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Gene expression analyses for AgSp1 and AgSp2 of A. trifasciata. Genes were each manually edited to 999 bp of the 3' ends for analyses. (A) Log number of normalized reads that align to AgSp1 and AgSp2 from aggregate gland tissue (agg), major ampullate gland tissue (maj) and fat body tissue (fat). Line ends correspond to statistical comparisons between tissues. (B) Transcripts per million (TPM) for AgSp1 and AgSp2 averaged across six aggregate gland tissue samples. Asterisks indicate significant q-and p-values (<0.05).
Source publication
An individual orb weaving spider can spin up to seven different types of silk, each with unique functions and material properties. The capture spiral silk of classic two-dimensional aerial orb webs is coated with an amorphous glue that functions to retain prey that get caught in a web. This unique modified silk is partially comprised of spidroins (...
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... identified the putative AgSp2 from cobweb weavers L. geometricus (accession#: MK138562) and L. hesperus (accession#: MK138563; Supplementary Fig. S5) after de novo assembly of online data sets (Supplementary Table S1). The assembly software was able to completely reconstruct AgSp2 in both species, with no evidence of truncation as seen in AgSp1. ...
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... data sequenced from A. trifasciata aggregate gland tissue to compare AgSp1 and AgSp2 expression, and from major ampullate silk gland fat body tissues as controls. AgSp1 and AgSp2 are among the most highly expressed genes within the aggregate glands, with significantly (q < 0.05) less expression detected in the major ampullate or fat body tissues (Fig. 5A). Within the aggregate glands, expression of AgSp1 and AgSp2 is not significantly different ( ...
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... repetitive motifs are notably similar between the two spidroins, and predicted protein alignments of the repeat motifs from these two spidroins supports paralogous origins (Fig. 3). Interestingly, AgSp2 is highly reduced in cobweb weavers (Supplementary Fig. S5). It is unknown if AgSp1 and AgSp2 interact with each other in any way to form the sticky capture glue, or what properties each imparts, particularly considering the differences between AgSp2 in orb web and cobweb weavers. ...
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... expansion regions are also found in pyriform spidroins , and QQ motifs have been shown to allow self-aggregation of these silk proteins into fibers (Geurts et al. 2010). The coding sequence of AgSp2 is dramatically reduced in Latrodectus cobweb weavers, consisting of less than 2000 bp (Supplementary Fig. S5). Differences between orb weaving and cobweb weaving pyriform spidroins have also been noted, including a loss of the QQ motifs ( Chaw et al. 2017). ...
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... identified the putative AgSp2 from cobweb weavers L. geometricus (accession#: MK138562) and L. hesperus (accession#: MK138563; Supplementary Fig. S5) after de novo assembly of online data sets (Supplementary Table S1). The assembly software was able to completely reconstruct AgSp2 in both species, with no evidence of truncation as seen in AgSp1. ...
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... data sequenced from A. trifasciata aggregate gland tissue to compare AgSp1 and AgSp2 expression, and from major ampullate silk gland fat body tissues as controls. AgSp1 and AgSp2 are among the most highly expressed genes within the aggregate glands, with significantly (q < 0.05) less expression detected in the major ampullate or fat body tissues (Fig. 5A). Within the aggregate glands, expression of AgSp1 and AgSp2 is not significantly different ( ...
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... repetitive motifs are notably similar between the two spidroins, and predicted protein alignments of the repeat motifs from these two spidroins supports paralogous origins (Fig. 3). Interestingly, AgSp2 is highly reduced in cobweb weavers (Supplementary Fig. S5). It is unknown if AgSp1 and AgSp2 interact with each other in any way to form the sticky capture glue, or what properties each imparts, particularly considering the differences between AgSp2 in orb web and cobweb weavers. ...
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... expansion regions are also found in pyriform spidroins , and QQ motifs have been shown to allow self-aggregation of these silk proteins into fibers (Geurts et al. 2010). The coding sequence of AgSp2 is dramatically reduced in Latrodectus cobweb weavers, consisting of less than 2000 bp (Supplementary Fig. S5). Differences between orb weaving and cobweb weaving pyriform spidroins have also been noted, including a loss of the QQ motifs ( Chaw et al. 2017). ...
