Egg Case Silk Research Articles

1H, 15N and 13C resonance assignments of eggcase silk protein 3.

Spider silk is a high-performance biomaterial known for its outstanding combination of strength and flexibility. Among the six distinct types of spider silk, eggcase silk stands out as it is exclusively produced from the tubuliform gland, playing a specialized role in offspring protection. In the spider species Latrodectus hesperus, eggcase silk is spun from a large spidroin complex, including the major silk component tubuliform spidroin 1 (TuSp1) and at least six different minor silk components. One of these minor components is eggcase protein 3 (ECP3), a small silk protein of 11.8kDa that lacks the typical spidroin architecture. ECP3 shows very limited homology to all known spidroins. In this study, we report nearly complete backbone and side-chain resonance assignments of ECP3 as a basis for studying the structural mechanisms involved in eggcase silk formation.

Spider silk as a potential antibiotic substitute

Studies were undertaken on the antibacterial activity of spider silk against bacterial strains, using egg case silk of Parawixia dehaani (Doleschall, 1859) and web silk of Pholcus phalangioides (Fuesslin, 1775). Both silk types inhibited gram negative bacteria more than gram positive bacteria. The egg case silk of P. dehaani showed more antimicrobial activity than the web silk of P. phalangioides. The egg case silk brought about 26.05 per cent inhibition against E. coli, compared to a 22.7 per cent inhibition for B. subtilis. A linear association was found between the volume of silk extract and the percentage of inhibition.The percentage of inhibition against E. coli increased from 5.13 to 26.05 per cent as the volume rose from 20 to 100 µl. in P. dehaani.

Long-period fiber grating humidity sensor based on spider silks

We propose a long-period fiber grating (LPFG) humidity sensor based on two kinds of spider silks. Both spider dragline silk (SpDS) and spider egg-case silk (SpECS) for sensor preparation are produced by the Araneus ventricosus. SpDS can contract up to half the original length in the axial direction when the ambient relative humidity (RH) increases due to its supercontraction property. However, the length of SpECS axial contraction is much smaller than that of SpDS as RH increases, because SpECS does not have the supercontraction property like SpDS. We fabricated a LPFG by irradiating a single-mode fiber with a high-frequency CO2 laser. One end of LPFG is fixed by ultraviolet glue, and the other end is inserted into the quartz round tube without fixing. Therefore, the unfixed end of LPFG serves as the movable end to ensure that the direction of movement of LPFG is controllable. In addition, SpDS and SpECS exert pull forces on LPFG in two opposite directions, and the overall structure forms an X shape. When the ambient RH increases, the length of SpDS becomes shorter due to the supercontraction property. It changes the structure curvature of LPFG, leading to the transmission spectrum shift. When the RH decreases, SpECS provides a recovery pull, like a spring, that restores SpDS to a taut state when it relaxes. The curvature of LPFG is reduced, and the transmission spectrum shifts in the opposite direction. Therefore, we monitor the RH by observing the wavelength shift of the transmission spectrum. Our sensor achieves humidity detection with − 0.2039 nm/%RH sensitivity in the 50%RH-80%RH range. Additionally, we can adjust the amount and length of the two spider silks to increase the maximum detection humidity range closer to 100% RH at the expense of reduced sensitivity. The proposed humidity sensor has the advantages of reversibility, good repeatability, and environmental friendliness.

Critical role of minor eggcase silk component in promoting spidroin chain alignment and strong fiber formation

Significance Our manuscript is focused on the structural and functional role of minor silk component TuSp2 in spider eggcase silk formation. We present the structure of TuSp2 core domain and reveal how this domain renders main component TuSp1 more favorable for molecular chain alignment, which is critical for strong fiber formation. We further designed a series of biomimetic minispidroin complexes and showed that the artificial silk fiber can surpass its native counterpart in strength significantly. This study represents a distinct advance in understanding the critical role of the minor component in silk formation. The strategy developed in this study also throws light on spinning other types of artificial silk fibers with predictable physical properties.

1H, 15N and 13C resonance assignments of a repetitive domain of tubuliform spidroin 2.

