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.

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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

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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

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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 construction of the sperm web, the male pumps sperm from its gonopore

(Eberhard and Huber 2010). After exiting through the gonopore, the sperm is taken up inductively by the copulatory organs, referred to as the palpal bulbs (Eberhard and Huber

2010). During copulation, the male uses its palpal organs to deposit a spermatophore into the female’s spermathecae via the gonopore (Levi 1967; Foelix 1996).

The reproductive anatomy generally observed in most species of female spiders includes the external genital opening, referred to as the epigynum, the primary genital opening or gonopore, the spermathecae, and the paired ovaries (Foelix 1996; Michalik et al. 2005). The gonopore is located near the epigynum on the ventral surface of the spider’s abdomen (Foelix 1996). The opening of the gonopore leads to the oviduct, a common duct that connects the gonopore to the spider’s ovaries (Foelix 1996). The

3 seminal receptacles branched off the common duct that leads from the gonopore to ovaries (Foelix 1996). Spider ovaries are located within the abdominal cavity. The ovary is comprised of epithelial cells (Morishita et al. 2003), a cell type found throughout the body, which are often arranged in sheet formation (Vaughan and Trinkhaus 1966).

A continuous distribution of oocyte sizes is observed within the ovary, with the largest oocytes being the most developed (Choi and Moon 2003; Morishita et al. 2003;

Pourie and Trabalon 2003). Spider oocytes protrude into the abdomen, but remain attached to the ovarian epithelium by a thin, stem-like structure called the pedicel

(Denardi et al. 2004). The pedicel acts as an anchor to maintain oocyte attachment to the ovarian wall during oogenesis, and may also assist in development by providing proteins to the developing oocyte (Denardi et al. 2004; de Oliveira et al. 2005).

Oocyte Development

Oocyte development follows a pattern that is consistent amongst an array of egg- laying (oviparous) organisms such as fish, insects, reptiles, and arachnids (Lamison et al.

1991; Tyler and Sumpter 1996; Morishita et al. 2003; Denardi et al. 2004, Hernández-

Franyutti et al. 2005; Palacios- Vargas and Alberti 2009; Talarico and Zeck-Kapp 2009;

Machado-Santos et al. 2015). For many species, the first phase of oogenesis is the pre- vitellogenic phase, and the pre-vitellogenic oocytes are small, yolkless, and unfertilized

(Lamison et al. 1991; Tyler and Sumpter 1996; Morishita et al. 2003; Denardi et al. 2004;

Hernández-Franyutti et al. 2005; Talarico et al. 2009; Machado-Santos 2015). The next phase in oocyte development is vitellogenesis (McPherson 1982; Raikhel 1987) during which the oocyte undergoes significant cytoplasmic changes associated with the accumulation of yolk (Figure 1), which are present in the oocyte in the form of yolk

4 granules. Vitellogenesis is the process of yolk protein incorporation into the oocyte and is the primary event responsible for the voluminous growth of oocytes seen across multiple species (Tyler and Sumpter 1996). After the vitellogenic phase, oocytes exit the body during a process known as oviposition. Fertilization of the oocyte occurs after oocyte final maturation and oviposition, however whether fertilization occurs internally or externally varies depending on the species (Gist and Jones 1989; Sasanami et al. 2013).

The vitellogenic phase occurs in stages, with each stage having particular traits

(Dennis and Bradley 1989; Talarico et al. 2009; Higuchi et al. 2016). Yolk granules are first observed in the early vitellogenic stage (Brinton and Oliver 1971; Tyler and Sumpter

1996; Pourie and Trabalon 2003; Denardi et al. 2004; de Oliveira et al. 2005; Kawakami et al. 2009; Talarico et al. 2009). The yolk granules seen in early vitellogenesis are small, and sparsely distributed throughout the ooplasm (Figure 1) (Talarico et al. 2009). The basal lamina and vitelline membrane are also first observed during early vitellogenesis

(Figure 1). As vitellogenesis progresses, the oocyte increases in volume as yolk granules fill the ooplasm (Figure 1) (Brinton and Oliver 1971; Choi and Moon 2003; Denardi et al.

2004; Kawakami et al. 2009; Talarico et al. 2009). The basal lamina becomes more prominent during the late vitellogenic stage, and the space between the basal lamina and the plasma membrane widens (Talarico et al. 2009). By late vitellogenesis, the ooplasm is dense with large yolk granules (Talarico et al. 2009), mitochondria, and endoplasmic reticulum.

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Figure 1. Summary of changes in ultrastructure that occur during oocyte development in the tickspider, Pseudocellus boneti. In arachnids, oocyte development occurs in three phases: pre-vitellogenic, vitellogenic, and post-vitellogenic. In Pseudocellus boneti, the developmental phases can be further broken down into stages: primary vitellogenesis (II), secondary vitellogenesis (III), and tertiary vitellogenesis (IV). Note that lipid and protein yolk are absent from the pre-vitellogenic oocyte. Yolk does not begin to accumulate until the oocyte enters the vitellogenic phase. A vitelline membrane is absent in the pre- vitellogenic phase, but begins to appear in the primary vitellogenic stage (II). The outer vitelline membrane has not fully formed, which is depicted as dark ovals between the inner vitelline membrane and the basal lamina. The vitelline membrane is fully formed at the post-vitellogenic phase, depicted as a dark, bold line. Modified from Talarico et al. (2009).

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Yolk Composition

In vertebrates, such as fish, amphibians, and chickens (Tyler and Sumpter 1996;

Sappington and Raikhel 1998), and invertebrates such as insects, nematodes

(Schupachand Wieschaus 1991; Sappington and Raikhel 1998), crustaceans (Shafir et al.

1992; Lee and Chang 1997), and arachnids (Brinton and Oliver 1971; Morishita et al.

2003; de Oliveira et al. 2005), lipids and proteins are stored within yolk granules within the cytoplasm of the oocyte. The yolk granules that form during vitellogenesis will be used later on to support embryonic development (Sappington and Raikhel 1998).

The primary yolk protein found in the yolk granules, or yolk bodies as referred to in fish and insects (Sappington and Raikhel 1998; McMillon 2007; Nation 2008), is vitellin (Vn) (Ferenz 1989; Fan and Katagiri 1998; Ghekiere et al. 2006; Sonenshine and

Roe 2014), a lipo-glyco-carotenoprotein (Shafir et al. 1992). The beginning of yolk granule accumulation signals the end of the pre-vitellogenic stage, and the beginning of the vitellogenic stage, however for this process to initiate, the vitellin protein requires that its precursor protein, vitellogenin, be imported into the oocyte (Tyler and Sumpter 1996;

Sappington and Raikhel 1998; Sonenshine and Roe 2013).

Vitellogenin (Vtg), the precursor protein of vitellin (Tyler and Sumpter 1996), is synthesized in the liver of vertebrates (Tyler and Sumpter 1996; Sappington and Raikhel

1998), in the adipose cells of insects (Sappington and Raikhel 1998) and in the ovary of crustaceans (Kunkel and Nordin 1985). In arachnids, vitellogenin is synthesized in adipose cells (Kawakami et al. 2009) and potentially from the pedicel cells (Denardi et al.

2004; de Oliveira et al. 2005). Once synthesized, Vtg is exocytosed, and released into the blood plasma of vertebrates (Tyler and Sumpter 1996; Grant and Hirsh 1999), or into the

7 hemolymph of invertebrates, such as arachnids (Kawakami et al. 2009) and insects

(Sappington and Raikhel 1998). From the blood or hemolymph, Vtg is transported into the oocyte by receptor-mediated endocytosis (Raikel 1987; Shafir et al. 1992; Tyler and

Sumpter 1996; Sappington and Raikhel 1998).

Receptor Mediated Endocytosis of Yolk Proteins

Receptor-mediated endocytosis occurs in all eukaryotic organisms, and is essential for life-sustaining biological processes such as nutrient uptake, host defense, and regulation of cell signaling (Grant and Hirsh 1999). Receptor-mediated endocytosis is used during oocyte development to import yolk proteins, such as Vtg, into the oocyte

(Sappington and Raikhel 1998). During receptor-mediated endocytosis, Vtg forms a receptor-protein complex with a membrane-bound Vtg-receptor protein on the oocyte’s surface (Sappington and Raikhel 1998; Nation 2008). The receptor-protein complex dips into the oocytes surface, creating clathrin-coated pits on the cytoplasmic side (Nation

2008). Clathrin-coated pits manifest as small (0.1 μm diameter) invaginations on the surface of the cell’s plasma membrane, and when the Vtg-receptor binds to its ligand the clathrin-coated pit buds off from the oocyte’s plasma membrane, forming a coated vesicle (Stahl and Schwartz 1986; Opresko and Wiley 1987; Ferenz 1989).