Citations
... It has been reported that spider silk proteins undergo post-translational modifications (PTMs), including glycosylation (Tillinghast et al. 1992;Sponner et al. 2007;Choresh et al. 2009;Stellwagen and Renberg 2019). However, specific details regarding the type of glycosylation in these proteins remain limited. ...
Spider dragline silk stands out as a remarkable biomaterial, representing one of nature's toughest fibres. Its strength rivals that of many synthetic fibres used commercially, rendering it applicable across various industrial and medical domains. However, its widespread utilisation requires cost-effective mass production. Biotechnology presents a promising avenue for achieving this goal, particularly through the production of recombinant dragline silk proteins in transgenic plant systems. This study aimed to assess the feasibility of producing one key protein component of dragline silk, MaSp1, from the western black widow spider, Latrodectus hesperus, (LhMaSp1) in the moss Physcomitrella (Physcomitrium patens). Here, we present the successful recombinant production of spider silk protein containing both the N-and C-terminal domains (NTD and CTD) of LhMaSp1 protein in moss cells. NTD and CTD are necessary domains for protein assembly in spider silk. The production of recombinant LhMaSp1 protein in Physcomitrella was performed in shake flasks and in five-litre photobioreactors and the correct synthesis of LhMaSp1 was proven via mass spectrometry. Keywords Bioreactor, bryotechnology, dragline silk protein, plant-made protein, smart materials, spidroins Key message We report the first successful plant-produced recombinant spider silk key protein component containing both the N-and the C-terminal domain.
... What remains unclear is what differences in biochemical composition-glycoproteins, LMMCs, or both-distinguish moth specialists from generalists. Intriguingly, across a wide range of species spidroin genes have a similar structure, suggesting that glycoproteins may not account for the differences among spiders in catching moths [22,32,33]. In contrast, generalist spiders "fine-tune" their LMMCs for maximal adhesion in their native environmental humidity [31]. ...
Orb-weaver spiders produce upwards of seven different types of silk, each with unique material properties. We focus on the adhesive within orb-weaving spider webs, aggregate glue silk. These droplets are composed of three main components: water, glycoproteins, and a wide range of low molecular mass compounds (LMMCs). These LMMCs are known to play a crucial role in maintaining the material properties of the glycoproteins, aid in water absorption from the environment, and increase surface adhesion. Orb-weavers within the Cyrtarachninae subfamily are moth specialists and have evolved glue droplets with novel material properties. This study investigated the biochemical composition and diversity of the LMMCs present in the aggregate glue of eight moth-specialist species and compared them with five generalist orb-weavers using nuclear magnetic resonance (NMR) spectroscopy. We hypothesized that the novel drying ability of moth-specialist glue was accompanied by novel LMMCs and lower overall percentages by silk weight of LMMCs. We measured no difference in LMMC weight by the type of prey specialization, but observed novel compositions in the glue of all eight moth-catching species. Further, we quantified the presence of a previously reported but unidentified compound that appears in the glue of all moth specialists. These silks can provide insight into the functions of bioadhesives and inform our own synthetic adhesives.
... In contrast to other spider silk proteins, silk glue is a viscoelastic, amorphous, wet material that responds to environmental conditions [102]. It is noteworthy that the glue droplets of cobweb weavers demonstrate greater firmness and resilience compared to those of most of their orb web-weaving counterparts [103]. ...
... The coding sequences of proteins in aqueous silk glue represent one of the largest groups of silk protein genes documented to date [102]. Predominantly, the sequence is composed of two distinct repeat motifs, measuring 387 bp and 339 bp, interspersed by glutamine-rich regions [102]. ...
... The coding sequences of proteins in aqueous silk glue represent one of the largest groups of silk protein genes documented to date [102]. Predominantly, the sequence is composed of two distinct repeat motifs, measuring 387 bp and 339 bp, interspersed by glutamine-rich regions [102]. In contrast to other spider silk proteins, the glue proteins exhibit pentameric QPGSG repeats, bearing a striking resemblance to preserved modular elements in mammalian elastin, an elastomeric protein interacting with collagen [105]. ...