Spider silk is renowned for its excellent mechanical properties. Among six types of silk and one silk glue produced by different abdominal glands for various purposes, tubuliform (eggcase) silk is unique due to its high serine and low glycine content. Eggcase silk is spun from at least two spidroins, tubuliform spidroin 1 (TuSp1) and TuSp2. TuSp1 and TuSp2 were identified as the major and the minor components in tubuliform glands, respectively. TuSp2 consists of multiple repetitive (RP) domains with short terminal tails and shares very limited homology to all known spidroins.Here we report backbone and side chain resonance assignments of TuSp2-RP as a basis for structural and functional studies on eggcase silk formation.

Self-assembly of tubuliform spidroins driven by hydrophobic interactions among terminal domains

Spider silk has remarkable physical and biocompatible properties. Investigation of structure-function relationship and self-assembly process of spidroins is necessary for uncovering the mechanism of silk fiber formation. Nevertheless, how the terminal domains initiate self-assembly of soluble tubuliform spidroins to form solid eggcase silk is still not fully understood. Here we investigate the roles of both terminal domains of tubuliform spidroin 1 (TuSp1) in the silk fiber formation. We found that interactions among the terminal domains drive rapid TuSp1 self-assembly and fiber formation, which is insensitive to pH changes from 6.0 to 7.0. These interactions also contribute to the spidroin chain alignment in fiber formation upon shear-force exposure. Structural analysis and site-directed mutagenesis identified eight critical surface-exposed residues involved in hydrophobic interactions among terminal domains. Spidroins with single-point mutations of these residues fail to form intermediate micelle-like structures. The structural docking model indicates that multiple terminal domains of TuSp1 may interact with each other based on hydrophobic interactions and surface complementarity, which may lead to forming the surface of the micelle-like structure. Our results provide new insights into the structural mechanism of eggcase silk formation and the basis for designing and producing novel biomaterials derived from spider eggcase silk.

Mesoscale structures in amorphous silks from a spider\u2019s orb-web

Of the 7–8 silk fibers making up an orb-web only the hierarchical structural organization of semicrystalline radial fibers -composed of major ampullate silk- has been studied in detail, given its fascinating mechanical features. While major ampullate silk’s nanofibrillar morphology is well established, knowhow on mesoscale (> 50–100 nm) assembly and its contribution to mechanical performance is limited. Much less is known on the hierarchical structural organization of other, generally less crystalline fibers contributing to an orb-webs’ function. Here we show by scanning X-ray micro&nanodiffraction that two fully amorphous, fine silk fibers from the center of an orb-web have different mesoscale features. One of the fibers has a fibrillar composite structure resembling stiff egg case silk. The other fiber has a skin–core structure based on a nanofibrillar ribbon wound around a disordered core. A fraction of nanofibrils appears to have assembled into mesoscale fibrils. This fiber becomes readily attached to the coat of major ampullate silk fibers. We observe that a detached fiber has ripped out the glycoprotein skin-layer containing polyglycine II nanocrystallites. The anchoring of the fiber in the coat suggests that it could serve for strengthening the tension and cohesion of major ampullate silk fibers.

Two novel tubuliform silk gene sequences from Araneus ventricosus provide evidence for multiple loci in genome

Spiders produce a diversity of silk fibers from multiple morphologically distinct silk glands for specific tasks, and each silk type primarily composed of one or more particular silk proteins encoded by silk gene family members believed to generated by duplication and divergence of ancient silk genes. Egg case silks spun from tubuliform glands are used to construct the tough outer structure of egg cases, are important for their reproduction. Here we present two novel complete TuSp1 sequences from orb weaving spider Araneus ventricosus. Alignment of the two spidroin iterated repeats showed both extreme intragenic homogenization. The pairwise Ka/Ks analysis revealed the terminal and repetitive regions for three TuSp1 loci including the reported TuSp1 gene are all under purifying selection. Phylogenetic analysis showed the two new TuSp1 variants could derive from recent duplication events.