Once inside the ooplasm, the clathrin coat separates from the vesicle containing the receptors and Vtg, and the uncoated vesicles fuse together. The newly formed uncoated vesicles are known as endosomes (Grant and Hirsh 1999; Nation 2008). A decrease in the endosome’s pH causes Vtg to dissociate from the Vtg receptor (Grant and

Hirsh 1999). The clathrin and receptors are sent back to the oocyte’s surface where they are reused to import more Vtg into the cell (Nation 2008).

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In somatic cells, imported macromolecules are ultimately digested by lysosomes, but yolk proteins such as Vtg are alternatively sent to yolk bodies for processing and storage (Sappington and Raikhel 1998; Nation 2008). A yolk body is a particular type of lysosome whose digestive function is paused until embryogenesis (Sappington and

Raikhel 1998). Endosomes combine with other endosomes into larger transitional yolk granules, and the yolk proteins within the endosomes are now called vitellins (Nation

2008). Vitellins are added continuously added to the transitional yolk granules until the yolk granule become mature (Nation 2008).

Although vitellin is a lipoprotein and the most abundant yolk protein found within the oocyte, it is not a significant transporter of lipids into the oocyte. Only about 5% of the oocyte’s total lipids are transported by Vn (Tufail and Takeda 2008). A majority of the lipids that comprise the oocyte’s lipids granules seen during oogenesis are not found naturally in the oocyte, so they must be transported from areas of the body where triglycerides are stored, such as the fat body (insects and arachnids) (Schneider 2008;

Tufail and Takeda 2012; Entringer et al. 2013). The oocyte receives most of its lipids from lipophorin, a lipoprotein that carries lipids from the fat stores to the oocyte (Benoit et al. 2011; Fruttero et al. 2011; Tufail and Takeda 2012; Leyria et al. 2014). Similar to vitellogenin, lipophorin is transported from the hemolymph to the oocyte by receptor- mediated endocytosis (Schneider 2008; Tufail and Takeda 2008; Parra-Peralbo and Culi

2011; Ravikumar et al. 2011). Once endocytosed, the lipids are stored as lipid yolk granules within the oocyte and are used later on to fuel embryonic development (Tufail and Takeda 2008).

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In addition to yolk granule accumulation within the oocyte, an extracellular matrix, or basal lamina, forms during the early stage of the vitellogenic phase of development and surrounds the oocyte’s outer surface (Beijnink et al. 1984; Smiley and

Cloney 1985; Giorgiet al. 1991; Hummel et al. 2004; Talarico et al. 2009). The components that make up the basal lamina originate from the ovarian epithelial cells, and extend from the ovarian epithelium and pedicel to the oocyte, encapsulating the entire ovary-pedicel-oocyte complex (Irving-Rodgers et al. 2004; Talarico et al. 2009).

Once yolk granule accumulation ceases, vitellogenesis is complete, and the oocyte is characterized as post-vitellogenic. In arachnids, chickens, fish, sea squirts, and amphibians, the post-vitellogenic oocyte has a granular appearance due to the abundance of yolk granules, a prominent basal lamina, and fully developed vitelline membrane

(Figure 1) (Tyler and Sumpter 1996; Gilbert 2000; Morishita et al. 2003; Denardi et al.

2004; de Oliveira et al. 2005; Mann and Mann 2008; Kawakami et al. 2009; Talarico et al. 2009; Kvarnryd et al. 2011; Schilling et al. 2015). The purpose of the vitelline membrane is not fully understood, although one potential function is to provide structural support to the oocyte during oviposition (Morishita et al. 2003).

Oviposition of Post-vitellogenic Oocytes

In oviparous species, oviposition occurs when oogenesis is complete (Morishita et al. 2003; Lubzens et al. 2010; Ostrovskij 2013). In the black widow spider, it is hypothesized that oviposition begins when the post-vitellogenic oocytes enter into the oviduct and ends when the last mature oocyte exits the gonopore. In arachnids and insects, the oviduct exists as a paired structure, one from each ovary, which branch from their respective ovary and merge together to form a common duct which connects to the

10 gonopore (Büning 1994; Foelix 1996). In some species of reptiles, fish, and amphibians only one ovary has an oviduct (Romer and Parsons 1977; Girling 2002; Cisint et al. 2014) and it is not directly connected to the ovary. For the species of reptiles, fish, and amphibians who go through a process of internal fertilization, oocytes are fertilized in the infundibulum, a funnel that catches oocytes as they are released from the ovary (Romer and Parsons 1977; Girling 2002; Cisint et al. 2014; Jacob 2015). Spiders lack an infundibulum but also go through a process of internal fertilization (Suzuki,1995; Foelix

1996; Kaster and Jakob 1997; Giribet 2003; Burger et al. 2006, Capinera 2008; Řezáč

2009; Huber and Dimitrov 2014; Lopardo and Hormiga 2015). Oocytes in spiders most likely become fertilized as they move through the oviduct past the seminal receptacles

(Dallai et al. 2012).

Oviposition is complicated in organisms that have an anatomy in which oocytes develop outside the ovarian lumen yet must make their way into the oviduct for oviposition (Choi and Moon 2003; Morishita et al. 2003; Denardi et al. 2004; de Oliveira et al. 2005; Talarico et al. 2009). It was observed in dissected gravid black widow spiders that oocytes were detached from their pedicels and appeared to be free floating in the hemocoel (Ouriel, personal observation.). If it is the case that fully developed oocytes are physically separated from the ovarian wall, then the question becomes how the oocytes enter into the oviduct for oviposition. Studies in other species of spider have not reached a consensus for the path the ova travel to the gonopore during oviposition (Sadana 1970;

Morishita et al. 2003).

Two models have been proposed for the movement of oocytes into the oviduct in spiders. The first model proposes that ova do not detach from their pedicel, but rather use

11 their pedicel attachment as a pathway into the oviduct (Brinton and Oliver 1971;

Morishita et al. 2003). In the second model proposed by Sadana (1970), the ova detach from the pedicel and locate an opening in the wall of the oviduct. The assumption is that the ovarian wall becomes progressively thinner during the reproductive cycle until holes appear, although it was not further elaborated how the ovarian epithelium changes to form such holes (Sadana 1970).

My study investigated oocyte development and the path of post-vitellogenic oocytes as they moved from the abdomen to the gonopore during oviposition. It was determined that the pre-vitellogenic phase in the black widow spider was similar to those in other spiders, and black widow spider oogenesis exhibited some unique characteristics during the vitellogenic phase. However, the exact mechanism for how oocytes traverse the abdomen to the gonopore remains unresolved.

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CHAPTER 2

METHODS

Spider Collection and Maintenance

Female black widow spiders, Latrodectus hesperus, were collected in June and

July of 2014 and 2015 in Riverside and Fullerton, California, United States. Each spider was housed in an individual plastic container (14 cm diameter, 5 cm height) with a cardboard retreat and maintained at a constant 27° C in a growth chamber with a 14-hour day and 10-hour night cycle. L. hesperus were fed on a diet of juvenile mealworms

(Tenebrio molitor). Spiders were fed one mealworm every two weeks.

Spiders that were observed to produce an egg case in the laboratory were designated as “mated” spiders, while those that completed their final adult molt in the laboratory were designated “unmated” spiders. Spider tissue obtained during oviposition are designated as “oviposition” spiders. A total of five unmated spiders and four mated spiders (one gravid, three oviposition) were used for this study.

Collection and Fixation of Spider Ovarian Tissue

All spiders (mated, unmated, and oviposition) were anesthetized by exposure to

CO2 gas for one minute, or until movement ceased. After anesthesia, the spider was euthanized by making a cut between the second and third walking legs perpendicular to the long axis of the cephalothorax. The posterior cephalothorax with attached abdomen was placed in 0.1M NaPO4 buffer (pH 7.4) and the abdomen was dissected away from the

13 remaining cephalothorax. A small number of oviposition spiders (n=3) were euthanized while eggs were being expelled in an attempt to determine the path taken by the oocytes during oviposition.

