Aciniform spidroin, a constituent of egg case sacs and wrapping silk fibers from the black widow spider Latrodectus hesperus

Spiders produce high performance fibers with diverse mechanical properties and biological functions. Molecular and biochemical studies of spider egg case silk have revealed that the main constituent of the large diameter fiber contains the fibroin TuSp1. Here we demonstrate by SDS-PAGE and protein silver staining the presence of a distinct approximately 300-kDa polypeptide that is found in solubilized egg case sacs. Combining matrix-assisted laser desorption ionization tandem time-of-flight mass spectrometry and reverse genetics, we have isolated a novel gene called AcSp1-like and demonstrate that its protein product is assembled into the small diameter fibers of egg case sacs and wrapping silks from the black widow spider, Latrodectus hesperus. BLAST searches of the NCBInr protein data base using the amino acid sequence of AcSp1-like revealed similarity to AcSp1, an inferred protein proposed to be a component of wrapping silk. However, the AcSp1-like protein was found to display more nonuniformity in its internal iterated repeat modules than the putative AcSp1 fibroin. Real time quantitative PCR analysis demonstrates that the AcSp1-like gene displays an aciniform gland-restricted pattern of expression. The amino acid composition of the fibroins extracted from the luminal contents of the aciniform glands was remarkably similar to the predicted amino acid composition of the AcSp1-like protein, which supports the assertion that AcSp1-like protein represents the major constituent stored within the aciniform gland. Collectively, our findings provide the first direct molecular evidence for the involvement of the aciniform gland in the production of a common fibroin that is assembled into the small diameter threads of egg case and wrapping silk of cob weavers.     

Material properties of cobweb silk from the black widow spider Latrodectus hesperus.

We present the material analysis of scaffolding silk from the cobweb of the black widow spider Latrodectus hesperus. 30 strands were tested from the webs of nine spiders. Strands were stretched at 0.211 mm/s as force and extension were recorded. Cross-sectional area was measured under 1000 x oil-immersion light microscopy. The stress strain curve shows that cobweb silk is a distinct material from other known spider silks. The average breaking point for this cobweb silk is 1.1 +/- 0.5 GPa at 0.22 +/- 0.05 strain. All samples increased stiffness as they were stretched, but to different extents. Variation in stiffness might be due to differential crystallization or alignment of the silk proteins during stretching.     

Molecular and mechanical properties of major ampullate silk of the black widow spider, Latrodectus hesperus

Molecular and material properties of major ampullate silk were studied for the cobweb-building black widow spider Latrodectus hesperus. Material properties were measured by stretching the silk to breaking. The strength was 1.0 +/- 0.2 GPa, and the extensibility was 34 +/- 8%. The secondary structure of the major ampullate silk protein was studied using carbon-13 NMR spectroscopy. Alanine undergoes a transition from a coiled structure in pre-spun silk to a beta sheet structure in post-spun silk. We have also isolated two distinct cDNAs (both about 500 bp) which encode proteins similar to major ampullate spidroin 1 and 2 (MaSp1 and MaSp2). The MaSp1-like silk contains polyalanine runs of 5-10 residues as well as GA and GGX motifs. The MaSp2-like silk contains polyalanine runs of varying length as well as GPG(X)(n) motifs. L. hesperus major ampullate silk is more like major ampullate silk from other species than other L. hesperus silks.     

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

The 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.     

Ultrastructure of the major ampullate gland of the black widow spider,Latrodectus hesperus

Silk production in the spider occurs within specialized glands that are capable of the synthesis of large fibrous proteins and the post-translational processing of those proteins to form an insoluble fiber. The major ampullate gland of Latrodectus hesperus (black widow) is similar in morphology to those found in the Araneid spiders. The tail domain of this gland is highly protein synthetic, giving rise to a core, fibrous protein product. In addition to a storage function, the ampulla region also synthesizes and exports an electron dense material that appears to form a 'coat' surrounding the silk generated within the tail. The duct of the gland consists of at least two distinct cell types: one type contains 'honeycomb' vesicles of unknown function, while the other possesses elaborate apical microvilli that may be involved in the reabsorption of water and subsequent dehydration of the silk. As the silk product transits through these various stages of assembly, it can been seen to undergo a condensation or concentration, possibly reflecting the influence of both the shear forces induced by movement into the duct and the dehydration that is thought to occur there.     

