw tuft clasper, the spinning organs and the modification of cheliceral promargin are discussed.

Conservation of folding and association within a family of spidroin N-terminal domains

Web spiders synthesize silk fibres, nature’s toughest biomaterial, through the controlled assembly of fibroin proteins, so-called spidroins. The highly conserved spidroin N-terminal domain (NTD) is a pH-driven self-assembly device that connects spidroins to super-molecules in fibres. The degree to which forces of self-assembly is conserved across spider glands and species is currently unknown because quantitative measures are missing. Here, we report the comparative investigation of spidroin NTDs originating from the major ampullate glands of the spider species Euprosthenops australis, Nephila clavipes, Latrodectus hesperus, and Latrodectus geometricus. We characterized equilibrium thermodynamics and kinetics of folding and self-association using dynamic light scattering, stopped-flow fluorescence and circular dichroism spectroscopy in combination with thermal and chemical denaturation experiments. We found cooperative two-state folding on a sub-millisecond time scale through a late transition state of all four domains. Stability was compromised by repulsive electrostatic forces originating from clustering of point charges on the NTD surface required for function. pH-driven dimerization proceeded with characteristic fast kinetics yielding high affinities. Results showed that energetics and kinetics of NTD self-assembly are highly conserved across spider species despite the different silk mechanical properties and web geometries they produce.

Morphological and functional diversity of minor ampullate glands in spiders from the superfamily Amaurobioidea (Entelegynae: RTA clade)

Minor ampullate glands produce fibers that are involved in construction of the complex adhesive band for capturing prey, which is produced by particular cribellate spiders. Despite such a specific role, however, the glands persist even in species where production of cribellate capturing bands no longer occurs. In these species, minor ampullate fibers are instead used to reinforce major ampullate fibers in draglines and capturing webs. The fibers are also used in combination with the aciniform fibrils to make silk for bridging lines – airborne lines used by spiders to allow them to move to points on the substrate where these threads adhere. In this study, we compare the morphology of minor and major ampullate glands in related cribellate and ecribellate groups within spider families of the group traditionally termed the Amaurobioidea, which lies at the base of the RTA clade. We found that the minor ampullate glands are bifurcated in the cribellate members of this group, in particular in the representatives of the families Amaurobiidae, Titanoecidae, Desidae, Amphinectidae and Phyxelididae. In ecribellate representatives, the major ampullate glands are never bifurcated. We found irregularly branched minor ampullate glands in some representatives of the family Agelenidae. In other ecribellates, the glands are either unbranched or they are absent. Thus, bifurcation of the minor ampullate gland seems to be important in determining some aspect of cribellate capturing band formation that is as yet undetermined.

Duplication and concerted evolution of MiSp-encoding genes underlie the material properties of minor ampullate silks of cobweb weaving spiders

BackgroundOrb-web weaving spiders and their relatives use multiple types of task-specific silks. The majority of spider silk studies have focused on the ultra-tough dragline silk synthesized in major ampullate glands, but other silk types have impressive material properties. For instance, minor ampullate silks of orb-web weaving spiders are as tough as draglines, due to their higher extensibility despite lower strength. Differences in material properties between silk types result from differences in their component proteins, particularly members of the spidroin (spider fibroin) gene family. However, the extent to which variation in material properties within a single silk type can be explained by variation in spidroin sequences is unknown. Here, we compare the minor ampullate spidroins (MiSp) of orb-weavers and cobweb weavers. Orb-web weavers use minor ampullate silk to form the auxiliary spiral of the orb-web while cobweb weavers use it to wrap prey, suggesting that selection pressures on minor ampullate spidroins (MiSp) may differ between the two groups.ResultsWe report complete or nearly complete MiSp sequences from five cobweb weaving spider species and measure material properties of minor ampullate silks in a subset of these species. We also compare MiSp sequences and silk properties of our cobweb weavers to published data for orb-web weavers. We demonstrate that all our cobweb weavers possess multiple MiSp loci and that one locus is more highly expressed in at least two species. We also find that the proportion of β-spiral-forming amino acid motifs in MiSp positively correlates with minor ampullate silk extensibility across orb-web and cobweb weavers.ConclusionsMiSp sequences vary dramatically within and among spider species, and have likely been subject to multiple rounds of gene duplication and concerted evolution, which have contributed to the diverse material properties of minor ampullate silks. Our sequences also provide templates for recombinant silk proteins with tailored properties.

Structural Investigations of Supercontracted Spider Dragline Silk

Spider dragline silk is a semicrystalline polymer of great interest in materials science due to its unique mechanical properties and biocompatibility. Spun from the major ampullate glands, this type of silk possesses an exceptional combination of elasticity, strength and toughness. At the molecular level, spider silk is composed of fibrous proteins organized into crystalline nanodomains embedded in an amorphous matrix. In the presence of liquid water or high humidity, the amorphous phase is plasticized and its hydrogen bonding network disrupted, which results in the shrinking of the fiber up to 50% of its initial length. This phenomenon, known as supercontraction, is triggered by the entropic folding of the polypeptide chains resulting from the water-induced increase in chain mobility. However, disagreements exist in the literature as to whether a change in the secondary structure of the protein occurs or not during supercontraction. Furthermore, although it has already been established that supercontraction induces a disorientation of the molecular units, this effect has not been quantified yet. Therefore, we will investigate and quantify, for different species, the conformational and orientational variations of the silk proteins induced by supercontraction. To achieve our goals, we will use Raman spectromicroscopy, a technique that as long been proven to be a useful tool to probe silk. Moreover, the effects of drawing speed on the magnitude of supercontraction will also be examined.

