EVALUATION OF THE EXISTENCE OF CHEMOSENSORY GLOMERULI IN

Item Type Electronic Thesis; text

Authors Mortensen, Michael

Citation Mortensen, Michael. (2020). EVALUATION OF THE EXISTENCE OF CHEMOSENSORY GLOMERULI IN SPIDERS (Bachelor's thesis, University of Arizona, Tucson, USA).

Publisher The University of Arizona.

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EVALUATION OF THE EXISTENCE OF CHEMOSENSORY GLOMERULI IN

SPIDERS

By

MICHAEL SAMUEL MORTENSEN

______

A Thesis Submitted to The Honors College

In Partial Fulfillment of the Bachelors degree With Honors in

Neuroscience and Cognitive Science

THE UNIVERSITY OF ARIZONA

M A Y 2 0 2 0

Approved by:

______

Dr. Wulfila Gronenberg Department of Neuroscience

Abstract

Spiders sit at the forefront of fearful minds of people across the globe, but scientific knowledge of these creatures is surprisingly far from complete. For instance, the current body of work that describes a ’s olfactory capabilities is lacking, specifically in narrowing down the possible locations for receptors by which odors are sensed. To push knowledge in this area forward, this study analyzed the region of the CNS known as the suboesophageal ganglion, referenced in the work done by Babu & Barth (1984), in five species of spider in an attempt to determine if glomeruli, globular arrangements of synaptic connections typically underlying olfactory processing in most (as described in Hildebrand & Shepherd, 1997), could be found. In all species studied, glomeruli-like structures were identified in the regions of the suboesophageal’s leg ganglia which contain synaptic connections between the leg nerves and the CNS. This study presents evidence suggesting that olfactory receptors may be more abundant on the legs of spiders, and that the sense of smell may be more pronounced than previously thought.

Dedication

This body of work is lovingly dedicated to the Nephila clavipes who gracefully excluded herself from this study by means of ignoring her provided meal and instead choosing to eat so much dried glue that she choked and died.

Also dedicated to my lab instructor, Wulfila Gronenberg. Without him and the time he spent making countless revisions, this thesis would have been much shorter and vastly inferior in quality.

Table of Contents

Abstract ...... 2

Introduction ...... 5

Methods ...... 15

Results ...... 19

Discussion ...... 33

Acknowledgements ...... 38

Bibliography ...... 39

Introduction

General Introduction/Terminology

The phylum of arthropoda is home to several fascinating classes and orders of animals, with some of the most well-known being the order of araneae, belonging to the family arachnida. The term araneae refers, of course, to what is commonly known as a spider. Spiders come in a huge variety of families, such as the Agelenidae (funnel-web spider), Lycosidae (), and Salticidae (). These families, despite their differences, all hold several attributes that qualify them for classification as araneae, including the existence of eight legs, silk glands, and (fangs) (Foelix, 2011).

There exist a wide variety of spider families, with each having a species count ranging from around 100 to several thousand (reviewed in Foelix, 2011). As expected with such a wide range of families and species, there exist notable differences between them, clearly shown in hunting behavior. Spiders within the Araneidae family (orb-web spiders), which contains the

Nephila clavipes used in this study, take a more passive approach when it comes to finding a meal. These spiders prefer a strategy of patience (Nyffeler et al., 1994; Nyffeler, 1999) and the laying of careful traps with their webs. These web-weaving spiders will spin their web in a way that is most beneficial to trapping a certain type of prey, whether they (Kajak, 1965; Chacon

& Eberhard, 1980; all as cited by Nyffeler, 1999) crawl, or jump, and then simply wait for the time to strike to arrive. As such, web-weaving spiders are thought to rely on prey that is, in

order to fall into their web, capable of moving (Turnbull 1973, as cited by Nyffeler, 1999). Other spiders are known best as hunters, such as ones from the Lycosidae family (wolf spiders)

(Nyffeler et al., 1994), including the lenta and californica used in this study, or the Sparassidae family (giant crab spiders) (Airamé & Sierwald, 2000), including the Olios gigantius used in this study. Spiders from these families place an active approach to finding a meal, and have been thought to have a broader plate of prey to choose from, so to speak, due to this (Jackson & Tarsitano, 1993; Nyffeler et al., 1990; Turnbull 1973, as cited by Nyffeler,

1999). Interestingly, this broader plate has been observed to include other spiders (Wise 1993, as cited in Nyffeler, 1999).

