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Araneoid spiders use specialized sets of abdominal silk glands to produce up to seven different types of silks, each with diverse functional properties. At the time of these studies, fibroin eDNA sequences that encode egg case silk had not been reported in the literature. This study used conventional nucleic acid-nucleic acid screening of a eDNA library to isolate a novel gene, named tubuliform spidroin 1 , from the black widow spider Latrodectus hesperus. TuSp 1 was demonstrated to be selectively expressed in the tubuliform gland (the gland suspected for egg case silk production), and examination of the amino acid sequence revealed highly homogeneous repeats (184 amino acid ensemble repeats), a characteristic feature of fibroin sequences. Analyses of the ensemble repeats within the amino acid sequence of TuSp 1 revealed the lack of long stretches of polyalanine and glycine-alanine sub-repeats, which are commonly found in minor ampullate and major ampullate silks. Polyserine blocks and short polyalanine stretches were highly represented in the TuSp 1 amino acid sequence. Our data support the assertion that TuSp 1 represents the main constituent within egg case silk. This supposition is supported by the observation that the amino acid composition of raw egg case silk was strikingly similar to the amino acid composition predicted from the translated TuSp1 eDNA. Two additional constituents identified in black widow egg case, egg case protein 1 (ECP-1) and egg case protein 2 (ECP-2), were also partially characterized in this study. Using immunohistochemical approaches, we demonstrate that ECPs predominantly localize to the exterior of the large diameter fibers of egg cases. Additionally, these studies revealed smaller amounts of ECPs localized to the interior portion of the fibers. Collectively, these results support TuSp1 as the predominant fibroin within egg sacs as well as reveal a structural role for the ECPs, providing clues regarding the supramolecular structure of egg case fibers.

Spider silk is one of the most remarkable materials produced in the natural world and its strength and extensibility are legendary. To date, the majority of studies have been performed on the following silks: dragline, capture spiral, and eggcase silk. The primary goal of this study was to characterize a lesser known silk type, called wrapping (swathing) silk, from Latrodectus hesperus. My research focused on elucidating the fibroins that make up the swathing silk. Perfonning MS/MS analyses on solubilized wrapping silk fibroins digested with trypsin, we demonstrate that a novel fibroin named AcSp !-like is present within wrapping silk. Consistent with this finding, SEM analyses reveal that wrapping silk is a composite material, containing at least three different diameter silk fibers. By using scanning electron micrographs, along with amino acid composition analyses and MS/MS analyses, we demonstrate wrapping material contains the fibroins AcSp1-like, MiSp1-like, MaSp1 and MaSp2. These are the first studies to reveal that minor, major and acinifonn silks are constituents of wrapping material.