Spider silk protein, renowned for its excellent mechanical properties, biodegradability, chemical stability, and low immune and inflammatory response activation, consists of a core domain with a repeat sequence and non-repeating sequences at the N-terminal and C-terminal. In this review, we focus on the relationship between the silk structure and its mechanical properties, exploring the potential applications of spider silk materials in the detection of energetic materials.
... Until recently, the identities of aggregate proteins were unknown. Leading candidates were members of the spidroin gene family expressed in aggregate glands, AgSp1 and AgSp2 (Collin et al., 2016;Stellwagen and Renberg, 2019). We confirmed the presence of these two spidroins in aggregate glue droplets of the araneoid cobweb weaving family Theridiidae . ...
... In addition to proteins identified from the Proteome Discoverer searches, we added spidroins based on genome sequencing (Stellwagen and Renberg, 2019;Diaz et al., 2022). Because full-length spidroins are highly repetitive, including the full-length sequence in MaxQuant searches would either not complete or would result in very few peptide identifications due to repetitive sequences biasing the FDR calculations, since FDRs are based on scrambled peptides (Cox and Mann, 2008). ...
... Aggregate spidroins are abundant in the glue of both groups, suggesting an important role of these proteins in aggregate glue function. Argiope AgSp1 repeat motifs are longer and have more opportunities for glycosylation than cobweb weaver AgSp1 repeat motifs (Stellwagen and Renberg, 2019), although we found abundant glycosylation in both groups. AgSp2 is highly phosphorylated in the cobweb weaving house spider , while it is barely phosphorylated in Argiope. ...
Introduction
Orb web and cobweb weaving spiders in the superfamily Araneoidea are distinguished by their ability to make a chemically sticky aqueous glue in specialized aggregate silk glands. Aggregate glue is an environmentally responsive material that has evolved to perform optimally around the humidity at which a spider forages. Protein components and their post-translational modifications confer stickiness to the glue, but the identities of these proteins have not been described for orb web weavers.
Methods
Using biomechanics, gene expression data, and proteomics, we characterized the glue’s physical properties and molecular components in two congeners that live in different environments, Argiope argentata (dry southwest US) and Argiope trifasciata (humid southeast US).
Results
The droplets of A. argentata are less hygroscopic than those of A. trifasciata and have proportionately smaller viscoelastic protein cores, which incorporate a smaller percentage of absorbed water as humidity increases. Argiope argentata protein cores were many times stiffer and tougher than A. trifasciata protein cores. Each species’ glue included ~30 aggregate-expressed proteins, most of which were homologous between the two species, with high sequence identity. However, the relative contribution and number of gene family members of each homologous group differed. For instance, the aggregate spidroins (AgSp1 and AgSp2) accounted for nearly half of the detected glue composition in A. argentata, but only 38% in A. trifasciata. Additionally, AgSp1, which has highly negatively charged regions, was ~2X as abundant as the positively charged AgSp2 in A. argentata, but ~3X as abundant in A. trifasciata. As another example, A. argentata glue included 11 members of a newly discovered cysteine-rich gene family, versus 7 members in A. trifasciata.
Discussion
Cysteines form disulfide bonds that, combined with the higher potential for electrostatic interactions between AgSp1 and AgSp2, could contribute to the greater stiffness of A. argentata glue. The ability to selectively express different glue protein genes and/or to extrude their products at different rates provides a faster mechanism to evolve material properties than sequence evolution alone.
... 83 Also, spiders have evolved to produce a rather unexplored set of adhesive silks, namely pyriform adhesive silk and aggregate silk glue, which can outperform man-made cyanoacrylate-based glues in certain applications. 155,156 The mechanical properties such as adhesion, strength, toughness, and elasticity are highly tunable and uniquely altered by spiders depending on the environmental conditions and ecological needs. 164 For example, dragline silk is stiff and has a high ultimate tensile strength in dry conditions but supercontracts and becomes elastomeric in the hydrated state. ...
... While its sequence is less biased toward Gly, Pro, and Gln, Ala residues are almost absent from the mid-block ( Figure 3A-viii). 156 What makes the mid-block distinctly different from that of any other spidroin is that it has a substantially higher content of Ser and Thr. These are found to be prone to Oglycosylation and directly reflect the hygroscopic properties, as well as the stickiness of the aggregate glue. ...
