OVARIAN DEVELOPMENT IN THE WESTERN BLACK WIDOW SPIDER LATRODECTUS HESPERUS ____________________________________ A Thesis Presented to the Faculty of California State University, Fullerton ____________________________________ In Partial Fulfillment of the Requirements for the Degree Master of Science in Biological Science ____________________________________ By Wendy Ouriel Thesis Committee Approval: Merri Lynn Casem, Department of Biological Science, Chair Kristy Forsgren, Department of Biological Science Alison Miyamoto, Department of Bioligcal Science Fall 2016 ABSTRACT The western black widow spider, Latrodectus hesperus, is a venomous spider that is widely distributed throughout the western United States. Despite species prevalence, research on the black widow spider is sparse. Although notorious for her mating rituals, no studies have explored the reproductive physiology of the black widow spider, including oogenesis, pedicel maturation, and oviposition. This study determined that oogenesis in the black widow spider occurred in three distinct phases of development: pre-vitellogenic (Phase 1), vitellogenic (Phase 2), and post-vitellogenic (Phase 3). Yolk was absent from the oocyte during the pre-vitellogenic phase. Yolk granules are first observed during vitellogenesis (Phase 2), which was further broken down into early and late vitellogenic stages. Early and late vitellogenic oocytes were defined according to yolk granule volume. The post-vitellogenic oocyte (Phase 3) had grown to 10 times the size of its former pre-vitellogenic state. The ova were filled with yolk granules, and detached from their pedicel presumably in preparation for oviposition. The pedicel stalk showed signs of apoptosis during late vitellogenesis, and was detached from Phase 3 oocytes. It was hypothesized that ova migrate into the ovarian lumen through an opening in the ovarian wall. My study represents the first analysis of ovarian development in the western black widow spider. ii TABLE OF CONTENTS ABSTRACT ................................................................................................................... ii LIST OF TABLES ........................................................................................................ v LIST OF FIGURES ....................................................................................................... vi Chapter 1. INTRODUCTION ................................................................................................ 1 Spider Reproductive Biology................................................................................ 1 Oocyte Development ............................................................................................ 3 Yolk Composition ................................................................................................. 6 Receptor Mediated Endocytosis of Yolk Proteins ................................................ 7 Oviposition of Post-vitellogenic Oocytes ............................................................. 9 2. METHODS ........................................................................................................... 12 Spider Collection and Maintenance ...................................................................... 12 Collection and Fixation of Spider Ovarian Tissue................................................ 12 Specimen Preparation and Embedding ................................................................. 14 Specimen Sectioning and Staining ....................................................................... 14 Electron Microscopy ............................................................................................. 15 Light Microscopy and Image Analysis ................................................................. 15 3. RESULTS ............................................................................................................. 17 Oocyte Cytoarchitecture ....................................................................................... 17 Phase 1: The Pre-vitellogenic Oocyte ............................................................ 17 Phase 2: The Vitellogenic Oocyte ................................................................. 19 Phase 3: The Post-vitellogenic Oocyte .......................................................... 23 The Pedicel ........................................................................................................... 25 Ovipostion ............................................................................................................. 25 iii 4. DISCUSSION ....................................................................................................... 30 Oogenesis .............................................................................................................. 30 The Pedicel ........................................................................................................... 36 Oviposition............................................................................................................ 41 REFERENCES .............................................................................................................. 48 iv LIST OF TABLES Table Page 1. Summary of the phases of oocyte development in Latrodectus hesperus ........... 24 v LIST OF FIGURES Figure Page 1. Summary of changes in ultrastructure that occur during oocyte development in the tickspider, Pseudocellus boneti.................................................................. 5 2. Light micrographs of L. hesperus oocytes from unmated and mated spiders in pre-vitellogenic, vitellogenic, and post-vitellogenic phases of oocyte development ......................................................................................................... 20 3. Transmission electron micrographs of unmated L. hesperus oocytes in pre-vitellogenic and vitellogenic phases .............................................................. 22 4. Changes in the basal lamina and plasma membrane between the pre-vitellogenic and late vitellogenic phases/stages of unmated L. hesperus oocyte development ............................................................................................. 24 5. Electron micrographs of pedicel organization at the pre-vitellogenic and vitellogenic phases of oocyte development in L. hesperus .................................. 26 6. Whole abdomen section of L. hesperus at the beginning of oviposition ............. 28 7. Light micrographs of whole body section of L. hesperus ovarian tissue acquired during late oviposition .......................................................................... 35 8. Light micrographs of whole abdominal sections of L. hesperus approximately five hours after oviposition. ................................................................................. 29 9. SEM image of L. hesperus ovarian tissue depicting a vitellogenic oocyte, and pedicel scars or “stumps” on the ovarian wall ..................................................... 29 vi 1 Chapter 1 INTRODUCTION The western black widow spider (Latrodectus hesperus) (Chamberlin and Ivie 1935) is a common spider found in California (Vetter 2009), yet represents an open frontier for scientific research. The vast majority of the research into the black widow spider has focused on its venom (Isbister et al. 2003; Pescatori and Grasso 1994; Rauber 1983; Scheer et al. 1983; Ushkaryov et al. 2004) and its silk (Blackledge and Swindeman 2005; Blasingame et al. 2009; Casem et al. 1999; Casem et al. 2002; Heim et al. 2009; Lawrence et al. 2004; Shao and Vollrath 1999), leaving the other aspects of black widow spider biology open to discovery. To date, no studies have examined the reproductive biology of black widow spiders; therefore, our knowledge of black widow spider reproduction is based upon research from other genera of spiders, or other arachnids. The purpose of my thesis was to contribute to our understanding of the reproductive biology of spiders by investigating the process of oogenesis and oviposition in L. hesperus. Spider Reproductive Biology To the best of our knowledge, all spiders are dioecious, meaning there are distinct females and males within the species (Elgar 1991; Foelix 1996). Female and male black widow spiders are distinguishable during adulthood; in which the adult female is significantly larger compared to the adult male and has a red hourglass marking on her abdomen (Foelix 1996). During development, both females and males go through several 2 molts, a shedding of the exoskeleton, to reach sexual maturity (Townley et al. 1993; Foelix 1996; Vetter and Rust 2010). Since males are smaller, they require fewer molts, and reach sexual maturity sooner than the female (Nentwig and Aitchison-Benell 1987; Foelix 1996; Sebastian 2009). In both sexes, the reproductive organs, the ovaries and the testes, are paired structures found within the abdomen (Foelix 1996). Male spiders develop a pair of appendages called pedipalps, which are used for the storage and transfer of a spermatophore, a spermatozoa-containing capsule, during mating (Foelix 1996). Prior to mating, the male spider must first load its pedipalps with sperm, a process referred to as charging (Eberhard and Huber 2010). Pedipalp charging in male spiders begins with the construction of a sperm web woven from the spider’s silk (Levi 1967). The sperm web usually takes the form of a triangular structure (Foelix 1996), and can vary in intricacy depending on the species (Eberhard and Huber 2010). During