Classification and functional characterization of spidroin genes in a wandering spider, Pardosa pseudoannulata.

Spiders impress us with their sophisticated use of silk and the stunningly distinct silk proteins (spidroins) in each spider species. Understanding how silks and spidroins function and evolve within the spider world is one profound interest to expand our knowledge on spider evolution. Spidroins are characterized with the divergent repeat core region flanked with the relatively conserved N- and C-terminus. The structure and number of the repeats contribute to the unique mechanical properties of the spidroin and the silk. Spidroins have been intensively studied in web-weaver spiders, but information regarding their diversity in wandering spiders remains scarce. Here, twenty spidroin genes were identified in the pond wolf spider, Pardosa pseudoannulata, belonging to the retrolateral tibial apophysis (RTA) clade. These spidroins were categorized into four classes, including twelve ampullate spidroin (AmpSp), four aciniform spidroin (AcSp), one tubuliform spidroin (TuSp), one pyriform spidroin (PiSp), and two spidroin-like proteins. Multiple copies of the AmpSp and AcSp genes were tandemly arranged in a cluster within the genome, and the N-terminal domains and repetitive sequences of the proximately located spidroins were highly similar, suggesting that the spidroin genes diversified via tandem duplication. Only four types of morphologically distinct silk glands were found in P. pseudoannulata, namely Ma, Mi, Ac, and Pi glands, consistent with the glandular affiliation hypothesis that spidroins co-evolved with glandular specialization to fit species-specific needs. Expression profiling revealed that the single tubuliform spidroin (TuSp) gene was highly expressed in gravid females and two AcSp genes displayed synchronous expression. Knock-down of the TuSp gene via RNAi resulted in fragile and cracked eggsacs and prolonged the female pre-oviposition period, validating its importance in spider reproduction. The genome-scale characterization and functional study of spidroin genes allows associating the presence of specific spidroins with silk utility in P. pseudoannulata and will expand our knowledge of spider evolution.

Solution Structure of Tubuliform Spidroin N-Terminal Domain and Implications for pH Dependent Dimerization.

The spidroin N-terminal domain (NT) is responsible for high solubility and pH-dependent assembly of spider silk proteins during storage and fiber formation, respectively. It forms a monomeric five-helix bundle at neutral pH and dimerizes at lowered pH, thereby firmly interconnecting the spidroins. Mechanistic studies with the NTs from major ampullate, minor ampullate, and flagelliform spidroins (MaSp, MiSp, and FlSp) have shown that the pH dependency is conserved between different silk types, although the residues that mediate this process can differ. Here we study the tubuliform spidroin (TuSp) NT from Argiope argentata, which lacks several well conserved residues involved in the dimerization of other NTs. We solve its structure at low pH revealing an antiparallel dimer of two five-α-helix bundles, which contrasts with a previously determined Nephila antipodiana TuSp NT monomer structure. Further, we study a set of mutants and find that the residues participating in the protonation events during dimerization are different from MaSp and MiSp NT. Charge reversal of one of these residues (R117 in TuSp) results in significantly altered electrostatic interactions between monomer subunits. Altogether, the structure and mutant studies suggest that TuSp NT monomers assemble by elimination of intramolecular repulsive charge interactions, which could lead to slight tilting of α-helices.

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.

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).