Polymeric materials produced from fossil fuels have been intimately linked to the development of industrial activities in the 20th century and, consequently, to the transformation of our way of living. While this has brought many benefits, the fabrication and disposal of these materials is bringing enormous sustainable challenges. Thus, materials that are produced in a more sustainable fashion and whose degradation products are harmless to the environment are urgently needed. Natural biopolymers─which can compete with and sometimes surpass the performance of synthetic polymers─provide a great source of inspiration. They are made of natural chemicals, under benign environmental conditions, and their degradation products are harmless. Before these materials can be synthetically replicated, it is essential to elucidate their chemical design and biofabrication. For protein-based materials, this means obtaining the complete sequences of the proteinaceous building blocks, a task that historically took decades of research. Thus, we start this review with a historical perspective on early efforts to obtain the primary sequences of load-bearing proteins, followed by the latest developments in sequencing and proteomic technologies that have greatly accelerated sequencing of extracellular proteins. Next, four main classes of protein materials are presented, namely fibrous materials, bioelastomers exhibiting high reversible deformability, hard bulk materials, and biological adhesives. In each class, we focus on the design at the primary and secondary structure levels and discuss their interplays with the mechanical response. We finally discuss earlier and the latest research to artificially produce protein-based materials using biotechnology and synthetic biology, including current developments by start-up companies to scale-up the production of proteinaceous materials in an economically viable manner.
... Both genes are comprised of distinct repeats arranged in a stereotypical pattern. The occurrence of hierarchically organized repetitive units (i.e., ensemble repeats) has been found in other spidroins such as MaSp1 [75] and AgSp [99,100] suggesting they may be critical to the mechanical performance of these proteins. Similarly, comparison of the U. diversus sequences with Octonoba reveals shared repetitive features that may have functional significance. ...
Background
Spiders have evolved two types of sticky capture threads: one with wet adhesive spun by ecribellate orb-weavers and another with dry adhesive spun by cribellate spiders. The evolutionary history of cribellate capture threads is especially poorly understood. Here, we use genomic approaches to catalog the spider-specific silk gene family (spidroins) for the cribellate orb-weaver Uloborus diversus .
Results
We show that the cribellar spidroin, which forms the puffy fibrils of cribellate threads, has three distinct repeat units, one of which is conserved across cribellate taxa separated by ~ 250 Mya. We also propose candidates for a new silk type, paracribellar spidroins, which connect the puffy fibrils to pseudoflagelliform support lines. Moreover, we describe the complete repeat architecture for the pseudoflagelliform spidroin (Pflag), which contributes to extensibility of pseudoflagelliform axial fibers.
Conclusions
Our finding that Pflag is closely related to Flag, supports homology of the support lines of cribellate and ecribellate capture threads. It further suggests an evolutionary phase following gene duplication, in which both Flag and Pflag were incorporated into the axial lines, with subsequent loss of Flag in uloborids, and increase in expression of Flag in ecribellate orb-weavers, explaining the distinct mechanical properties of the axial lines of these two groups.
... In addition, the black widow spider is distinctive from those spiders that construct classic twodimensional aerial capture webs. The spider webs of black widow spiders are three-dimensional and are called cobwebs [19][20][21]. Therefore, genetic deciphering of black widow spider silk provides data and clues for the diversification of spiders. ...
Background:
The black widow spider has both extraordinarily neurotoxic venom and three-dimensional cobwebs composed of diverse types of silk. However, a high-quality reference genome for the black widow spider was still unavailable, which hindered deep understanding and application of the valuable biomass.
Findings:
We assembled the Latrodectus elegans genome, including a genome size of 1.57 Gb with contig N50 of 4.34 Mb and scaffold N50 of 114.31 Mb. Hi-C scaffolding assigned 98.08% of the genome to 14 pseudo-chromosomes, and with BUSCO, completeness analysis revealed that 98.4% of the core eukaryotic genes were completely present in this genome. Annotation of this genome identified that repetitive sequences account for 506.09 Mb (32.30%) and 20,167 protein-coding genes, and specifically, we identified 55 toxin genes and 26 spidroins and provide preliminary analysis of their composition and evolution.