The ovarian tissue of black widow spiders caught during oviposition was examined in situ by fixing whole, intact abdomens. Whole abdomens were fixed using

Bouin’s fixative or aqueous spider fixative (4% glutaraldehyde 2% formaldehyde) prior to exoskeleton removal. The fixation process first required that the whole abdomen be placed into a petri dish containing 0.1M NaPO4 buffer (pH 7.4). Whole abdomens were washed in the buffer, and placed in the aqueous spider fixative for 24 hours or aqueous

Bouin’s fixative (5% acetic acid, 9% formaldehyde, 0.9% acetic acid) for three days at

4° C. To remove the exoskeleton, a longitudinal cut was then made along the abdomen’s dorsal side using 3 mm cutting edge spring scissors. Dumont #5a forceps were used to gently peel the exoskeleton from the endosternite tissue, while taking care to keep the abdomen intact. The exoskeleton was discarded. The sample was dehydrated in ethanol using a standard ethanol series.

For unmated specimens and one gravid specimen, the ovarian tissue was dissected out of the abdomen prior to fixation. Spider abdomens were placed in a petri dish containing 0.1M NaPO4 buffer (pH 7.4) following euthanasia, and the abdomen was dissected. The abdomen was dissected by making a midline cut on the dorsal side, and the exoskeleton was separated from the endosternite tissue. Upon opening the abdomen, the ovarian tissue was located by looking for translucent tissue within the opaque yellow fatty tissue. Ovarian tissue was removed from the abdomen with Dumont #5a forceps, washed with three exchanges of 0.1M NaPO4 buffer and the sample was placed in

14 fixative (4% glutaraldehyde 2% formaldehyde) for 24 hours at 4° C. Once fixation was complete, the specimens underwent dehydration using a graded ethanol series.

Specimen Preparation and Embedding

After specimens were fixed, they were dehydrated in a graded series of ethanol

(Hayat 2012). Dehydration was conducted at 4° C (Hayat 2012). After the final immersion in 100% ethanol, the specimens were placed in propylene oxide for 10 minutes. Following immersion in propylene oxide, specimens were placed in a 1:1 propylene oxide and epoxy resin mixture for one day. When the specimens had sat in the propylene oxide and epoxy resin mixture for one day, then placed in 100% epoxy resin.

Infiltration with the epoxy resin took three days, with a resin change every 24 hours.

Once the infiltration step was complete, specimens were embedded into blocks with epoxy resin (Spurr 1969).

Whole abdomens or isolated ovarian tissue were placed into a mold for Epon embedding. It was determined that orienting the whole abdomen with the anal tubercle facing downwards into the mold was optimal for viewing the abdominal contents, especially the ovarian tissue and oocytes. There was no special orientation for the isolated ovaries in the mold. For polymerization of the epoxy resin, the molds were placed in a heated oven (67° C) for three days.

Specimen Sectioning and Staining

Epon embedded specimens were sectioned using an LKB Ultratome V ultramicrotome. 1µm thick sections for use with light microscopy were cut using a glass knife. It was necessary to trim the whole abdomen specimens in order to orient the region in which the ovaries were located with the block face of the knife. Serial sections were

15 cut in 50 µm increments, and sections transferred to glass slides in sequential order.

Sections transferred to a glass slide were stained with toluidine blue prior to viewing with the light microscope. Epon-embedded ovarian tissue was cut into 80 nm thick sections with a diamond knife using the LKB Ultratome V for use with the electron microscope.

Thin sections were placed onto 270 mesh copper grids and stained with 3% uranyl acetate and 3% lead citrate following standard protocols (Bozzola and Russell 1999).

Electron Microscopy

Ultrastructure of isolated ovarian tissue was examined using a Hitachi-7000 transmission electron microscope. Images were captured onto Kodak electron microscopy film, and developed in lab for analysis. The negatives obtained from developing the film were used to analyze the of the contents of the ooplasm (mitochondria, ribosomes, yolk), measure the size of the nucleus, and look for any indication of receptor-mediated endocytosis on the plasma membrane. The electron micrographs were used to analyze pedicel cell organelles (endoplasmic reticulum, mitochondria) and pedicel cell organization.

Light Microscopy and Image Analysis

Plastic-embedded specimens were observed using a Leica MZFLIII compound microscope. Specimens were observed using standard bright field and phase contrast light microscopy. Images were captured using a QImaging digital camera and QCapture Pro 7

2010 Software (QImaging) and analyzed using ImageJ software (Schneider et al. 2012).

Oocyte size, ooplasm contents, basal lamina, plasma membrane, and vitelline membrane were analyzed using ImageJ software. Prior to analysis the software was calibrated. Calibration was performed using a 1 mm stage micrometer. The micrometer

16 was photographed through the microscope at the same magnification as the specimens to be analyzed. Once the micrometer was photographed, the image was opened in ImageJ software. Using the software, a line was drawn from the first tick mark of the micrometer to the last tick mark (1 mm). The resulting scale was applied to all images under the same magnification. To apply scale bars to images under a different magnification, the above mentioned steps were repeated, but with the micrometer photographed under the changed magnification.

Estimates of the volume of pre-vitellogenic, vitellogenic, and post-vitellogenic oocytes were determined by measuring oocyte diameter. An average oocyte diameter was calculated by drawing three bisecting lines through the nucleus of the oocyte. The average of the three lines was determined, and halved to obtain the radius. The value for the radius was used to obtain the oocyte’s volume assuming the oocytes were spherical.

For this project 15 unmated spiders and five (three during oviposition, one before oviposition, one after oviposition) mated spiders were used. For each phase of development, a total of 30 pre-vitellogenic, 30 vitellogenic, and 18 post-vitellogenic oocytes were examined (Table 1).

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CHAPTER 3

RESULTS

Oocyte Cytoarchitecture

The progression of oogenesis in the black widow spider was characterized by examining ovarian tissue isolated from mated adult (gravid), unmated adult, and mated spiders caught during oviposition. Spiders that underwent their final molt to sexual maturity in the laboratory were used as a baseline to characterize oogenesis in the absence of mating and fertilization. These samples were compared to the ovarian tissue isolated from spiders that were observed to produce at least one viable egg case in captivity. Examination of the ovaries of unmated and mated L. hesperus revealed oocytes that could be sorted into the following three populations based on their distinct morphologies.

Phase 1: The Pre-vitellogenic Oocyte

The pre-vitellogenic oocyte was the smallest of all of the observed oocytes and was present in the ovaries of both unmated and mated spiders. The pre-vitellogenic oocytes (n = 30) observed in light micrographs of plastic embedded tissue had an average volume of 4.56 x 104 µm3 (Table 1) with the smallest volume granule measuring 1.60 x

104 µm3 and the largest volume granule measuring 4.69 x 105 µm3.

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Table 1: Summary of the phases of oocyte development in Latrodectus hesperus

Average Average Average Protein Oocyte Lipid Yolk Yolk Description of Phase Phase Volume Volume Volume Characteristics

Oocyte was anchored to the ovarian wall via its pedicel attachment There is a central 1: No protein 4.56 x 104 No lipid yolk nucleus. Ooplasm Pre-vitellogenic yolk µm3 present appears n = 30 present homogenous. There is no evidence of yolk granules (Figure 2a; Figure 3a).

The oocyte remains Early Early anchored to the (n = 34): (n = 14): 3 3 ovarian wall by 2.81 µm 0.66 µm pedicel attachment 2: 2.06 x 105 (Figure 2c). Lipid Vitellogenic µm3 and protein yolk n = 30 Late Late granules are present. (n = 34): (n = 14): Visible basal lamina 623.2 µm3 1.52 µm3 and vitelline membrane

At this stage, the oocyte detached from the pedicel and seen in the 3: hemocoel. The 1.40 x 108 Not Not Post-vitellogenic oocyte is fully µm3 measured Measured n = 18 developed, and ready for oviposition. The oocyte is at its largest in the post- vitellogenic phase.