Biology of a Spider

A good place to start before delving into the subject of this paper is to briefly explain the basic biology of a spider as laid out by Foelix (2011). The body of a spider consists of two sections strung together. The anterior section, called the prosoma, carries the central nervous system (CNS). The prosoma is also the location of four pairs of legs, a single pair of , which are used as feelers (and, in males, as copulatory organs), and a pair of chelicerae, which are used to bite. The posterior section, called the (or abdomen), instead deals with tasks such as silk production, reproduction, and digestion, amongst other things. The opisthosoma is also home to a spider’s , which are essential for web-building. The prosoma and opisthosoma are connected by a stalk called the pedicel. The primary focus of this paper lies in the CNS that sits inside the prosoma.

As can be imagined, the actual

CNS of a spider is quite different from that of a vertebrate. The most notable feature of a spider’s CNS is that its ganglia are fused into two parts that both lie in the prosoma.

These sections are known as the supraoesophageal and Figure 1: Diagram of Spider CNS. Left Figure: Dorsal view of the two sections of a spider (Tegenaria)’s body, with the CNS in the prosoma shown to extend via nerve bundles to suboesophageal ganglion (SOG) (Babu the opisthosoma. Right Figure: Lateral view of the CNS of a spider of the same species. SUPRA: supraoesopageal ganglion. SUB: subeosophageal gaglion. Eso: esophagous, & Barth, 1984). The which divides the two areas of the CNS. PG: palpal ganglion. 1-4: Leg ganglia. Image taken from Foelix 2011. (p119) supraoesophageal ganglion (the brain proper) sits anteriorly and dorsally to the SOG, and the two parts are divided by the spider’s esophagus (Babu & Barth, 1984; Foelix, 2011). The supraoesophageal ganglion is a higher order processing center, where information from visual and other sensory pathways are integrated

(Babu & Barth, 1984; Foelix, 2011). It comprises the protocerebrum (which supplies the eyes) and the deutocerebrum, which supply the chelicera (Babu & Barth, 1984; Foelix, 2011; Tanaka et al. 2013). The SOG is instead made up of the fused ganglia of the legs, pedipalps, and the abdominal ganglia (Babu & Barth, 1984; Foelix, 2011). The traditional term ‘suboesophageal ganglion’ is an unfortunate choice, as in other it refers to a more specific ganglion which only supplies the mouthparts. A less misleading term would be ‘fused post-oral ganglia’, but I will use the traditional term ‘suboesophageal ganglion’ because it is used by many of the relevant previous studies. As Babu & Barth (1984) and Foelix (2011) summarize, the ganglia

composing the SOG can still be recognized as individual neuromeres, each of which supplies a leg (or ). The neuromeres are separated ventrally but fused dorsally in the SOG. Each neuromere contains areas known as neuropils where synaptic connections are located. It is here that the CNS receives incoming sensory fibers and gives rise to outgoing axons that originate from large motoneurons whose cell bodies reside in the peripheral cell body cortex, which lies below the central neuropil (Gronenberg, 1989; 1990; Milde & Seyfarth; 1988; all as cited in Foelix 2011; Babu & Barth, 1984).

Senses of a Spider

Perhaps the most striking and well understood aspects of a spider’s biology are their mechanical senses based on mechanoreceptors, which react to mechanical stimuli from the surrounding environment, such as vibrations and touch. A spider’s mechanoreceptors include the hair sensilla, which take the form of either the abundant, simplistic tactile hairs or scarce, complex filiform hairs, and the slit sense organs (Foelix, 2011). The tactile hairs can serve a multitude of purposes, from sensory functions to practical mechanical functions. Depending on the species, these hairs can sense external stimuli such as touch, can be used as a comb to assist with trapping prey, or can be used as a brush to help with cleaning (Foelix & Jung, 1978;

Huber & Fleckenstein, 2008; Berland, 1932; all as cited in Foelix 2011). The filiform hairs, also known as trichobothria, are only found clustered on particular areas of the legs (Foelix, 2011).