Comprehensive Proteomic Analysis of Spider Dragline Silk from Black Widows: A Recipe to Build Synthetic Silk Fibers.

The outstanding material properties of spider dragline silk fibers have been attributed to two spidroins, major ampullate spidroins 1 and 2 (MaSp1 and MaSp2). Although dragline silk fibers have been treated with different chemical solvents to elucidate the relationship between protein structure and fiber mechanics, there has not been a comprehensive proteomic analysis of the major ampullate (MA) gland, its spinning dope, and dragline silk using a wide range of chaotropic agents, inorganic salts, and fluorinated alcohols to elucidate their complete molecular constituents. In these studies, we perform in-solution tryptic digestions of solubilized MA glands, spinning dope and dragline silk fibers using five different solvents, followed by nano liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) analysis with an Orbitrap Fusion™ Tribrid™. To improve protein identification, we employed three different tryptic peptide fragmentation modes, which included collision-induced dissociation (CID), electron transfer dissociation (ETD), and high energy collision dissociation (HCD) to discover proteins involved in the silk assembly pathway and silk fiber. In addition to MaSp1 and MaSp2, we confirmed the presence of a third spidroin, aciniform spidroin 1 (AcSp1), widely recognized as the major constituent of wrapping silk, as a product of dragline silk. Our findings also reveal that MA glands, spinning dope, and dragline silk contain at least seven common proteins: three members of the Cysteine-Rich Protein Family (CRP1, CRP2 and CRP4), cysteine-rich secretory protein 3 (CRISP3), fasciclin and two uncharacterized proteins. In summary, this study provides a proteomic blueprint to construct synthetic silk fibers that most closely mimic natural fibers.

The effects of seasonal changes on the molecular weight of Nephila clavata spider silk

The molecular weight of spider proteins contained in the major ampullate glands of spiders collected during different periods. The standard deviation is also indicated. A peak for molecular weight is observed in mid-autumn, which corresponds to the mating season.

Material properties of evolutionary diverse spider silks described by variation in a single structural parameter.

Spider major ampullate gland silks (MAS) vary greatly in material properties among species but, this variation is shown here to be confined to evolutionary shifts along a single universal performance trajectory. This reveals an underlying design principle that is maintained across large changes in both spider ecology and silk chemistry. Persistence of this design principle becomes apparent after the material properties are defined relative to the true alignment parameter, which describes the orientation and stretching of the protein chains in the silk fiber. Our results show that the mechanical behavior of all Entelegynae major ampullate silk fibers, under any conditions, are described by this single parameter that connects the sequential action of three deformation micromechanisms during stretching: stressing of protein-protein hydrogen bonds, rotation of the β-nanocrystals and growth of the ordered fraction. Conservation of these traits for over 230 million years is an indication of the optimal design of the material and gives valuable clues for the production of biomimetic counterparts based on major ampullate spider silk.

Proteomic Evidence for Components of Spider Silk Synthesis from Black Widow Silk Glands and Fibers.

Spider silk research has largely focused on spidroins, proteins that are the primary components of spider silk fibers. Although a number of spidroins have been characterized, other types of proteins associated with silk synthesis are virtually unknown. Previous analyses of tissue-specific RNA-seq libraries identified 647 predicted genes that were differentially expressed in silk glands of the Western black widow, Latrodectus hesperus. Only ∼5% of these silk-gland specific transcripts (SSTs) encode spidroins; although the remaining predicted genes presumably encode other proteins associated with silk production, this is mostly unverified. Here, we used proteomic analysis of multiple silk glands and dragline silk fiber to investigate the translation of the differentially expressed genes. We find 48 proteins encoded by the differentially expressed transcripts in L. hesperus major ampullate, minor ampullate, and tubuliform silk glands and detect 17 SST encoded proteins in major ampullate silk fibers. The observed proteins include known silk-related proteins, but most are uncharacterized, with no annotation. These unannotated proteins likely include novel silk-associated proteins. Major and minor ampullate glands have the highest overlap of identified proteins, consistent with their shared, distinctive ampullate shape and the overlapping functions of major and minor ampullate silks. Our study substantiates and prioritizes predictions from differential expression analysis of spider silk gland transcriptomes.

Probing the Impact of Acidification on Spider Silk Assembly Kinetics.

Spiders utilize fine adjustment of the physicochemical conditions within its silk spinning system to regulate spidroin assembly into solid silk fibers with outstanding mechanical properties. However, the exact mechanism about which this occurs remains elusive and is still hotly debated. In this study, the effect of acidification on spider silk assembly was investigated on native spidroins from the major ampullate (MA) gland fluid excised from Latrodectus hesperus (Black Widow) spiders. Incubating the protein-rich MA silk gland fluid at acidic pH conditions results in the formation of silk fibers that are 10-100 μm in length and ∼2 μm in diameter as judged by optical and electron microscope methods. The in vitro spider silk assembly kinetics were monitored as a function of pH with a (13)C solid-state MAS NMR approach. The results confirm the importance of acidic pH in the spider silk self-assembly process with observation of a sigmoidal nucleation-elongation kinetic profile. The rates of nucleation and elongation as well as the percentage of β-sheet structure in the grown fibers depend on the pH. These results confirm the importance of an acidic pH gradient along the spinning duct for spider silk formation and provide a powerful spectroscopic approach to probe the kinetics of spider silk formation under various biochemical conditions.