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Pre-vitellogenic oocytes were characterized by their pedicel attachment (Figure

2a; Figure 5), the absence of stained intracellular granules within the ooplasm and the appearance of a central nucleus in some sections (Figure 2a). The ooplasm was rich with mitochondria, which appeared to cluster around the nuclear membrane (Figure 3b). The average nuclei volume for pre-vitellogenic oocytes (Figure 2a) was 8.19 x 103 m3, and

8.18 x103 m3 (Figure 3b) The oocytes at the pre-vitellogenic phase had a thin basal lamina surrounding the plasma membrane (Figure 4b). Endoplasmic reticulum and lysosomes were not observed in the ooplasm.

Phase 2: The Vitellogenic Oocyte

Vitellogenic oocytes were present in both unmated and mated spiders (Figure 2b;

Figure 2c; Figure 4a). Similar to the pre-vitellogenic oocyte, the vitellogenic oocyte remained anchored to the ovarian wall through the attachment to the pedicel (Figure 3a).

The vitellogenic oocyte had an average volume of 2.06x 105 µm3 (n = 30) (Figure 2c;

Table 1). The vitellogenic oocytes exhibited lipid and protein yolk accumulation as indicated by toluidine blue staining of intracellular granules. The staining properties of toluidine blue differentiated between protein yolk (minimal staining), and lipid yolk (dark staining). The ooplasm of the vitellogenic oocyte had a granular appearance that was not observed in pre-vitellogenesis (Figure 2b; Figure 2c). As vitellogenesis progressed, oocyte size increased along with increased lipid and protein granule quantity and volume

(Figure 3c; Figure 3d).

Early and late vitellogenic oocytes were defined according to yolk granule volume (Table 1). Early vitellogenic oocytes had scattered lipid and protein yolk granules that were small; early vitellogenic lipid granules (n = 34) had an average volume of

20

Figure 2. Light micrographs of L. hesperus oocytes from unmated and mated spiders in pre- vitellogenic, vitellogenic, and post-vitellogenic phases of oocyte development. All specimens fixed using spider fixative. 2a: Oocytes in the pre-vitellogenic phase. Oocytes exhibited homogenous staining and large nuclei relative to the oocyte’s size. 2b: Early vitellogenic oocytes exhibited spherical yolk granules scattered throughout the ooplasm. A thin basal lamina was also visible at the early vitellogenic stage. 2c: Vitellogenic oocyte from an unmated spider anchored to the ovarian wall by pedicel attachment. 2d: Post-vitellogenic oocyte from a mated spider. The ooplasm was filled with yolk granules. The oocyte more than doubled in size. BL = basal lamina, G = yolk granule, LY = lipid yolk, N = nucleus, O1 = pre-vitellogenic oocyte, O2 = vitellogenic oocyte, O3 = post-vitellogenic oocyte, OL = ovarian lumen, OT = ovarian tissue, P =pedicel, PY = protein yolk

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2.81µm3, with the smallest granule measured having a volume of 0.24 µm3 and the largest having a volume of 498.1 µm3 (Figure 3c). Early vitellogenic protein granules

(n = 14) had an average volume of 0.66 µm3 with the smallest granule having a volume of 0.21 µm3, and the largest protein granule having a volume of 1.48 µm3 (Figure 3c). A population of late vitellogenic oocytes contained large lipid granules (n = 34) having an average volume of 623.2µm3 with the smallest lipid granule measuring at a volume of

2.31 µm3 and the largest having a volume of 2031.4 µm3 (Figure 3c; Figure 4a). The protein yolk granules (n = 14) found in the late vitellogenic oocytes were larger on average than those measured in the early vitellogenic oocytes, with an average volume of

1.52 µm3, and the smallest protein yolk granule measuring 0.10 µm3 (Figure 3c). For both early and late vitellogenic oocytes, lipid yolk granules appeared in greater quantities than protein yolk granules (Figure 2b; Figure 2c; Figure 3c; Figure 3d).

The surface of the vitellogenic oocyte consisted of a basal lamina on top and a plasma membrane underneath (Figure 3c; Figure 4b; Figure 4c). The basal lamina was thinner in the early vitellogenic oocytes and thicker in the late vitellogenic oocytes

(Figure 4c). The basal lamina appeared to cover both the oocyte, and its pedicel attachment (Figure 3a). The vitelline membrane, a space between the basal lamina and plasma membrane, widened during the subsequent stages of vitellogenesis (Figure 4b;

Figure 4c). The plasma membrane had coated pits along its surface (Figure 4b; Figure

4c). The coated pits seen on the plasma membrane were more prominent in the late vitellogenic stage (Figure 4c). Coated vesicles were also observed near the plasma membrane, in the oocyte’s interior during late vitellogenesis (Figure 4c).

22

Figure 3. Transmission electron micrographs of unmated L. hesperus oocytes in pre-vitellogenic and vitellogenic phases. Dissected ovarian tissue. 3a: Pre-vitellogenic (Phase 1) oocyte. The ooplasm has a granular appearance. A thin basal lamina was observed surrounding the oocyte and pedicel. 3b: Pre- vitellogenic (Stage 1) oocyte sectioned through the nucleus. Mitochondria were observed in clusters around the nuclear envelope 3c: Early vitellogenic (Stage 2) oocyte. Lipid accumulation was seen as electron-dense granules throughout the ooplasm. There is a narrow space between the basal lamina and plasma membrane. The ooplasm is dense with lipid and protein yolk granules. The oocyte had increased in size. 3d: Late-vitellogenic oocyte. Numerous large lipid and protein yolk granules were in the ooplasm. BL = basal lamina, G = granules, LY= lipid yolk granule, N = nucleus, O2 = vitellogenic oocyte, O2E = early vitellogenic oocyte, O2L = late vitellogenic oocyte P = pedicel attachment, PM = plasma membrane, PY = protein yolk granule, Y = yolk granules

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Phase 3: Post-vitellogenic Oocyte

The post-vitellogenic oocyte was found only in mated spiders that were gravid

(Figure 2d) or in oviposition (Figure 6; Figure 7). The average volume for post- vitellogenic oocytes was 1.40 x 108 µm3 (n = 18). The smallest oocyte had a volume of

3.72 x 108 µm3 and the largest oocyte had a volume of 2.33 x 109 µm3. The post- vitellogenic oocyte was densely packed with lipid and protein yolk granules (Figure 2d;

Figure 6; Figure 7). Another key characteristic of the post-vitellogenic oocyte was its prominent basal lamina (Figure 2d). The density of the yolk granules was too great, and prevented measurement of individual yolk granules. Therefore, the volume of the yolk granules could not be determined for the post-vitellogenic oocytes.

24

Figure 4. Changes in the basal lamina and plasma membrane between the pre-vitellogenic and late vitellogenic phases/stages of unmated L. hesperus oocyte development. Whole abdomens fixed using spider fixative. 4a: Light micrograph field of view containing oocytes in the pre-vitellogenic, early vitellogenic, and late vitellogenic phases. Area in box is shown at higher magnification below 4b: Electron micrograph of the pre-vitellogenic oocyte pictured in 4a (solid line). 4c: Electron micrograph of the late vitellogenic oocyte pictured in 4a (dotted line box). The basal lamina had thickened from 0.161 μm (4b) to 0.175 μm. The space between the basal lamina and plasma membrane had also widened to 2.5 μm. Note the presence of clathrin-coated pits. BL = basal lamina, CP = coated pits, CV = coated vesicle, VL = Vitelline membrane, LY = lipid yolk, MV = microvilli, O2E = early vitellogenic oocyte, O2L = late vitellogenic oocyte, PV = pre-vitellogenic oocyte, PY = protein yolk.

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The Pedicel

The pedicel of a pre-vitellogenic oocyte had a different appearance from the pedicel connecting a vitellogenic oocyte to the ovarian wall (Figure 5a; Figure 5b).

Pedicel cells of a pre-vitellogenic oocyte were not arranged in an organized manner

(Figure 5a). The pedicel attached to the vitellogenic oocyte was narrower than when attached to a pre-vitellogenic oocyte (Figure 5). The pedicel stalk at the pre-vitellogenic phase (Figure 5a) measured 73 μm in diameter at its narrowest point. For vitellogenic oocytes, the pedicel cells exhibited a more organized appearance, arranging into a lamellar stack, and the entire pedicel structure (Figure 5b) narrowed to 24.2 μm in diameter. Pedicel attachment was only observed for pre-vitellogenic (Figure 2a), and vitellogenic oocytes (Figure 2c). Pedicel attachment was not observed for post- vitellogenic oocytes (Figure 2d; Figure 6; Figure 7).