The most important quality of these filiform hairs is their incredibly high sensitivity to air currents, which can even be used to detect an insect’s beating wings (Barth & Höller, 1999, as

cited in Foelix, 2011). This sensitivity has been shown to elicit different behavior in different types of spiders; when a hunting spider detects a fly above them, they will leap to attack it

(Brittinger, 1998; as cited in Foelix, 2011). Web-weaving spiders instead utilize the detection of beating wings to assume a defensive stance (Klärner & Barth, 1982, as cited in Foelix, 2011). Slit sense organs are found embedded in a spider’s exoskeleton, but are most commonly found on the legs (Foelix, 2011). They serve as stress or strain receptors, and are receptive to a wide range of mechanical stimuli such as those that result from gravity or vibrations (Barth, 1976;

Barth, 1985; all as cited in Foelix, 2011). There exist several different types of slit sense organs, and exact function and purpose of them differ between types. For example, a type of slit sense organ that is highly receptive to vibrational stimuli is the metatarsal lyriform organ (Liesenfeld,

1961; Walcott & Van der Kloot, 1959; all as cited in Foelix, 2011). It is important to note that vibration detection is an essential part of a spider’s ability to hunt, regardless of their hunting method. Spiders that actively hunt for their prey utilize mechanical vibration, such as that found on a disturbed water’s surface, to know when to trigger an attack (Williams, 1979; Bleckmann,

1982; all as cited in Foelix, 2011). Web spiders such as orb weavers that wait until prey finds its way into their waiting web also utilize vibrations, as the prey stuck in the web produce specific vibrations serving as the spider’s cue to leave their hiding spot and attack (Klärner and Barth,

1982; Liesenfeld, 1961; all as cited in Foelix, 2011).

Another major focus in the realm of spider research (and therefore, understanding) is their sense of vision, which varies between families. We shall compare the passive web- weaving spiders and the aggressive hunting spiders. Despite what might be thought about their

visual capabilities due to their nature, web-weaving spiders have been observed to act in a way implying they do utilize a sense of vision. For example, the orb weaver Araneus sexpunctatus appears to use the fading light of the day to decide when to leave its shelter (Homann, 1947, as cited in Foelix 2011). Hunting spiders, in general, seem to utilize even more of their sense of vision, to the point of complete dependence. A jumping spider (from the family Salticidae) has been observed to lose its hunting prowess when kept in a dark environment (Jackson, 1977, as cited in Foelix 2011). Jumping spiders specifically have been the subject of extensive vision tests and as a result, much is known about their visual capabilities. In a strange way, a jumping spider’s vision is not that different from our own. Of course, unlike us, a spider usually has eight eyes. These can be classified (Homann, 1928; Land, 1985; all as cited in Foelix 2011) into either main eyes, which are also known as the anterior median eyes (AME) due to their location at the front of the spider (Barth, 2002, as cited in Foelix, 2011), and secondary eyes, whose structure and function often varies between families (Homann 1950; Homann, 1952; all as cited in Foelix,

2011). In a jumping spider, the main eyes have a larger density of visual cells in the fovea, or central region of the eye, when compared to the peripheral region of the eye. This implies that, like our own, the area of the retina known as the fovea is essential for visual acuity (Homann,

1928; Land, 1969; all as cited in Foelix, 2011). Additionally, the retina of a jumping spider can move and track targets, compensating for their tiny visual field (Scheuring, 1914, as cited in

Foelix, 2011). On the contrary, the most notable feature of the secondary eyes of a jumping spider is their substantial visual field, which is used to detect movement, much like our own peripheral vision (Duelli, 1978; Komiya et al, 1988). The visual field of a jumping spider’s secondary eye overlaps with the visual field from their other secondary eyes, which is what

allows a jumping spider to accurately estimate distances and make the dramatic leaps for which they are known (Homann, 1928, as cited in Foelix 2011).

Olfaction

Olfaction is an essential function of a huge number of animals that exist in the world, and can serve a large number of purposes. For example, the ability to detect and process odors allows an to react to signals that may indicate danger, such as a fire, or signals that may indicate something more positive, such as food (Ache & Young, 2005). Allelochemicals, or odors from another species, can serve to assist with important functions such as territorial marking and pollination (Whittaker & Feeny, 1971, as cited in Ache & Young, 2005). Additionally, odors known as pheromones function as identifiers of sorts for an individual animal, signifying social status and sex (Shorey, 1976; Vandenburg, 1983; all as cited in Ache & Young, 2005).

The way animals detect and process odors is complex but, importantly and most interestingly, largely similar between a wide range of species (Ache & Young, 2005). It all starts with odor signals themselves, which are compounds (odorants) or, far more often, a mixture of odorants that are, in the context of insects and land-based vertebrates, volatile and airborne.

