<p>Researchers have long assumed that black widow coloration functions as a warning signal to avian predators. However adult female black widow coloration does not resemble typical warning coloration in two distinct ways. First, black widows are less colorful than most other documented aposematic species. Second, the hourglass shape of an adult female varies both between species and within a site. Here I examine the ultimate causes and consequences of North American black widow coloration. </p><p>In chapter two I present data that suggest that black widow coloration not only functions as an aposematic signal to avian predators, but has also been selected to be inconspicuous to insect prey. In choice experiments with wild birds, I found that the red-and-black coloration of black widows deters potential predators: wild birds were ~3 times less likely to attack a black widow model with a red hourglass than one without. Using visual-system appropriate models, I also found that a black widow's red-and-black color combo is more apparent to a typical bird than typical insect (Euclidean color distance ~2.2 times greater for birds than insects). Additionally, an ancestral reconstruction revealed that red dorsal coloration is ancestral in black widows and that at some point some North American black widows lost their red dorsal coloration (while maintaining the ventral hourglass). Behaviorally, differences in red dorsal coloration between two North American species are accompanied by differences in microhabitat that affects how often a bird will view a black widow's dorsal region. All observations are consistent with a cost-benefit tradeoff of being conspicuous to potential predators while being inconspicuous to prey. I suggest that avoiding detection by prey --- combined with Müllerian mimicry --- may help explain why red-and-black aposematic signals occur frequently in nature.</p><p>In chapter three, I examine the variation in hourglass shape. Classical aposematic theory predicts near uniformity in warning signal appearance because a uniform signal is easier to learn to avoid than a variable signal. However the shape of the hourglass of North American black widows appears to vary both within and between sites in ways that are inconsistent with classical aposematic theory. Using 133 black widows of three different species from nine sites across the United States, I quantified the variation in hourglass shape and examined how Müllerian mimicry, species type, and condition each influenced hourglass shape. A principle components analysis revealed that 84.5% of the variation in hourglass shape can be explained by principle components 1, 2, and 3, which corresponded to hourglass size (PC1), the separation between hourglass parts (PC2), and the slenderness of the hourglass (PC3). Both a black widow's condition and species significantly predicted hourglass shape; however I found no support for localized Müllerian mimicry within different geographical regions. My results suggest a relaxed role for selection on hourglass shape. I discuss several hypotheses that could explain the variation in hourglass morphology including that potential predators may avoid any red markings rather than an exact shape (categorical rather than continuous perception).</p><p>In chapter four I expand on my work from chapter two to examine the eavesdropper's perspective on private communication channels. Private communication may benefit signalers by reducing the costs imposed by potential eavesdroppers such as parasites, predators, prey, or rivals. It is likely that private communication channels are influenced by the evolution of signalers, intended receivers, and potential eavesdroppers, but most studies only examine how private communication benefits signalers. Here, I address this shortcoming by examining visual private communication from a potential eavesdropper's perspective. Specifically, I ask if a signaler would face fitness consequences if a potential eavesdropper could detect its signal more clearly. By integrating studies on private communication with those on the evolution of vision, I suggest that published studies find few taxon-based constraints that could keep potential eavesdroppers from detecting most hypothesized forms of visual private communication. However, I find that private signals may persist over evolutionary time if the benefits of detecting a particular signal do not outweigh the functional costs a potential eavesdropper would suffer from evolving the ability to detect it.</p> / Dissertation

Spiders are able to spin a variety of silk types for various purposes, each with their own unique properties. The mechanical properties of spider silk out-perform the mechanical properties of many man-made materials we use today, including tensile steel, KevlarTM, and nylon. To further understand the proteins the silks are made of and how they are synthesized in the silk glands, transcriptional and proteomic analysis was conducted. Transcriptional regulation of silk genes was investigated to determine how and why several silk proteins are transcribed into mRNA products together in the same gland. The tubuliform gland is one of the major contributors of egg case silk production. The mRNA of major ampullate spidroins 1 and 2 (MaSp1, MaSp2) and tubuliform spidroin 1 (TuSp1) is found in the tubuliform glands, but not all are translated into proteins for egg case silk purposes. To understand why not all of the transcribed mRNA products are not being translated into proteins, the promoter sequences of MaSp1, MaSp2, and TuSp1 were aligned and found to contain an E-Box site. Several constructs containing the cDNA of the promoter sequences and cDNA of bHLH transcription factors were built to test transcriptional regulation of MaSp1, MaSp2, and TuSp1. Proteomic analysis of egg case silk and the tubuliform glands was also conducted to identify further proteins synthesized in the tubuliform glands and to determine which of these proteins are ultimately incorporated into the egg case silk fibers by MS/MS analysis. Multiple silk proteins were identified within the tubuliform glands and incorporated into the egg case fibers, suggesting silks are composite fibers of multiple spidroins.

No description available.