Pedicel cells attached to pre-vitellogenic and vitellogenic oocytes contained mitochondria and endoplasmic reticulum (Figure 5a; Figure 5b). The mitochondria appeared to alter their distribution within the cytoplasm as development progressed, similar to what was observed in the oocyte (Figure 5a; Figure 5c).

A basal lamina enclosed both the pedicel cells and attached oocyte (Figure 5). The basal lamina extended from the pedicel to the oocyte as a continuous structure. Microvilli were present in the area where the pedicel and the pre-vitellogenic oocyte contact (Figure

5c). Lateral membrane interdigitation was observed between adjacent pedicel cells not attached directly to the oocyte (Figure 5a).

Pedicel cell morphology also varied depending on the location of the cell. Pedicel cells that contacted the oocyte had a squamous morphology, and pedicel cells that were

26

Figure 5. Electron micrographs of pedicel organization at the pre-vitellogenic and vitellogenic phases of oocyte development in L. hesperus. All specimens fixed using spider fixative. 5a: Pre-vitellogenic phase pedicel. Pedicel cells contain mitochondria, and large elongated nuclei. Lateral membrane interdigitations were seen among pedicel cells that only contacted each other 5b: Pedicel of a late vitellogenic oocyte. The pedicel cells were arranged in lamellar fashion. Rough endoplasmic reticulum was more apparent. 5c: Pedicel attachment to a pre-vitellogenic phase oocyte. Microvilli contained within the space between the oocyte and pedicel. Note that the basal lamina, which surrounded the oocyte’s exterior, continued to the pedicel. BL=basal lamina, Ec = epithelial cell LMI = lateral membrane interdigitation, M = mitochondria, MV = microvilli, N = nucleus, O1 = pre-vitellogenic oocyte, OW = ovarian wall, P = pedicel

27 only in contact with other pedicel cells, or the ovarian epithelium had a rounded morphology (Figure 5b). The pedicel cells that were in contact with the oocyte had nuclei were also considerably smaller and flatter than the nuclei of pedicel cells adjacent to other pedicel cells, or the ovarian epithelium.

The pedicel attached to the vitellogenic oocyte was narrower than when attached to a pre-vitellogenic oocyte (Figure 5).

Oviposition

L. hesperus displayed a predictable pattern for the timing and frequency of egg clutch production. The interval between the production of a clutch of eggs was 45 days on average (Ouriel, personal observation). The reliable nature of egg laying activity provided an approximate timeframe for monitoring spiders for oviposition. However, knowing precisely when oviposition would occur proved to be a daunting task, resulting in the small sample size of spiders caught during transport of the oocytes.

The abdomens of the spiders caught during oviposition were fixed at the onset of oviposition (early oviposition) (Figure 6) 20 minutes after oviposition began (late oviposition) (Figure 7), or five hours after the completion of oviposition (Figure 8). For the spider in late oviposition, the stores of post-vitellogenic oocytes were nearly depleted, and only pre-vitellogenic oocytes, and one vitellogenic oocyte were visible (Figure 7a;

Figure 7b; Figure 8). This is in contrast to the spider in early oviposition (Figure 6) whose post-vitellogenic oocytes filled the abdomen. When post-vitellogenic oocytes are still present in the abdomen, they appeared to be separate from the pre-vitellogenic oocytes (Figure 6; Figure 7). The post-vitellogenic oocytes appeared to be separate from the pre-vitellogenic oocytes by a membrane compartment.

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Figure 6. Whole abdomen section of L. hesperus at the beginning of oviposition. Whole abdomen prepared using Bouin’s fixative. The post-vitellogenic oocytes (ova) measured 1.08 ± 0.58 mm3 in volume (n = 7). Ova and pre-vitellogenic oocytes are separate from each other. The post-vitellogenic oocytes are contained within their own membrane-bound compartment. Pedicels were seen attached to the membrane bound compartment (arrowhead). Note that no vitellogenic oocytes are visible. H = heart, MA = major ampullate silk gland, O1 = pre-vitellogenic oocyte, O3 = post-vitellogenic oocyte, T = tubuliform silk gland.

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Figure 7. Light micrographs of whole body section of L. hesperus ovarian tissue acquired during late oviposition. Whole abdomen fixed using Bouin's fixative. 7a: Multiple oocytes in pre-vitellogenic phase of development were observed. There was only one vitellogenic, two post-vitellogenic oocytes observed during late oviposition. Post-vitellogenic oocytes depicted were not spherical, which may be an artifact or due to the plane of sectioning. 7b: Increased magnification of the specimen is shown in 7a (boxed selection). The space between the mature ova and pre-vitellogenic oocytes was more apparent. He = hemocoel, O1=pre-vitellogenic oocyte, O2 = vitellogenic oocyte O3 = post-vitellogenic oocyte, OW = ovarian wall.

Figure 8. Light micrographs of whole abdominal sections of L. hesperus approximately five hours after oviposition. Whole abdomen fixed using spider fixative. 8a: Sections taken about 1.4 mm deep into abdomen (diagram shown). One end of the major ampullate gland was visible in the section. Only pre-vitellogenic oocytes were visible in this post-oviposition spider. Note the empty space (**). 8b: Specimen depicted in Figure 8a, sectioned 450 μm deeper into the abdomen. The section cut directly through the major ampullate gland. Note that the ovarian tissue enclosed the space where the pre- vitellogenic oocytes were found (asterisk). Oocytes are observed on both sides of the enclosed space (**). H = heart, MA = major ampullate gland, O1 = pre-vitellogenic oocyte

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CHAPTER 4

DISCUSSION

Oogenesis

All embryonic development, regardless of the species, begins with an unfertilized egg. The unfertilized egg, or oocyte, must undergo specific developmental changes for it to become a mature organism. For an oocyte to become a mature organism, the oocyte must first undergo oogenesis. Previous research on the reproductive biology of other genera of spiders indicates that oogenesis occurs in the following successive phases: the pre-vitellogenic phase, the vitellogenic phase, and the post-vitellogenic phase (Nentwig and Aitchison-Benell 1987; Choi and Moon 2003; Morishita et al. 2003; Pourie and

Trabalon 2003). Although the data has shown that the basic progression of oogenesis is similar amongst the spiders that have been studied (Morishita et al. 2003; Pourie and

Trabalon 2003; Jędrzejowska and Kubrakiewicz 2010), no research has been conducted to survey the process in L. hesperus, leaving the cellular aspects of its reproductive biology open for discovery.

Changes accompanying L. hesperus oocyte development were consistent with the changes observed in the oocytes of other oviparous species (McPherson 1982; Raikhel

1987; Dennis and Bradley 1989; Tyler and Sumpter 1996; Morishita et al. 2003; Pourie and Trabalon 2003; Denardi et al. 2004; Talarico et al. 2009; Jędrzejowska and

Kubrakiewicz 2010), and a similar criterion was used to describe oogenesis in L.

31 hesperus. Staging criteria for L. hesperus oogenesis included three phases: pre- vitellogenic (Phase 1), vitellogenic (Phase 2), and post-vitellogenic (Phase 3). The phases used for my staging criteria aligned with the published accounts of oogenesis in ticks

(Denardi et al. 2004; Saito et al. 2004; de Oliveira et al. 2005 2007), mites (Talarico et al.

2009), and spiders (Morishita et al. 2003; Pourie and Trabalon 2003). The phases of oogenesis in L. hesperus were identified based on specific changes that occurred to the oocyte as development progressed (Table 1). It was observed that the oocyte, originally small and yolkless (Phase 1), increased in size as it gradually accumulated yolk (Phases 2 and 3). There were two subpopulations of oocytes observed within Phase 2 that varied in oocyte volume, and the volume of lipid and protein yolk. To further categorize the subpopulation of vitellogenic oocytes based on the differences in oocyte and yolk granule volume, Phase 2 was broken down into stages: early vitellogenic and late vitellogenic

(Pourie and Trabalon 2003; Talarico et al. 2009). The contents of the ooplasm altered during all phases of oogenesis, the pedicel attached to the oocyte matured (Phases 1 and

2) and eventually detached (Phase 3), and the plasma membrane showed evidence of receptor-mediated endocytosis (Phases 1 and 2).