Odors are detected by odorant receptors (ORs) (Ache & Young, 2005), and the ORs of spiders are thought be housed in hair sensilla known as pore hairs, although the sensilla appear to be rare among spiders when compared to insects (Foelix, 1985). The few that have been observed have been found on the legs of a an opilionid (Foelix 1976; Gnatzy & Dumpert, personal

communication with Foelix, 1978; all as cited in Foelix, 1985), which is a close relative of a spider, and one was found on the dorsal side of the tarsus in the spider Gradungula (Foelix,

1985). The amount of different olfactory receptor genes that exist between animals has been observed to have a huge amount of variability, with humans having roughly 388 genes (Niimura

& Nei, 2003, as cited in Ache & Young, 2005) while a dog has around 1070 (Quignon et al., 2003, as cited in Ache & Young, 2005). However, the number of genes in each species is thought to have changed over time in accordance with the theory of evolution and the ever-adjusting need for olfaction for survival (Ache & Young, 2005). Humans were observed to have around 414 pseudogenes (Niimura & Nei, 2003, as cited in Ache & Young, 2005), or functional genes with an inactivating mutation, and dogs were found to have 230 pseudogenes (Quignon et al., 2003;

Glusman et al., 2001; all as cited in Ache & Young, 2005).

The neuronal pathway by which animals use to process olfactory information is relatively straightforward. As stated above, an odorant first interacts with an odorant receptor.

These ORs are composed of G protein-coupled receptors that respond to the compound(s) and send a signal downstream via a signal cascade (Ache & Young, 2005; Buck & Axel, 1991, as cited in Ache & Young, 2005). This sends a signal to the first synaptic relay, being the olfactory bulb in vertebrates, the olfactory lobe in crustaceans, and the antennal lobe in insects (Chase &

Tolloczko, 1986; Bellonci, 1883; all as cited in Ache & Young, 2005). The neurons that originate from the respective olfactory organ terminate in glomeruli, which are a globular structure composed of both these axon terminals as well as local interneurons and projection neurons that lead to the next synaptic level (Pinching & Powell, 1971; Tolbert & Hildebrand, 1982; all as

cited in Ache & Young, 2005). All olfactory receptor neurons that express the same OR project to unique glomeruli that are responsible for representing one particular odorant or small group of chemical compounds. Different odors are thus represented by different combinations of glomeruli being activated (Hildebrand & Shepherd, 1997). The projection neurons then finally lead to either a mammal’s olfactory cortex or an ’s lateral protocerebrum and corpora pendunculata (Ache & Young, 2005).

Several behavioral studies show evidence that a spider can detect and react to chemical stimuli, with several different applications of this sensitivity to olfaction having been discovered and detailed. Virgin female web-weaving spiders appear to produce an attractive pheromone that is detected by male spiders, who then seek out the source of the pheromone (Peckham &

Peckham, 1887, as cited in Foelix 2011; Uhl, 2013). Several species of spider appear to use olfactory cues to determine where a good habitat for optimal feeding may lie (Uhl, 2013).

Similarly, Pardosa milvina, a wolf spider, was shown to detect chemical cues emitted by its predator, Hogna helluo (Schonewolf et al., 2006; Persons et al., 2001a; Persons et al., 2001b;

Persons et al., 2002; Lehmann et al 2004; all as cited in Uhl, 2013). Spiders such as Latrodectus hesperus were observed to choose artificial habitats that formerly belonged to crickets, a prey of some spiders, over similar habitats that were not previously occupied by crickets (Johnson &

Johnson, 2011, as cited in Uhl, 2013). From these studies, it is clear that spiders have the capability for olfaction. However, the details of how this olfactory information is processed and the location of the exact odor receptors are currently unknown (Foelix, 2011). Currently, it is thought that olfaction is detected and processed via tarsal organs, which are small holes that

are positioned on the dorsal side of each leg in a segment called the tarsus (Blumenthal, 1935, as cited by Ehn & Tichy, 1994; Ehn & Tichy, 1994). It is thought that the tarsal organ is home to sensory cells known as hygroreceptors and thermoreceptors (Blumenthal, 1935, as cited by Ehn

& Tichy, 1994), which respond to changes in humidity and temperature respectively, with the hygroreceptors also responding to chemical stimuli (Ehn & Tichy, 1994). While these organs appear to respond to a selection of odors, such as those from acids, they do not respond to a variety of other odors that secrete from insects or other spiders (Ehn & Tichy, 1994; Foelix,

2011). Therefore, the current scientific understanding of a spider’s olfactory capabilities is currently incomplete. This study hopes to address this confusion by identifying chemosensory glomeruli in the SOG, which would provide further clues as to the location of the olfactory receptors.