Conclusions:
We present the first chromosome-level genome assembly of a black widow spider and provide substantial toxin and spidroin gene resources. These high-qualified genomic data add valuable resources from a representative spider group and contribute to deep exploration of spider genome evolution, especially in terms of the important issues on the diversification of venom and web-weaving pattern. The sequence data are also firsthand templates for further application of the spider biomass.
... The tail region contains GGQ, PGG, GPG and QGP motifs and polythreonine stretches on both ends. The C-terminal transition region has the same organization as the internal repeats (Stellwagen and Renberg, 2019). ...
... It is estimated that more than 80% of threonine residues in aggregate proteins are O-glycosylated (Tillinghast et al., 1992). In the first three subgroups of the AgSp1 protein high serine/threonine regions are seen which are likely glycosylated (Stellwagen and Renberg, 2019). Aggregate proteins in N. clavipes are reported as glycosylated proteins. ...
Spider silk threads have exceptional mechanical properties such as toughness, elasticity and low density, which reach maximum values compared to other fibre materials. They are superior even compared to Kevlar and steel. These extraordinary properties stem from long length and specific protein structures. Spider silk proteins can consist of more than 20,000 amino acids. Polypeptide stretches account for more than 90% of the whole protein, and these domains can be repeated more than a hundred times. Each repeat unit has a specific function resulting in the final properties of the silk. These properties make them attractive for innovative material development for medical or technical products as well as cosmetics. However, with livestock breeding of spiders it is not possible to reach high volumes of silk due to the cannibalistic behaviour of these animals. In order to obtain spider silk proteins (spidroins) on a large scale, recombinant production is attempted in various expression systems such as plants, bacteria, yeasts, insects, silkworms, mammalian cells and animals. For viable large-scale production, cost-effective and efficient production systems are needed. This review describes the different types of spider silk, their proteins and structures and discusses the production of these difficult-to-express proteins in different host organisms with an emphasis on plant systems.
... Several recombinant studies have demonstrated that functional silk fibers, albeit with diminished mechanical properties, can be produced from the transgenic expression of constructs containing only the terminal and limited repetitive sequence from a single spidroin (Adrianos et al., 2013;Brooks et al., 2008;Heidebrecht et al., 2015;Saric et al., 2021;Xia et al., 2010;You et al., 2018). Second, recent advances in genomic technologies have made possible the discovery of complete silk gene sequences, which were previously inaccessible (Babb et al., 2017;Kono et al., 2019;Sheffer et al., 2021;Stellwagen and Burns, 2021;Stellwagen and Renberg, 2019;Zhou et al., 2021). Genomic comparisons coupled with our biomechanical tests will allow us to understand Fig. 3. Velocity of aggregate glue spreading at >90% relative humidity (RH) and associated adhesion forces. ...
... changes in amino acid composition and how they affect glue behavior. Recent studies using long-read Oxford Nanopore (ON) data, one of which was focused on the bolas spider Mastophora phrynosoma, has revealed the full sequence and structure of aggregate spidroins (Stellwagen and Burns, 2021;Stellwagen and Renberg, 2019). This research has verified the presence of two primary copies, AgSp1 and AgSp2, and elucidated key aspects of intraspecific and interspecific variation. ...
... These genes have the largest coding regions recorded to date among spidroins and, like fiber-forming spidroins, are composed primarily of highly homogenized repetitive sequences. The amino acid translation of these repeat regions in both species is characterized by abundant threonine residues that are likely targets of O-linked glycosylation, a post-translational modification expected to enhance glue adhesive properties (Singla et al., 2018;Stellwagen and Renberg, 2019;Tillinghast et al., 1992). There is a sizable difference, however, in the repeat structure of these genes between M. phrynosoma and the orb-web weaving garden spider Argiope trifasciata. ...