The ovarian tissue of mated and unmated L. hesperus contained oocytes in phases one and two, of development (Figure 2, Figure 6), however, only the mated females were observed to have oocytes in the post-vitellogenic phase (Figure 2d, Figure 6). The absence of post-vitellogenic oocytes in unmated spiders was an indication that mating status may play a role in the later phases of oogenesis (Figures. 2a-c). The dependency of the progressions of oogenesis on mating status has been observed in the female giant house spider (Tegenaria atrica) where oogenesis pauses at the pre-vitellogenic phase in

32 unmated females (Trabalon et al. 1998). Oogenesis in the giant house spider remains in pre-vitellogenesis, which is earlier than observed in L. hesperus, which produce vitellogenic oocytes in unmated females (Figure 2b, Figure 2c). Trabalon et al., (1998) proposed that the reason why oogenesis cannot progress past the pre-vitellogenic phase is because unmated female spiders do not have the necessary hormones to continue the process. Oogenesis past the pre-vitellogenic stage in the giant house spider therefore requires hormonal impetus provided by the male, and will stay paused until mating occurs (Trabalon et al. 1998; Pourie and Trabalon 2003). L. hesperus was capable of producing vitellogenic oocytes without mating, and may produce enough sex hormones on her own, permitting oogenesis to continue to the vitellogenic phase without pause until late vitellogenesis. Post-vitellogenic oocytes were only observed in mated black widow spiders, which can produce egg clutches from a past mating event. The sperm stored within the spermathecae may play a role in hormonal control of oogenesis past the late vitellogenic stage by circulating the hormones to the ovaries during oogenesis via the spiders open circulatory system.

20-hydroxyecdysone (20E) is an ecdysteroid hormone found in insects and arachnids that acts as both a molting and sex hormone (Pondeville et al. 2008; Pourie and

Trablon 2003; Gabrieli et al. 2014). When acting as a sex hormone 20E can induce oogenesis past the pre-vitellogenic stage (Pourie and Trabalon 2003) by stimulating Vtg synthesis in the fat body, and its exocytosis to the hemolymph (Friesen and Kaufman

2004).

In the mosquito, Anopheles gambiae 20E received from the male during mating has a three-fold effect, it promotes vitellogenesis past the pre-vitellogenic phase by

33 stimulating vitellogenin synthesis in the fat body, it promotes vitellogenin exocytosis from the fat body to the hemolymph, and prohibits further copulation after the first mating event by lowering the female’s receptivity to insemination (Gabrieli et al. 2014).

Similar to the black widow spider and other arthropods, Anopheles gambiae stores sperm after a mating event, and can produce multiple egg clutches from that stored sperm.

However, unlike many species of arthropod, Anopheles gambiae does not copulate with multiple males throughout its lifetime (Gabrieli et al. 2014). After the mosquito has its first mating 20E gradually reduces the female’s receptivity to future copulation, possibly due to a combination of factors such as a change in sexual behavior and modifications to the reproductive organs (Gabrieli et al. 2014). The loss of sexual receptivity has also been noted in Drosophila, who have been observed to be less sexually responsive after mating, albeit only temporarily (Kubli and Bopp 2012).

Just as observed in the mosquito and Drosophila, the giant house spider’s

(Tenegaria atrica) sexual behavior was also influenced by 20E levels (Trabalon et al.

2005). But unlike those other arthropods whose sexual receptivity declined with increased 20E, the giant house spider became more receptive to mating after treatment with the hormone (Trabalon et al. 2005). Researchers noted that in addition to having low sexual receptivity (42%), females not treated with 20E also engaged in sexual cannibalism more frequently than the control (Trabalon et al. 2005). After 20E treatment, female sexual receptivity boosted to 100% and sexual cannibalism did not occur

(Trabalon et al. 2005). 20E in the giant house spider is needed for oogenesis (Pourie and

Trabalon 2003), but begins working during the mating phase of sexual reproduction when

34 hormone provokes the desire to mate in the female, and inhibits the urge to engage in sexual cannibalism (Trabalon et al. 2005).

20E levels can be elevated from diet. It has been noted in our lab that mated black widow spiders produce an egg clutch within an interval of 45 days between clutches and approximately 10 days post-feeding, which may be the result of elevated 20E levels received from their food (Pondeville et al. 2008; Baldini et al. 2012; Gabrieli et al. 2014).

Mosquitoes also have elevated 20E levels from a bloodmeal diet (Pondeville et al. 2008;

Baldini et al. 2012; Gabrieli et al. 2014), although it was not discussed whether 20E levels were elevated because of the 20E content in the diet, or because the diet itself raised nutrient levels within the organism, permitting oogenesis to proceed to the next phase. In male mosquitoes 20E is synthesized in the accessory glands, and transferred to the female within the spermatophore during mating (Baldini et al. 2012) In female mosquitoes 20E is synthesized in the ovaries and fat body regardless if it is the result of a blood meal, or from mating, however the levels of 20E are highest when received from a male during copulation (Gabrieli et al. 2014). The combination of 20E received from feeding and mating therefore provides the impetus to promote oogenesis and oviposition in mosquitoes.

Vitellogenesis in L. hesperus occurred in stages which were categorized based on the changes in yolk granule volume (Table 1) (Figure 1, Figure 2, Figure 3, Figure 4).

Vitellogenic stages were also noted in the giant house spider (Pourie and Trabalon 2003) and the spider mite (Talarico et al. 2009), and just as in these organisms, the early vitellogenic stage in the black widow spider saw that the lipid yolk granules were at their smallest volume (Table 1) (Figure 2b, Figure 4a). By the late vitellogenic stage, the lipid

35 granules were significantly larger in volume (Table 1) (Figure 2c, Figure 4a) (Pourie and

Trabalon 2003; Talarico et al. 2009). I observed a large variation in yolk granule size between early and late phase vitellogenic oocytes (Table 1). Pourie and Trabalon (2003) attribute the vitellogenic stages to high levels of 20E, which increased female specific proteins, such as Vtg in the hemolymph and ovaries. Although my study did not perform

SDS page to investigate the proteins (such as Vtg) in L. hesperus ovaries or hemolymph,

I did observe similar vitellogenic staging to Pourie and Trabalon (2003), and was able to delineate between early and late vitellogenic oocytes based on yolk granule size. The vitellogenic stages seen in the giant house spider and the black widow may be due to similar circumstances, specifically the synthesis and endocytosis of Vtg as a result of raised 20E levels. However, it remains uncertain why oogenesis pauses at different phases for different spiders.

Yolk granules cannot grow any further when there are no more yolk proteins such as vitellogenin in the hemolymph to be endocytosed into the cell. Oocytes will therefore be at their largest volume when the importation of yolk proteins ceases. A lack of vitellogenin in the hemolymph will occur when the protein ceases to be synthesized in the fat body. One of 20E’s roles is regulating vitellogenin synthesis, and when 20E levels are low, vitellogenin will not be synthesized (Baldini et al. 2012). 20E levels were found to be at high enough levels to initiate Vtg synthesis following a meal and mating, but these levels were only elevated for a few days, indicating that the majority of yolk granule growth occurs within days of feeding or mating (Baldini et al. 2012). After several days

20E levels decline, along with the amount of Vtg being synthesized (Baldini et al. 2012).

36

When Vtg is no longer synthesized and imported into the oocyte, the vitellogenic phase has ended, and the next phase in oogenesis is post-vitellogenesis.

The Pedicel

The ovarian-pedicel system is not unique to black widow spiders, or even to spiders. A similar structure has been observed in ticks (Brinton and Oliver 1971; Denardi et al. 2004; de Oliveira et al. 2007) scorpions (Nath 1925; Yamakazi and Makioka 2001), fish (Erickson and Pikitch 1993; Morris et al. 2011), and chickens (Clauer 2016). In ticks, mites, and scorpions the pedicel’s role is thought to be to maintain oocyte attachment to the ovarian wall during oogenesis (Nath 1925; Brinton and Oliver 1971; Yamakazi and

Makioka 2001; Denardi et al. 2004; de Oliveira et al. 2007). Just as in ticks, mites, and scorpions, the pedicel in L. hesperus also appears to function to maintain oocyte attachment to the ovarian wall. In ticks, the pedicel has also been found to play a role in oocyte development by being a site of Vtg synthesis (de Oliveira et al. 2007), but the ability to synthesize Vtg has yet to be examined in the pedicels of L. hesperus.