Facile Fabrication of Microspheres and Microcapsules from a Recombinant Spider Eggcase Silk Protein for Drug Delivery

Spider silk is renowned for its exceptional mechanical properties, and compatibility and biodegradability for various functional applications, such as drug delivery. By a facile HFIP (1,1,1,3,3,3-hexafluoro-2-propanol)-on-Oil method, microspheres and microcapsules were assembled from a recombinant spider eggcase silk protein. Doxorubicin (Dox) can be loaded into microspheres or microcapsules before or after their preparation, while maintaining morphological integrity. The isoelectric point of the silk proteins was 4.8, which affected the drug loading of silk spheres for different pH media. For enzyme-assisted drug delivery, Dox-loaded silk spheres effectively released the drug under enzymatic degradation by addition of proteinase K.

Objective equilibriometric quantification of individual locomotor behaviour as a translational pharmacodynamic biomarker in schizophrenia

Orb-web spiders produce at least seven different protein-based silk that exhibit different mechanical properties. Egg case silk is spun by tubuliform glands and used to form the outer shell of egg case. To date, only two complete cDNA sequences have been reported for tubuliform spidroin (TuSp, also known as CySp for cylindrical spidroin). Here, we report the complete genomic DNA sequence for a tubuliform spidroin (TuSp1) gene from orb-web weaving spider species Araneus ventricosus, which was obtained by using a long distance PCR approach. The primary component of Araneus ventricosus TuSp1 gene contains a single large exon (5763 bp) and encodes 1921 amino acid residues dominated by 9 tandem repeats flanked by conserved non-repetitive N- and C-terminal domains. These repeats (each 176–155 amino acids long) found in Araneus ventricosus TuSp1 are highly conserved (>96% identical at amino acid level).

Modular Assembly of a Conserved Repetitive Sequence in the Spider Eggcase Silk: From Gene to Fiber.

Spider silk features extraordinary toughness in combination with great biocompatibility and biodegradability, fascinating researchers to prepare artificial silk fibers inspired from the natural art of spinning. In addition to C- and N-terminal domain, a repeat unit from Lactrodectus mactans spider eggcase silks displays substantial sequence conservation across species. Herein, we attempt to spin the engineered tubuliform spidroin 1 (eTuSp1) by microfluidics in a mode of modular assembly comprising the genetic construction, micellar formation, phase separation, and further solidification. Based on the conserved gene sequence, a unique amphiphilic behavior was predicted and then verified by combined techniques of dynamic light scattering, transmission electron microscopy, and synchrotron radiation X-ray diffraction to reveal the formation of micelle-like structure. Through the employ of biomimetic microfluidic devices, desolvation of eTuSp1 was simplified by the nonsolvent induced phase separation in place of the conventional ions exchange and acidification. Both controlled by protein concentrations and flow rate ratios, silk fibers were assembled similar to these reported in other studies of spheres/spherical aggregates observed as intermediates. Because of the applied shear and elongational flow in microfluidic systems, these intermediates were forced to form fibrillar assemblies accompanied by the conformational transformation from α-helix to β-sheet. The resultant mechanical properties were investigated in response to the change of secondary structures and morphologies during spinning process. This work studies the sequence-structure-property relationship, providing comprehensive and systematic insight into the design rational on the preparation of artificial silk fibers from microscale to macroscale.

Facile preparation of recombinant spider eggcase silk spheres via an HFIP-on-Oil approach

Versatile spider silk proteins have been prepared by various methods in morphology of spheres for functional applications. Inspired from natural spinning process, a facile approach for the fabrication of silk spheres is described. Distinct from the traditional emulsification method, silk spheres were assembled as rapidly as 10 s by using the HFIP-on-Oil method without any surfactants and agitation used. Notably, a series of factors, such as evaporation rate of HFIP, polarity and molecular weight of oils play central roles on the final silk morphologies. With regard to the increase of protein concentrations, the average dimension and size distribution of silk spheres were both increased. Together with present study, silk spheres prepared by other methods were summarized for comparison in drug delivery applications. As a proof-of-concept, silk spheres loaded with Rhodamine B and Doxorubicin were investigated for the potential proteinase-enhanced drug delivery. On the extracellular environment, ethanol-mediated silk spheres exhibited higher resistance against enzymatic degradation of proteinase K when compared with pristine spheres. Under fluorescent detection by the aid of CLSM, proteinase-enhanced release behaviors were further demonstrated through in-vitro experiments within Hela cells. The facile fabrication of spheres with tunable β-sheets establishes a fascinating platform for functional silk-based applications.