Methods

Animal Collection/Dissection

Six spiders, each of three species (Hogna lenta, Tigrosa georgicola and Nephila clavipes), were bought from an online supplier (tarantulaspiders.com). One specimen each of Olios giganteus and Aphonpelma chalcodes were caught locally in Tucson, Arizona, courtesy of Dr.

Wulfila Gronenberg. The spiders were fed small crickets and given water biweekly. When it came time for dissection, the spider was briefly placed in a freezer or on ice and monitored until it appeared to be knocked out (but not dead). Its legs, pedipalps, and abdomen were then removed with iridectomy scissors. Spiders were dissected under fixative (see below) or in phosphate buffered saline (PBS). The CNS was then carefully extracted from the spider’s prosoma with the use of forceps and small scissors to sever any internal attachments. After extraction, the brain was placed in a vial with fixative (either 4% paraformaldehyde or 2%

Glutaraldehyde and 2% paraformaldehyde in PBS) and left on a shaker for at least a day. In the event of an inability to process the brain shortly thereafter, the brain was rinsed with (PBS) and stored in a fridge in 0.05M cacodylate buffer.

CNS Processing (Osmium Protocol)

Once the CNS was ready for processing, it was removed from its current buffer and rinsed in water before being stained in 1% aqueous osmiumtetroxide solution for 1-3 hours

(depending on CNS size) at 4oC and for an additional 30 – 60 minutes at room temperature. It was then repeatedly rinsed in water and placed stored in 50% ethanol for approximately 20 minutes or until further processing. To dehydrate the CNS, ethanol was then replaced with dimethylpropane for another 20 minutes. After the allotted time, the brain was rinsed twice with 100% acetone for roughly 30 minutes each. Once the rinsing was complete, the brain was placed in a 50% Spurr’s plastic embedding medium and 50% acetone mixture and left again for

6-12 hours. The mixture was then changed to a 90% acetone 10% Spurr’s mixture and left for another 12 hours. The next day, the CNS were placed in pure, 100% Spurr’s medium and left to sit for another day. All these steps were performed on a shaker/rotator to facilitate solution and plastic medium penetration. The CNS was then embedded in a small, cylindrical, plastic cap

(Beem® capsule). The capsule, containing the 100% Spurr's as well as the CNS, were then placed in an oven and heated for 16hours at 70oC to allow the plastic to harden. Once completely hardened, the “block” of Spurr’s and the CNS was removed from the plastic tube and sectioned on a sliding microtome. The section thickness slightly varied among preparations, but averaged around 10 µm microns. The sections were finally mounted onto a microscope slide. These slides were analyzed using an Axioskop microscope (Zeiss) and pictures were taken using an attached black and white microscope camera (SPOT FLEX, Diagnostic Instruments). Pictures of sections were taken and exported via a USB drive.

Image Processing

Images were processed through usage of the programs Adobe Photoshop and Fiji

(ImageJ). Due to the size of the CNS in each section and the small field of view due to the desired microscope magnification, several pictures of each section had to be taken in order to get a full picture. Photoshop was used to stitch these pictures together into one full image, as well as blend together sections that had become warped or were uneven. Images were then imported into ImageJ and the tools available allowed for the counting of glomeruli. Data was exported to Microsoft Excel to make the figures detailing the data.

Glomeruli Counting

Glomeruli were counted manually by hand. To determine what was and what was not a glomerulus, certain criteria were established. In order to be considered a glomerulus, the structure had to be: 1) relatively circular or oval-ish in shape; 2) have a clearly defined globular shape that are not closely mixed in with other cells; 3) possess a relatively uniform and similar size to others, to a difference of around 5 um. If a structure was identified but did not meet these criteria, then it was excluded from the count. Neuropil that were observed to be elongated were assumed to be mechanosensory neuropil and were excluded from the count. A

final note is that, due to technical limitations and difficulties clearly discerning the desired structures, any glomeruli counts should be considered rough estimates.

Results

Overall Structure of the Subesophageal Ganglion

The overall organization of the SOG is shown via Fig. 1 and 3A. As detailed in Babu &

Barth (1984), in the dorsal regions the SOG comprise longitudinal tracts connected by commissural tracts. More ventrally, the SOG is segregated into four separate leg neuromeres and the posterior-most abdominal neuromere on either side. Each leg neuromere in turn receives sensory input from the respective leg and supplies the leg muscles through the leg nerve, which carries sensory and motor nerve fibers (Milde & Seyfarth; 1988; Gronenberg,

1989; 1990; all as cited in Foelix 2011; Babu & Barth, 1984). As further mentioned in Babu &

Barth (1984), on either side another neuromere linked to the spider’s pedipalp is found more anteriorly than the first leg neuromere (‘PG’ in Fig. 1, not shown in Fig. 3A). At the ventral side and the periphery of each of these neuromeres are the cell bodies of motor neurons and interneurons supplying the neuropils, with the cell bodies of the sensory neurons residing in the legs in their respective sensilla.