Although present in insects and arachnids, a function for the pedicel beyond anchoring the oocyte to the ovarian wall has neither been established or thoroughly explored. To investigate the pedicel in L. hesperus the pedicel cells were examined at the

EM level and found that pedicel cells associated with L. hesperus pre-vitellogenic and vitellogenic oocytes contained rough endoplasmic reticulum and mitochondria (Figure

5a-b). Similar to what was discovered in L. hesperus, researchers also found rough endoplasmic reticulum and mitochondria in the pedicel cells of the tick, Amblyomma triste, and concluded that the pedicel is involved in Vtg synthesis (de Oliveira et al.

2007). However, the presence of protein processing and energy producing organelles in

37 the pedicel’s cells does not provide strong support that the pedicel is producing and exporting Vtg to the oocyte. It is possible that the endoplasmic reticulum and mitochondria are only supporting pedicel cell homeostasis, and have no role in supporting oogenesis. To investigate further, an experiment such as immunocytochemical staining for Vtg should be performed, which would show if Vtg originates in the pedicel, and is transported to the oocyte.

No study has conclusively determined the exact mechanism for how pedicel detachment from the oocyte occurs, but my findings in L. hesperus, and the findings of one research study on the tick, Rhipicephalus sanguineus (de Oliveira et al. 2005), suggest that the pedicel cells gradually deteriorate during vitellogenesis (Figure 5b). The pedicel’s cells, when attached to a pre-vitellogenic oocyte (Figure 5a, Figure 5c), were markedly different than the cells of a pedicel attached to a vitellogenic oocyte (Figure

5b). Signs of deterioration began during vitellogenesis and included a loss of endoplasmic reticulum and a smaller nucleus (Figure 5b). A loss of endoplasmic reticulum means that the cell cannot synthesize proteins (rough ER), assemble and metabolize lipids (smooth ER), or produce some hormones, like 20E (Beaulaton et al.

1984). A shrunken nucleus means condensation of genetic material, which impedes upon the cells ability to function properly (Kumar et al. 2007). de Oliveira et al. (2007) also noted a decrease in the size of nuclei in pedicel cells attached to later phase oocytes, in addition to a loss of endoplasmic reticulum and mitochondria, which was abundant in earlier phases. The observed difference in the pedicel as the oocyte progresses from the pre-vitellogenic phase to the vitellogenic phase may be the result of apoptosis, causing

38 the pedicel cells to go through functional decline as oogenesis proceeds past the vitellogenic phase.

Apoptosis is a distinct method of cell death that occurs in all multicellular organisms (Wilczek 2005; Elmore 2007). Apoptosis is also referred to as “programmed cellular death” or “cell suicide” because the process is initiated by an external stimulus, and triggers the cell to begin the process of self-destruction. Programmed cell death is a necessary aspect of healthy development and aging (Proskuryakov et al. 2003; Elmore

2007). Examples of apoptosis during development include the removal of webbed tissue during human embryo development to create fingers and toes and the removal of a tadpole’s tail during metamorphosis (Potten and Wilson 2004). Apoptosis can be identified when the cell shows the following signs: cell shrinkage, crowding of organelles in the cytoplasm, chromatin condensation (pyknosis), DNA fragmentation and degradation within the nuclear envelope, cellular membrane blebbing, tearing apart of the cell into apoptotic bodies, and phagocytosis of the apoptotic bodies (Saraste and Pulkkii

2000; Elmore 2007).

Apoptosis differs from necrosis, another form of cell death, in which cells die pre- maturely from infection, injury, or lack of blood flow to bodily tissues (Proskuryakov et al. 2003). Unlike apoptosis, which occurs through a tightly regulated series of signals, necrosis is unregulated, and following rupture of the cell membrane, the organelles and other cellular material uncontrollably spill into the extracellular space (Proskuryakov et al. 2003). When the cellular material leaks into the extracellular space, an inflammatory response initiates, blocking phagocytes from removing the necrotic cells. In the case of gangrene, where tissue dies from insufficient blood flow or infection, surgical

39 intervention is often required due to phagocyte’s inability to remove dead cells (Eke

2000).

Apoptosis could be the mechanism for post-vitellogenic oocyte detachment from their pedicel. During my experiment, only post-vitellogenic oocytes detached from their pedicel (Figure 2d, Figure 6, Figure 7) were observed. To ensure that pedicel detachment was an accurate depiction of oogenesis, intact spider abdomens were used to prevent an artifact pedicel detachment. Additionally, some gravid spiders were dissected prior to fixation, and ova were seen to spill out from the abdomen, suggesting that they had lost their pedicel attachment. In the same images where post-vitellogenic oocytes were visible, pedicel-attached pre-vitellogenic oocytes were also observed, making it unlikely that the pedicels were too small to be observed under the given magnification. However, given the oocyte’s large size in comparison to the pedicel, it is possible that with the pedicels are obscured behind the oocyte. Membrane interdigitations, a tight interlocking of cells, were noted among the oocyte’s basal lamina and the pedicel (Figure 5a). The interdigitations appear to keep the oocyte and pedicel closely attached, and may require apoptosis to detach the oocyte (Pavelka and Roth 2010). The pedicel also appears shrunken following oocyte detachment, providing further support for apoptosis (Figure

9). The pedicels, albeit shrunken, were not completely gone. Remnants of the pedicel were still left behind much like some skin is left in between fingers and toes during apoptosis in embryogenesis.

The effects of apoptosis also extend to the basal lamina; whose degradation occurs in response to pedicel cell death (Steller 2015). It was originally thought that basal lamina deterioration triggered apoptosis because this led to a loss of cellular adhesion, but

40 was later disregarded after it was observed that blocking basal lamina degradation did not inhibit apoptosis (Steller 2015). Rather, basal lamina deterioration seems to be triggered by matrix metalloproteinases (MMP), enzymatic proteins secreted by apoptotic cells that can degrade extracellular matrix proteins (Steller 2015). The deterioration of the basal lamina following apoptosis of the pedicel cells may explain why no remnant basal lamina was observed in the abdomen following oviposition (Figure 8), however it is possible that the basal lamina goes with the oocyte following detachment.

To test for apoptosis, an assay can be performed. There are multiple ways to perform an apoptosis assay because the apoptotic process occurs through a complex signaling cascade, which physically alters the cell, and utilizes several different proteins in the process (Elmore 2007). For a future experiment to test for apoptosis in the L. hesperus pedicel cells, I would choose an assay that focused on the cytomorphological alterations (DNA fragmentation, cellular membrane blebbing, presence of apoptotic bodies, and phagocytosis of the apoptotic bodies) that result from apoptosis because distinct changes were observed to the pedicel cell following oocyte detachment. The most notable change observed was a shrunken nucleus (Figure 5b). Additionally, the pedicel narrows (Figure 5b), loses endoplasmic reticulum (Figure 5b) and has a “stump-like” appearance, suggesting that it is no longer functional (Figure 9). To investigate apoptosis in L. hesperus, early vitellogenic ovarian tissue containing oocyte-detached pedicel cells would be observed under the TEM. If apoptosis is complete, the cells would have been phagocytosed by phagocytes, and would no longer be observable (Erwig and Henson

2008). However, there are particular cytomorphological changes apparent in the beginning phases of apoptosis that are observable, and can be imaged inside the cell at

41 the EM level (Elmore 2007). The cytomorphological changes to be examined include an electron-dense nucleus, nuclear fragmentation, and large translucent vacuoles (Elmore

2007).

Figure 9. SEM image of L. hesperus ovarian tissue depicting a vitellogenic oocyte, and pedicel scars or “stumps” on the ovarian wall. Taken by Dr. Casem, personal communication. PS = pedicel scar.

Oviposition

After vitellogenesis, the next step is for the ova to exit the body during oviposition. When oviposition begins, ova are pushed out of the body through the gonopore, but the exact path from abdomen to gonopore remains unclear. Two models explain the possible migration of post-vitellogenic oocytes from the abdomen to the

42 gonopore. In the first model, which has been observed in the brown recluse spider

(Loxosceles intermedia), an oocyte transits through the lumen of its pedicel and into the oviduct (Morishita et al. 2003). Evidence supporting this model comes from images obtained from a gravid brown recluse depicting a post-vitellogenic oocyte squeezing through pedicel cells, which have rearranged to create an opening to the ovarian lumen

(Morishita et al. 2003). The oocyte dramatically alters its shape to squeeze through the narrow pedicel opening, which appears to put the oocyte at risk for rupture. Although ova migration directly through the pedicel into the oviduct is a straight-forward explanation for how oviposition occurs, similar images to Morishita et al. (2003) were not obtained in this experiment. At no point in my observations was an oocyte observed squeezing through pedicel cells. The brown recluse’s post-vitellogenic oocytes were also smaller

(100-150 m diameter) compared to the post-vitellogenic oocytes found in L. hesperus.

