Experimental alterations of morphological traits during development can reveal life history tactics and resource allocation patterns. I examined effects of amputation of a structure directly involved with mating compared to one that is less associated with fitness. I amputated one of the paired external copulatory organs (palps) of juvenile male black widow spiders Latrodectus hesperus), and compared changes in life history traits and fitness to males with amputated legs and controls. I show that palps are more likely to be regenerated than legs, smaller juveniles are more likely to regenerate, and mating success is adversely affected in all adults that suffered early amputation. The pre-existing relationship between juvenile size and development was a critical determinant of regeneration, however no life history costs of regeneration were evident. I conclude that plasticity in life history facilitates regeneration, and that this may mask trade-offs involved in compensatory effects at the population level.

Experimental alterations of morphological traits during development can reveal life history tactics and resource allocation patterns. I examined effects of amputation of a structure directly involved with mating compared to one that is less associated with fitness. I amputated one of the paired external copulatory organs (palps) of juvenile male black widow spiders Latrodectus hesperus), and compared changes in life history traits and fitness to males with amputated legs and controls. I show that palps are more likely to be regenerated than legs, smaller juveniles are more likely to regenerate, and mating success is adversely affected in all adults that suffered early amputation. The pre-existing relationship between juvenile size and development was a critical determinant of regeneration, however no life history costs of regeneration were evident. I conclude that plasticity in life history facilitates regeneration, and that this may mask trade-offs involved in compensatory effects at the population level.

No description available.

Although numerous spider fibroins have been reported, no known silk coating peptides have been discovered. We provide the first biochemical evidence for a spider coating peptide, called SCP-2, found on gumfooted lines, scaffolding joints and egg cases. The presence of this spider coating peptide on the fibers is supported by MS/MS analysis. Using quantitative real-time PCR analysis, we also demonstrate that SCP-2 has a flagelliform-restricted mRNA pattern of expression. Molecular modeling of the SCP-2 amino acid sequence predicts it adopts an alpha-helical structure that is amphipathic in nature. SCP-2, which can be extracted from fibers using water, is hypothesized to influence the mechanical properties of the silk fibers as well as serve a protective function for the threads. Based upon the restricted pattern of expression of SCP-2, our findings reveal novel insight regarding the glandular function of the flagelliform gland in . cob weaving spiders, suggesting it produces aqueous coating materials that are deposited on a wide range of different silk types. In addition, in an attempt to advance our understanding regarding silk gene transcription, our lab has developed the first antibody against the bHLH factor SGSF. SGSF has been implicated as a potential transcriptional regulator of silk gene transcription in spiders. Development of the anti-SGSF antibody was accomplished via the overexpression and purification of a fusion protein in bacteria, which consisted of the C-terminal region of SGSF fused to thioredoxin. Purified SGSF fusion proteins were injected into rabbits and the polyclonal antiserum was collected and tested by western blot analysis to determine the specificity of the immunological reagent. Western blot analyses revealed the anti-SGSF antiserum was capable of recognizing bacterially expressed SGSF in an efficient manner. Collectively, these studies lay the groundwork for future investigations involving the use of the antibody to determine the role of SGSF in silk transcription.

Silks from araneoid spiders have become an active area of research for material scientists, biochemists, and molecular biologists. Mechanical properties of spider silk such as elasticity, tensile strength, and toughness make the manufacturing of silk for medical sutures, body armor, ropes and other synthetic material applications great possibilities. The difficulties of having a black widow spider farm to harvest silk, due to their cannibalistic nature, make recombinant expression of silk proteins a fundamental goal of spider silk research. In order to express silk fibers, cDNAs encoding the corresponding silk fiber products must first be isolated and identified. One of the first steps in gene identification relies on the identification of the proteins in the silk fibers. No previous study has demonstrated the molecular constituents of gumfooted lines. In the course of this research, the core fibroins in the gumfooted lines were identified to be members of the Major Ampullate Spidroin family (MaSp), using mass spectrometry. This research was the first to identify the core fibroins of the gumfooted lines. Novel peptide fragments from solubilized gumfooted lines were acquired from manual de novo MSIMS sequencing after in-gel tryptic digestion. These peptide fragments showed post-translational modifications consistent with glycosylation, which aligns with the reported chemical properties of glue proteins. Novel peptide sequences were also acquired from the attachment discs as well as novel scanning electron microscopy images and reveal, for the first time, the physical attributes and molecular properties of threads attached to the surface of an immobilized structure. This study was the first to identify the molecular constituents of the attachment discs.

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.

Large volumes of antisera were generated against Apis melli/era venom with which to develop a novel, platform technology for the inexpensive production of anti venoms. The ovine sera contained high levels of specific antibodies which neutralised the myotoxic, phospholipase A2 and in vivo activities of the venom. Methods of processing the antisera to provide Fab or F(ab')2 were investigated. F(ab')2 was thought to be clinically advantageous and, by determining the conditions necessary for the preferential breakdown of Fc and serum components other than F(ab')2' it was possible to avoid salt precipitation. Diafiltration was then used to remove most of the unwanted small fragments and anion-exchange chromatography to remove any remaining acidic impurities such as pepsin and large aggregates. The F(ab')2 was -97% pure and the yield - 199 per L of serum. This is the first specific therapy for mass envenoming by European or Africanised bees. Spiders of the genus Latrodectus (black widows) are distributed widely and about 2,500 bites are reported annually in the USA. The neurotoxic effects of the venom were studied on the isolated phrenic nerve diaphragm preparation. Low venom concentrations (ImgIL) were stimulatory while high concentrations (IOmg/L) caused nerve blockade which was potentiated by increased calcium levels. Although effective, the Merck antivenom, which is unprocessed horse serum, causes unacceptable risks. The second purpose of this project was to prepare an improved Latrodectus spider antivenom using the new platform technology. Different immunisation schedules were studied to optimise the humoral immune response. Sheep immunised with 2mg La. hesperus venom produced the highest levels of specific antibodies as assessed by ELISA, using the isolated nerve diaphragm preparation or in vivo in mice. The new process provided a pure F(ab')2 antivenom retaining 78% of the original antisera ED so neutralising power and was - twice as effective as the Merck antivenom.

Thesis (M.S.)--Air Force Institute of Technology, 2006. / "AFIT/GLM/ENS/06-08." "March 2006." Includes bibliographical references (leaves 217-219). Also available online via the Defense Technical Information Center website (http://stinet.dtic.mil/).