Egg case protein-1. A new class of silk proteins with fibroin-like properties from the spider Latrodectus hesperus

Spiders produce multiple types of silk that exhibit diverse mechanical properties and biological functions. Most molecular studies of spider silk have focused on fibroins from dragline silk and capture silk, two important silk types involved in the survival of the spider. In our studies we have focused on the characterization of egg case silk, a third silk fiber produced by the black widow spider, Latrodectus hesperus. Analysis of the physical structure of egg case silk using scanning electron microscopy demonstrates the presence of small and large diameter fibers. By using the strong protein denaturant 8 M guanidine hydrochloride to solubilize the fibers, we demonstrated by SDS-PAGE and protein silver staining that an abundant component of egg case silk is a 100-kDa protein doublet. Combining matrix-assisted laser desorption ionization tandem time-of-flight mass spectrometry and reverse genetics, we have isolated a novel gene called ecp-1, which encodes for one of the protein components of the 100-kDa species. BLAST searches of the NCBInr protein data base using the primary sequence of ECP-1 revealed similarity to fibroins from spiders and silkworms, which mapped to two distinct regions within the ECP-1. These regions contained the conserved repetitive fibroin motifs poly(Ala) and poly(Gly-Ala), but surprisingly, no larger ensemble repeats could be identified within the primary sequence of ECP-1. Consistent with silk gland-restricted patterns of expression for fibroins, ECP-1 was demonstrated to be predominantly produced in the tubuliform gland, with lower levels detected in the major and minor ampullate glands. ECP-1 monomeric units were also shown to assemble into higher aggregate structures through the formation of disulfide bonds via a unique cysteine-rich N-terminal region. Collectively, our findings provide new insight into the components of egg case silk and identify a new class of silk proteins with distinctive molecular features relative to traditional members of the spider silk gene family.     

First record of the western black widow spider Latrodectus hesperus Chamberlin & Ivie, 1935 (Araneae: Theridiidae) in South Korea

Two individuals of Latrodectus hesperus Chamberlin & Ivie were newly found in munitions storage at military facilities in Daegu, South Korea. A female was first discovered on August 31, 2018, and followed by another female along with three egg sacs on September 20, 2018. These spiders were identified based on morphological characters and DNA barcode sequence analysis. Additional surveys were made around the initial detection site to examine the range and spread of this species, but no additional individuals were found.     

A crustacean-specific neurotoxin from the venom of the black widow spider Latrodectus mactans tredecimguttatus

A method of the isolation of a crustacea-specific neurotoxin from the venom of the Latrodectus mactans tredecimguttatus spider by means of ion exchange chromatography on Mono Q and Mono S columns and hydrophobic chromatography on Phenyl-Superose column has been developed. LD50 of the toxin has been elucidated.     

Protein and amino acid composition of silks from the cob weaver, Latrodectus hesperus (black widow)

The silks from the cob weaving spider, Latrodectus hesperus (black widow), have been examined with the goal of expanding our understanding of the relationship between the protein structure and mechanical performance of these unique biomaterials. The scaffolding, dragline and inner egg case silks each appear to be distinct fibers based on mole percent amino acid composition and polypeptide composition. Further, we find that the amino acid composition of dragline and egg case silk are similar to the analogous silks produced by orb weaving spiders, while scaffolding silk may represent a novel silk. The black widow silks are comprised of multiple high molecular weight polypeptides, however, the egg case and scaffolding silks also contain some smaller polypeptides.     

Electron microscopy of alpha-latrotoxin from the venom of the black widow spider Latrodectus mactans tredecimguttatus]

Two-dimensional crystals of alpha-latrotoxin from the venom of black widow spider (Latrodectus mactans tredecimguttatus) were studied by the negative staining electron microscopy. Two-dimensional crystals were obtained by adsorption of the protein solution with a high Mg2+ concentration on carbon-coated electron microscopy grids. The crystals were about 0.4 mkm in size, had the unit cell parameters: a = b = 15.55 nm, gamma = 90 degrees, p4 plane group symmetry. The contour map of a stain-excluding region of such crystals was calculated by the Fourier-filtering procedure at about 4 nm resolution. The calculation of molecular weight of the unit cell, with the symmetry p4 taken into account, showed that alpha-latrotoxin particles, revealed by negative staining, consisted of 4 or 8 protomers.     

The black widow spider genus Latrodectus (Araneae: Theridiidae): phylogeny, biogeography, and invasion history

The spider genus Latrodectus includes the widely known black widows, notorious because of the extreme potency of their neurotoxic venom. The genus has a worldwide distribution and comprises 30 currently recognized species, the phylogenetic relationships of which were previously unknown. Several members of the genus are synanthropic, and are increasingly being detected in new localities, an occurrence attributed to human mediated movement. In particular, the nearly cosmopolitan range of the brown widow, Latrodectus geometricus, is a suspected consequence of human transport. Although the taxonomy of the genus has been examined repeatedly, the recognition of taxa within Latrodectus has long been considered problematic due to the difficulty associated with identifying morphological features exhibiting discrete geographic boundaries. This paper presents, to our knowledge, the first phylogenetic hypothesis for the Latrodectus genus and is generated from DNA sequences of the mitochondrial gene cytochrome c oxidase subunit I. We recover two well-supported reciprocally monophyletic clades within the genus: (1) the geometricus clade, consisting of Latrodectus rhodesiensis from Africa, and its is sister species, the cosmopolitan L. geometricus, and (2) the mactans clade containing all other Latrodectus species sampled, including taxa occurring in Africa, the Middle East, Iberian Peninsula, Australia, New Zealand, and North and South America. Recovery of the geometricus and mactans clades is consistent with previous designations of species groups within the genus based on female genitalic morphology. All L. geometricus sampled, consisting of specimens from Africa, Argentina, North America, and Hawaii, were recovered as a strongly supported monophyletic group with minimal amounts of genetic divergence, corroborating the hypothesis that human transport has recently expanded the range of this species.     