Spider silks are protein-based fibers that possess remarkable mechanical properties. Major ampullate silk, also referred to as dragline silk, is renowned for its high tensile strength, extensibility and toughness. Dragline silk is produced from a liquid spinning dope that undergoes chemical and physical changes during extrusion. To date, no proteins that participate in the assembly process of major ampullate silk proteins have been identified. The goal of this project is the identification of such protein products. De novo sequencing of peptides from in pollution tryptic digestion of black widow spider dragline silk identified several novel peptides that were not derived from the full-length primary sequences of the major ampulate fibroins, MaSpl and MaSpl. One of the peptides corresponded to a region within a translated cDNA retrieved from a library constructed from silk-producing glands.

Spiders spin a wide variety of different silk types with different biological functions that are known for their extraordinary mechanical properties. Dragline silk has predominantly captured the interest of researchers because it exhibits high tensile strength and toughness while maintaining its elasticity. This thesis has focused on the characterization of a family of small molecular weight proteins recently discovered in dragline silk. These proteins were discovered in the western black widow spider, Latrodectus hesperus, and have been termed Cysteine-Rich Proteins (CRPs) due to their high conserved cysteine content. CRP family members were used in protein-protein interaction studies to determine if there is any interaction with the major ampullate spidroins (MaSps). After affinity chromatography and co-expression studies in bacteria, there were no detectable interactions between the CRPs and MaSp1. Further studies which could be an important role in the natural silk assembly process. Further protein interaction studies in different salt and pH conditions can further determine the function of the CRPs in dragline silk formation.

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 this study we have focused on the characterization of egg case silk, a third silk fiber produced by the black widow spider, Latrodectus hesperus , whose DNA coding sequences have not been reported. Based upon solubility differences in 8 M guanidine hydrochloride, it is demonstrated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and silver staining that the egg case silk is relatively complex at the molecular level, containing a large number of proteins with differing molecular weights. Protein components of egg case silk with a size about 100 kDa were obtained by a solubilization time course study, which indicates these proteins are likely to be embedded in the silk filament. Peptides in these 100 kDa proteins were released by tryptic in-gel and in-solution digestion. The peptides were sequenced using a MALDI tandem TOF mass spectrometer. Some of the de novo sequences were confirmed using a linear ion trap mass spectrometer equipped with a nanospray ion source. Combining the peptide sequences obtained, reverse genetics was employed to trace silk genes encoding proteins containing these de novo peptides. Three silk protein coding sequences were successfully discovered, which encode silk proteins named 3B, T1 and ECSP-1, respectively. 3B and T1 show the standard fibroin protein pattern. Amino acid repeat patterns were observed in these two silk clones. But the amino acid compositions of 3B and T1 show differences with the total amino acid composition of egg case silk, and also, the peptide sequences cannot be found in the primary amino acid sequences of 3B and T1. ECSP-1 protein represents one of the egg case silk proteins with a size of about 100 kDa. A number of peptide sequences obtained by mass spectrometric de novo sequencing were successfully located in ECSP-1's primary amino acid sequence. Sequence analysis demonstrates ECSP-1 represents a new class of silk proteins, with fibroin-like properties. The expression pattern of ecsp-1 is largely restricted to the tubuliform gland inside of the L. hesperus spider, with lower levels detected in the major and minor ampullate glands, which also confirms the identity of ECSP-1. It is also demonstrated that ECSP-1 assembles into higher aggregate structures through the formation of disulfide bonds. Peptide sequences from silk proteins from the Tarantula spider Grammostola rosea were also obtained. These sequences will be beneficial in obtaining genes encoding the silk from this spider species.

Spider silk has outstanding mechanical properties, displaying high tensile strength and extensibility. The unique combination of strength and great extensibility make it one of the toughest materials in the world. Of the seven different spider silks, dragline silk, the lifeline silk of the spider, represents one of the most renowned fiber types that has extraordinary properties. As a result, many labs across the globe are racing to manufacture synthetic dragline silk fibers. With the production of synthetic dragline silk fibers, there are unlimited commercial applications. In this study, we developed several codon-optimized MaSp1 minifibroin constructs for recombinant protein expression in bacteria. These recombinant MaSp1 minifibroin constructs were engineered to contain the N-terminal domain (NTD), different copies of internal block repeats (ranging from 2 to 64 copies of 35 amino acid blocks), and the C-terminal domain (CTD). The NTD and CTDs were derived from the natural cDNA sequences of black widow spiders, while the internal block repeats were generated from synthetic DNA fragments that were codon-optimized for expression in Escherichia coli . Different numbers of internal block repeats were created using a specialized seamless cloning strategy. By applying this seamless cloning strategy, we successfully multimerized MaSp1 block repeats that approach the natural fibroin size. Moreover, through the construction of a customized NTD-CTD spidroin construct, multimerized block repeats from any fibroin can be rapidly inserted to facilitate minifibroin protein expression in bacteria. Overall, this strategy as well as the created vectors, should help advance the silk community in the production of synthetic silk fibers that have properties that more closely resemble natural fibers.