Egg Case Silk Gene Sequences from Argiope Spiders: Evidence for Multiple Loci and a Loss of Function Between Paralogs.

Spiders swath their eggs with silk to protect developing embryos and hatchlings. Egg case silks, like other fibrous spider silks, are primarily composed of proteins called spidroins (spidroin = spider-fibroin). Silks, and thus spidroins, are important throughout the lives of spiders, yet the evolution of spidroin genes has been relatively understudied. Spidroin genes are notoriously difficult to sequence because they are typically very long (≥ 10 kb of coding sequence) and highly repetitive. Here, we investigate the evolution of spider silk genes through long-read sequencing of Bacterial Artificial Chromosome (BAC) clones. We demonstrate that the silver garden spider Argiope argentata has multiple egg case spidroin loci with a loss of function at one locus. We also use degenerate PCR primers to search the genomic DNA of congeneric species and find evidence for multiple egg case spidroin loci in other Argiope spiders. Comparative analyses show that these multiple loci are more similar at the nucleotide level within a species than between species. This pattern is consistent with concerted evolution homogenizing gene copies within a genome. More complicated explanations include convergent evolution or recent independent gene duplications within each species.

From EST to novel spider silk gene identification for production of spidroin-based biomaterials

A cDNA library from a pool of all the seven silk glands from a tropical spider species was constructed. More than 1000 expressed sequence tag (EST) clones were created. Almost 65% of the EST clones were identified and around 50% were annotated. The cellular and functional distribution of the EST clones indicated high protein synthesis activity in spider silk glands. Novel clones with repetitive amino acid sequences, which is one of the most important characteristics of spider silk genes, were isolated. One of these clones, namely TuSp2 in current research, contains two almost identical fragments with one short C-terminal domain. Reverse transcription (RT) PCR and expression analysis showed that it is expressed in the tubuliform gland and involved in eggcase silk formation. Furthermore, its single repetitive domain can be induced to form various types of materials, including macroscopic fibers, transparent film and translucent hydrogel. This study implies promising potentials for future identification of novel spidroins and development of new spidroin-based biomaterials.

Characterization of full-length tubuliform spidroin gene from Araneus ventricosus

Orb-web spiders produce at least seven different protein-based silk that exhibit different mechanical properties. Egg case silk is spun by tubuliform glands and used to form the outer shell of egg case. To date, only two complete cDNA sequences have been reported for tubuliform spidroin (TuSp, also known as CySp for cylindrical spidroin). Here, we report the complete genomic DNA sequence for a tubuliform spidroin (TuSp1) gene from orb-web weaving spider species Araneus ventricosus, which was obtained by using a long distance PCR approach. The primary component of Araneus ventricosus TuSp1 gene contains a single large exon (5763bp) and encodes 1921 amino acid residues dominated by 9 tandem repeats flanked by conserved non-repetitive N- and C-terminal domains. These repeats (each 176–155 amino acids long) found in Araneus ventricosus TuSp1 are highly conserved (>96% identical at amino acid level).

Candidate egg case silk genes for the spider Argiope argentata from differential gene expression analyses.

Orb-web weaving spiders produce a variety of task-specific silks from specialized silk glands. The genetics underlying the synthesis of specific silk types are largely unknown, and transcriptome analysis could be a powerful approach for identifying candidate genes. However, de novo assembly and expression profiling of silk glands with RNA-sequencing (RNAseq) are problematic because the few known gene transcripts for silk proteins are extremely long and highly repetitive. To identify candidate genes for tubuliform (egg case) silk synthesis by the orb-weaver Argiope argentata (Araneidae), we estimated transcript abundance using two sequencing methods: RNAseq reads from throughout the length of mRNA molecules, and 3' digital gene expression reads from the 3' region of mRNA molecules. Both analyses identified similar sets of genes as differentially expressed when comparing tubuliform and nonsilk gland tissue. However, incompletely assembled silk gene transcripts were identified as differentially expressed because of RNAseq read alignments to highly repetitive regions, confounding interpretation of RNAseq results. Homologues of egg case silk protein (ECP) genes were upregulated in tubuliform glands. This discovery is the first description of ECP homologues in an araneid. We also propose additional candidate genes involved in synthesis of tubuliform or other silk types.