Repeatability but no short-term plasticity in the web architecture of Latrodectus hesperus western black widow spiders (Araneae: Theridiidae)

A spider's web is an extended phenotype and a direct product of behavior. Variation in web architecture may be the result of individual differences in genotype, environment, or experience. We analyzed variation in the web architecture of western black widow spiders, Latrodectus hesperus, by testing two hypotheses: (a) intrinsic individual differences and (b) plasticity according to experience of the site of prey capture. We used treatments manipulating the site of prey capture experienced by the spiders: sheet only, gum-footed lines only, and alternating patterns between sheet and gum-footed lines equally. We found considerable consistent individual variation (repeatability of r = .56) in one web component (gum-footed lines) and negligible plasticity in either component. We discuss our results in terms of the potential for the design of these web components to respond to selection, and of potential additional causes of variation in extended phenotypes.     

Gumfooted lines in black widow cobwebs and the mechanical properties of spider capture silk

Orb-weaving spiders produce webs using two types of silk that have radically different mechanical properties. The dragline silk used to construct the supporting frame and radii of the web is stiff and as strong as steel, while the capture spiral is much weaker but more than ten times as extensible. This remarkable divergence in mechanical properties has been attributed to the aqueous glue that coats the capture spiral, which is thought to decrease capture spiral stiffness and increase its extensibility. However, discerning the effect of the aqueous glue on fiber performance is complicated because dragline silk and the capture spiral are assembled from different proteins, which may also affect mechanical performance. Here, we use the sticky gumfooted lines of black widow cobwebs to test the effect of the addition of aqueous glue on the mechanical properties of dragline silk. We also surveyed orb-webs spun by a broad range of species for bundles of looped silk. Such bundles, termed windlasses, have been thought to increase capture spiral extensibility by "paying out" additional lengths of silk. Our results suggest that neither plasticization of silk by aqueous glue nor excess silk in windlasses can by themselves account for the remarkable extensibility of orb-weaver capture silk compared to other spider silks. This argues that the unique amino acid motifs of the flagelliform fibroins that constitute the core of the capture spiral play an essential role in capture silk's extreme extensibility.     

Structure of tryptic fragments of a neurotoxin from black widow spider venom

The N-terminal amino acid sequence of a neurotoxin from the venom of Latrodectus mactans tredecimguttatus (alpha-latrotoxin) was determined. Latrotoxin was subjected to the tryptic cleavage and total or partial amino acid sequences of 25 peptides were established. In total the tryptic fragments contained 252 amino acid residues. Essential structural information on cloning of the latrotoxin structural gene was obtained.     

Predation strategy of the brown widow spider Latrodectus geometricus Koch, 1841 against prey with different defensive mechanisms

Journal of Ethology - Predators, such as spiders, may modulate their predatory behavior according to the defensive mechanisms of their prey. This study analyzed the prey capture behavior of...     

Novel molecular and mechanical properties of egg case silk from wasp spider, Argiope bruennichi

Araneoid spiders use specialized abdominal glands to produce up to seven different protein-based silks/glues that have various mechanical properties. To date, the fibroin sequences encoding egg case fibers have not been fully determined. To gain further understanding of a recently reported spider silk protein gene family, several novel strategies were utilized in this study to isolate two full-length cDNAs of egg case silk proteins, cylindrical silk protein 1 (CySp1, 9.1 kb) and cylindrical silk protein 2 (CySp2, 9.8 kb), from the wasp spider, Argiope bruennichi. Northern blotting analysis demonstrated that CySp1 and CySp2 are selectively expressed in the cylindrical glands. The amino acid composition of raw egg case silk was closely consistent with the deduced amino acid composition based on the sequences of CySp1 and CySp2, which supports the assertion that CySp1 and CySp2 represent two major components of egg case silk. CySp1 and CySp2 are primarily composed of remarkable homogeneous assemble repeats that are 180 residues in length and consist of several complex subrepeats, and they contain highly homologous C-termini and markedly different N-termini. Our results suggest a possible link between CySp1 and CySp2. In addition, comparisons of stress/strain curves for dragline and egg case silk from Argiope bruennichi showed obvious differences in ultimate strength and extensibility, and similarities in toughness.