Smaller post-vitellogenic oocytes may be capable of squeezing through their pedicel, but in L. hesperus, whose post-vitellogenic oocytes measured significantly larger (Table 1)

(Figure 6) than the brown recluse, it does not seem likely that the relatively tiny pedicel could be used as a transport to the oviduct.

In the second model, oocyte transit occurs from the hemocoel to an opening in the ovarian epithelium. Sadana (1970) proposed that ova detach from their pedicel, and migrate towards an opening in the ovarian wall. For ova to migrate into the oviduct

Sadana (1970) stated that the ovarian wall becomes thinner during the reproductive cycle, forming holes leading to the ovarian lumen. However, whether ovarian thinning occurs by decreasing the number of epithelial cells, or the morphology of the ovarian wall alters with the reproductive cycle was not discussed. Although it is not certain how the ovarian

43 wall would create openings of sufficient size for mature oocytes, it is necessary for oocytes to detach from their pedicel to get through the ovarian wall openings. In the

American house spider, Achaearanea tepidariorum, Suzuki (1995) described post- vitellogenic oocytes in the lumen of the ovary, which is where fertilization was said to occur. However, it was not discussed how the oocyte makes its way into the ovarian lumen.

Oviposition in L. hesperus is more consistent with the second model for oviposition, with the greatest support being that no post-vitellogenic oocytes were observed to be attached to a pedicel (Figure 2d, Figure 6, Figure 7), reducing the possibility that the pedicel is used as a vessel for oocytes to get to the oviduct. Consistent with the second model of oviposition, pedicel detachment may occur for ova to move through the hemocoel to an opening in the ovarian wall. It was assumed that ova resided in the hemocoel prior to oviposition, but to confirm the abdomen was examined during and after oviposition (Figure 6, Figure 7, Figure 8). Comparing the abdomen during and after oviposition showed where the ova remained until they left the body, and what the abdomen looked like when its stores of ova have been depleted. I observed ova in the hemocoel (Figures 7a-b) and an empty space in the location where ova were found prior to oviposition (Figures 8a-b). For ova to occupy the hemocoel, pedicel detachment must occur, and therefore transport through the pedicel during oviposition was not observed in

L. hesperus. While in the hemocoel, I observed post-vitellogenic oocytes in a membrane- bound compartment, which may function to corral the post-vitellogenic oocytes out of the body during oviposition. A membrane-bound compartment permits oocytes to exit the

44 body as one unit, and is more efficient than having free-floating oocytes gradually make their way to an opening in the ovarian wall.

Ova movement from the hemocoel to an opening in the ovarian wall (leading to the oviduct and then to the gonopore) is more complicated than ova movement through the pedicel, because the hemocoel offers no direct route into the oviduct. If oocytes were not restricted to moving into the oviduct through the pedicel, but rather moved through the hemocoel (following pedicel detachment, as observed in post-vitellogenic oocytes depicted in Figure 6) into an opening in the ovarian wall, then it remains uncertain how ova are innately capable of moving in the correct direction. It also remains uncertain whether such holes in the ovarian wall exists. Some research has suggested that the ovarian wall thins during oviposition, creating holes for oocytes to pass through into the oviduct (Sadana 1970), but ovarian wall thinning, or holes was not observed in L. hesperus.

Whether fertilization occurs externally or internally in spiders is still up for debate. Foelix (1996) describes fertilization occurring in the uterus externus, and states that sperm nuclei begin their migration to the center of the oocyte during oviposition.

Suzuki (1995) reports that fertilization in the American house spider, Achaearanea tepidariorum, occurs internally prior to oviposition. Suzuki (1995) examined the oocytes found in the ovarian lumen and noted a “nucleus-like” body within the oocytes. The

“nucleus-like body” described was sperm nuclei, and indicative that fertilization was occurring internally. The findings of Suzuki (1995) suggest that sperm housed within the spermathecae are activated prior to oviposition, and differs from Foelix who postulated that sperm are activated during oviposition. I have not examined sperm nuclei in L.

45 hesperus oocytes before or after oviposition, so it still remains uncertain whether fertilization in the black widow occurs internally or externally. The absence of sperm nuclei in the post-vitellogenic oocyte may indicate that fertilization had not occurred, which would imply that fertilization occurs externally after oviposition.

In conclusion, my study is the first comprehensive analysis of oogenesis in the black widow spider, Latrodectus.hesperus. I determined that oogenesis occurs through three phases of development: pre-vitellogenesis, vitellogenesis, and post-vitellogenesis, which was consistent with what has been observed in other arachnids (Morishita et al.

2003; Pourie and Trabalon 2003; Denardi et al. 2004; de Oliveira et al. 2005; Saito et al.

2005; Talarico et al. 2009). The vitellogenic phase in the black widow is further broken down into two stages: early vitellogenesis and late vitellogenesis similar to what has been observed in mites (Talarico et al. 2009), ticks (Denardi et al. 2004; de Oliveira et al.

2005; Saito et al. 2005), and other spiders (Pourie and Trabalon 2003). I observed evidence for receptor-mediated endocytosis by presence of clathrin-coated pits on the surface of the oocyte (Figure 4). Similar to what was observed in other spiders, as vitellogenesis progressed in L. hesperus, yolk granules accumulated within the oocyte, and were at their largest quantity and volume in late vitellogenesis. Also, during vitellogenesis, the black widow spider, and other spider species (Morishita et al. 2003;

Pourie and Trabalon 2003) gained a prominent basal lamina and vitelline membrane.

Unlike other species of spider that pause oogenesis at pre-vitellogenesis when unmated

(Pourie and Trabalon 2003), the black widow spider is capable of carrying oogenesis to the vitellogenic phase prior to mating. Although I did not investigate hormones specifically, I suspect that if I were to compare the unmated black widow spider to other

46 unmated spider species, the black widow spider may have higher amounts of 20E naturally occurring in the body prior to mating. If there was extra 20E in L. hesperus, then it may be responsible for my observations that oogenesis pauses later, at the vitellogenic phase, and not at the pre-vitellogenic phase.

The pedicel’s function in the black widow spider is similar to other arachnids because it serves to maintain oocyte attachment to the ovarian wall during oogenesis, however additional functions of the pedicel may vary among species. In the tick,

Amblyomma triste, the pedicel may also function as a source of Vtg proteins for the oocyte during vitellogenesis (de Oliveira et al. 2007). In the brown recluse, the pedicel may function as a transport during oviposition (Morishita et al. 2003). I have not investigated for Vtg proteins originating from the pedicel, or observed black widow spider oocyte’s traveling through the pedicel to get to the oviduct, but propose that these areas be explored in future research. I also propose that an apoptosis assay be performed on pedicel cells to determine if L. hesperus oocytes naturally detach from the pedicel.

The main question that remains unanswered is how do oocytes travel from the hemocoel, to the oviduct during oviposition. To investigate oocyte migration during oviposition, three challenges must be overcome. The first challenge is obtaining spider samples while the spider is laying eggs, the second challenge is determining how oocytes get into the ovarian lumen, and the third challenge is knowing what an oocyte in the ovarian lumen looks like. For future research, I propose a study that focuses solely on L. hesperus oviposition, and gathers samples only from spider caught while laying eggs. In this study, the anatomy of the spider needs to be mapped out so that investigators can deduce whether oocytes are in the ovarian lumen, or the hemocoel. Questions that should

47 be addressed in a study on oviposition should include, is there an opening in the ovarian epithelium, and if so, what would this look like? Does pedicel detachment occur, or do oocytes use the pedicel as a transport to the ovarian lumen? What would an oocyte in the ovarian lumen look like, and how can this be differentiated from an oocyte in the hemocoel? Knowledge of oocyte migration in L. hesperus would be beneficial to research in other arachnids such as ticks, mites, and scorpions, because the same question of how an oocyte goes from the hemocoel to the oviduct remains unanswered.

48

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