Ancient properties of spider silks revealed by the complete gene sequence of the prey-wrapping silk protein (AcSp1)

Abstract

Spider silk fibers have impressive mechanical properties and are primarily composed of highly repetitive structural proteins (termed spidroins) encoded by a single gene family. Most characterized spidroin genes are incompletely known because of their extreme size (typically >9 kb) and repetitiveness, limiting understanding of the evolutionary processes that gave rise to their unusual gene architectures. The only complete spidroin genes characterized thus far form the dragline in the Western black widow, Latrodectus hesperus. Here, we describe the first complete gene sequence encoding the aciniform spidroin AcSp1, the primary component of spider prey-wrapping fibers. L. hesperus AcSp1 contains a single enormous (∼19 kb) exon. The AcSp1 repeat sequence is exceptionally conserved between two widow species (∼94% identity) and between widows and distantly related orb-weavers (∼30% identity), consistent with a history of strong purifying selection on its amino acid sequence. Furthermore, the 16 repeats (each 371-375 amino acids long) found in black widow AcSp1 are, on average, >99% identical at the nucleotide level. A combination of stabilizing selection on amino acid sequence, selection on silent sites, and intragenic recombination likely explains the extreme homogenization of AcSp1 repeats. In addition, phylogenetic analyses of spidroin paralogs support a gene duplication event occurring concomitantly with specialization of the aciniform glands and the tubuliform glands, which synthesize egg-case silk. With repeats that are dramatically different in length and amino acid composition from dragline spidroins, our L. hesperus AcSp1 expands the knowledge base for developing silk-based biomimetic technologies.

Fig. 1.

Relationships among spider species used in this study based on hypotheses of Scharff and Coddington (1997), Coddington (2005), Ayoub, Garb, Hedin, et al. (2007), Kuntner et al. (2008), and Elices et al. (2009). Species abbreviations precede the species names. Major taxonomic groups are bracketed.

Fig. 2.

Amino acid compositions of complete Latrodectus hesperus AcSp1, MaSp1, and MaSp2. Three letter amino acid abbreviations are used.

Fig. 3.

Pairwise values of nonsynonymous substitutions per nonsynonymous sites (Ka; gray) and synonymous substitutions per synonymous sites (Ks; black) for intragenic comparisons among Latrodectus hesperus spidroin repeats or intergenic comparisons between L. hesperus and L. geometricus for corresponding regions of spidroins. Values shown for repeats are averaged across all pairwise comparisons.

Fig. 4.

Maximum likelihood tree of combined spidroin N- and C-terminal encoding regions. Species abbreviations are defined in figure 1 and supplementary table S2, Supplementary Material online. Thickened branches indicate relationships supported by >50% MP bootstrap replicates and >0.95 Bayesian posterior probability for both amino acids and nucleotides. Support values for numbered nodes are shown in supplementary table S3, Supplementary Material online. Rooting with the mygalomorph spidroin, B.c. fibroin1, resulted in the fewest inferred duplications and losses. Slashed lines indicate that the branch to B.c. fibroin1 was arbitrarily shortened. Dots indicate L. hesperus paralogs. Spidroins in bold had significant similarity to the L. hesperus AcSp1 repeat according to BLASTP. Major clades of spidroins are bracketed.

Fig. 5.

Maximum likelihood tree of expanded dataset of C-terminal region encoding nucleotides. Species abbreviations are defined in figure 1 and supplementary table S2, Supplementary Material online. Thickened branches indicate that the node is supported by >50% MP bootstrap replicates and >0.95 Bayesian posterior probability for both amino acids and nucleotides. Support values for numbered nodes are shown in supplementary table S4, Supplementary Material online. Rooting with the mygalomorph spidroins resulted in the fewest inferred duplications and losses. Dots indicate L. hesperus paralogs. Spidroins in bold had significant similarity to the L. hesperus AcSp1 repeat according to BLASTP. Major clades of spidroins are bracketed.