The toughest recorded spider egg case silks are woven into composites with tear-resistant architectures

In this communication, we report important preliminary evidence for possibly the toughest egg case silk threads recorded to date spun by the hermit spider, Nephilengys cruentata (G¯=193MJm−3). We further elucidate that the egg case itself is woven with a specialised repeat cross-weave that when subjected to tension, drives perpendicular-to-force threads to pile. This piling of threads constrains damage to small areas and retains the architectural integrity of the surrounding egg case material. We deduce that by having ultra-tough threads coupled to a tear resistant architecture, N. cruentata is able to protect its eggs from predators with a considerable level of effectiveness.

Alteration of tubuliform silk gland cytoarchitecture with the reproductive cycle of the Western black widow spider, Latrodectus hesperus

AbstractThe protein synthetic and secretory activity of spider tubuliform glands is known to be coordinated with the reproductive stage of the spider. For spiders that produce multiple egg cases, such as the black widow Latrodectus hesperus, this means that the cells that make up the tubuliform gland cycle from minimal to maximal silk protein synthesis and exocytosis as the spider transitions from early vitellogenesis to a gravid state and back. The impact of these transitions on the cells that form the tubuliform gland has yet to be characterized. The entire tubuliform gland undergoes an elastic deformation, doubling in size in response to the accumulation and depletion of egg case silk proteins within its lumen. Similarly, the diversity and organization of organelles within the cytoplasm of the secretory epithelial cells that make up the wall of the tubuliform gland change with the reproductive stage of the spider. Progression of a spider from early to late vitellogenesis is accompanied by decondensed nucleoli and distention of the rough endoplasmic reticulum, markers of protein synthetic activity. The presumed silk proteins that fill the lumen of the tubuliform gland of a gravid spider include a fibrous matrix with homogeneous spherical inclusions. These components are also present within the cytoplasm of the cell; however, only the fibrous material appears to be enclosed by membranous organelles. Transition of the tubuliform gland from peak silk synthesis back to a quiescent state is marked by the appearance of multivesicular bodies and organelles resembling phagophores and autophagosomes, suggestive of a role for autophagy in the process of recovery. The reproducible cellular dynamics of the tubuliform silk gland of the black widow spider makes it a potential model system for study of the regulation of silk gene expression, endomembrane transport, and exocytosis of silk proteins and autophagy.

Optically probing torsional superelasticity in spider silks

We investigate torsion mechanics of various spider silks using a sensitive optical technique. We find that spider silks are torsionally superelastic in that they can reversibly withstand great torsion strains of over 102−3 rotations per cm before failure. Among various silks from a spider, we find the failure twist-strain is greatest in the sticky capture silk followed by dragline and egg-case silk. Our in situ laser-diffraction measurements reveal that torsional strains on the silks induce a nano-scale transverse compression in its diameter that is linear and reversible. These unique torsional properties of the silks could find applications in silk-based materials and devices.

Intramolecular homologous recombination event occurred in the spider egg case silk gene CySp2 of wasp spider Argiope bruennichi

To gain further understanding of egg case silk proteins gene family, Zhao et al. (2006) isolated two full-length cDNAs for egg case silk proteins, cylindrical silk protein 1 (CySp1) and cylindrical silk protein 2 (CySp2), from the wasp spider, Argiope bruennichi. CySp2 was reported to contain no apparent Signal peptide sequences, and the CySp1-CySp2 complex, which would possess a signal peptide, would be transported across the endoplasmic reticulum and secreted to the Golgi. According to a report by Hayashi, genomic DNA sequencing is one approach that can be successfully utilized to retrieve 5′ ends of silk genes; using this method, we retrieved the 5’ end of CySp1. We found that CySp2 contained a typical signal peptide similar to that found in CySp1; thus, due to technical limitations, an artificial error had occurred in the CySp2 sequence reported by Zhao et al.