Morphological structures and extended phenotypes are made possible by materials that are encoded by the genome. Nearly all biomaterials are viscoelastic, which means that to understand performance, one must understand the strain rate-dependent properties of these materials in relevant ecological interactions, as the behavior of a material can vary dramatically and rapidly. Spider silks are an example of materials whose properties vary substantially intra- and inter-specifically. Here, we focus on aggregate silk, which functions as a biological adhesive. As a case study to understand how a material manifests from genome through organism to ecology, we highlight moth-specialist spiders, the Cyrtarachninae, and their glues as an ideal experimental system to investigate the relationship between genomics and ecologically variable performance of a biological material. There is a clear eco-evolutionary innovation that Cyrtarachne akirai and related species have evolved, a unique trait not found in other spiders, a glue which overcomes the scales of moths. By examining traditional orb-weavers, C. akirai and other subfamily members using biomechanical testing and genomic analysis, we argue that we can track the evolution of this novel bioadhesive and comment on the selection pressures influencing prey specialization. The importance of the ecological context of materials testing is exemplified by the poor performance of C. akirai glue on glass and the exceptional spreading ability and adhesive strength on moths. The genetic basis for these performance properties is experimentally tractable because spider silk genes are minimally pleiotropic and advances in genomic technologies now make possible the discovery of complete silk gene sequences.
... Trees produced using Mr. Bayes v3.2.7 and visualized with the iTOL web server expression of some of these genes is shown in Fig. 4. Genes with higher expression in Spiny Legs include protease inhibitors such as zonadhesins, as well as transferases and transporter proteins. We observed reduced expression in Spiny Legs of some genes with aggregate gland-specific expression in other spiders, including an N-acetylgalactosaminyltransferase (pp-GalNAc-T) [41] and a globin protein [40]. Other genes with lower expression in Spiny Legs include an aminotransferase, a semaphorin membrane protein, and a cathepsin endopeptidase. ...
... Consistent with the loss of aggregate glands, Spiny Legs exhibited reduced expression of genes with aggregate gland-specific expression in other spiders, including a pp-GalNAc-T, a type of transferase that participates in O-glycosylation [54]. O-glycosylation contributes to the adhesive qualities of aggregate glue [55], and pp-GalNAc-T is highly expressed in the aggregate glands of the orbweaver Argiope trifasciata [41]. The reduced expression of this gene in Spiny Legs accompanied by their loss of aggregate glands suggests that this gene could play a similar role in aggregate silk production in tetragnathids. ...
Background
A striking aspect of evolution is that it often converges on similar trajectories. Evolutionary convergence can occur in deep time or over short time scales, and is associated with the imposition of similar selective pressures. Repeated convergent events provide a framework to infer the genetic basis of adaptive traits. The current study examines the genetic basis of secondary web loss within web-building spiders (Araneoidea). Specifically, we use a lineage of spiders in the genus Tetragnatha (Tetragnathidae) that has diverged into two clades associated with the relatively recent (5 mya) colonization of, and subsequent adaptive radiation within, the Hawaiian Islands. One clade has adopted a cursorial lifestyle, and the other has retained the ancestral behavior of capturing prey with sticky orb webs. We explore how these behavioral phenotypes are reflected in the morphology of the spinning apparatus and internal silk glands, and the expression of silk genes. Several sister families to the Tetragnathidae have undergone similar web loss, so we also ask whether convergent patterns of selection can be detected in these lineages.
Results
The cursorial clade has lost spigots associated with the sticky spiral of the orb web. This appears to have been accompanied by loss of silk glands themselves. We generated phylogenies of silk proteins (spidroins), which showed that the transcriptomes of cursorial Tetragnatha contain all major spidroins except for flagelliform. We also found an uncharacterized spidroin that has higher expression in cursorial species. We found evidence for convergent selection acting on this spidroin, as well as genes involved in protein metabolism, in the cursorial Tetragnatha and divergent cursorial lineages in the families Malkaridae and Mimetidae.
Conclusions
Our results provide strong evidence that independent web loss events and the associated adoption of a cursorial lifestyle are based on similar genetic mechanisms. Many genes we identified as having evolved convergently are associated with protein synthesis, degradation, and processing, which are processes that play important roles in silk production. This study demonstrates, in the case of independent evolution of web loss, that similar selective pressures act on many of the same genes to produce the same phenotypes and behaviors.