Besides the cell bodies, each leg neuromere and the pedipalpal neuromere comprise different kinds of neuropil. Two neuropil regions can be more or less well distinguished from the rest of the neuropil by their texture and location: mechanosensory neuropil and glomerular, presumed chemosensory neuropil (Fig. 2A) (Anton & Barth, 1993). In a perfect preparation, these types of neuropil appear relatively distinct from each other and occupy different areas of

the neuromere. Mechanosensory neuropils seem to be composed of elongated regions (lamelli) and, in the osmium staining used in this study, appear to portrayed in a lighter grey. Glomeruli instead appear circular and accumulate a darker staining in these preparations, indicating a network of finer fibers as osmium stains cell membranes but not the axoplasm of the neurons.

However, the distinction between these differing neuropils is not always clear. Unlike the clearly distinguished globular structures, some sections displayed large masses of neuropil that greatly differed in area or structure from either mechanosensory neurons or glomeruli, and as such could not be classified as either. It is possible that these complex neuropils comprise the condensed glomeruli or mechanosensory neurons partly organized in a lamellar fashion, but that this kind of organization is obscured by other, less visibly organized neuronal processes.

Due to this ambiguity, these complex neuropil regions were largely not considered when counting glomeruli. A B

Figure 2: SOG Schematic Views: (A): Horizontal view of SOG leg ganglion. Demonstrates location of cell bodies andsome lamellar neuropil . (B): Saggital view of SOG, demonstrating the leg ganglia (AMG1-4) and neuropil (NP) location. ANT: Anterior position.

PST: Posterior position. Images taken from Babu/Barth 1984

Hogna lenta

The glomeruli identified in a sample of Hogna lenta were found to be most abundant in the 2nd leg, with an estimated glomerulus number of 177 (Fig. 3F). The size of glomeruli varied between diameters of approximately 7.3 µm and 12.1 µm. Interestingly, the actual structure of the leg neuropil differed between leg neuromeres. The 3rd left leg demonstrated two very distinct structures close to the ventral surface, one of which largely consisted of darker, globular structures, and the other consisting of lighter tissue (Fig. 3C). The 2nd left leg, the same depth from the ventral surface, instead showed three separate structures (Fig. 3B). The two structures found most posteriorly seem to contain structures that hold resemblance to glomeruli, much like the posterior structure in the adjacent leg as detailed prior. The anterior most structure matched the anterior most structure in the 3rd leg. More dorsally in the SOG, the neuropil that likely contained glomeruli shifted towards the lateral and medial tips of the neuromere (Fig. 3D and 3E). At a depth relatively close to the ventral surface, distance from the posterior lateral edge of the 2nd left leg ganglion to the neuropil region was 113 µm. Observing the same ganglion at 40 µm farther dorsally reveals that this distance has shrunk to 53 µm (Fig.

3D and 3E).

Figure 3: Hogna lenta: A) an overview of the SOG at a magnification of 10x. The leg neuromeres (L1-4 for left legs 1-4 and R1-4 for right legs 1-4) are labeled. B) A detailed close-up of the left second leg neuromere close to the ventral surface at a magnification of 20x. Note the three subdivisions of the neuropil, marked by asterisks. C) A detailed close-up of the left third leg neuromere at the same depth as Fig. 3B at a magnification of 20x. Note two subdivisions in the neuropil, marked by asterisks. D)

A detailed close-up of the left half of the SOG regions close to the ventral surface and a magnification of 10x. E) A detailed close- up of the same region 60 µm farther dorsal than D).The green circles show the shifted location of the darkened neuropil. F) An overview of the total chemosensory glomeruli that were counted in each individual leg neuromere. G) An overview of how the chemosensory glomeruli were found throughout sections starting from their earliest appearance. Yellow circles in (B) – (D) indicate glomerular neuropil regions

Tigrosa californica

The glomeruli identified in a sample of Tigrosa californica were found to be most abundant in the 1st leg neuromere, with a glomeruli number estimate of 78 (Fig. 4C). The greatest number of glomeruli were found more ventrally than dorsally (Fig. 4D). The size of glomeruli varied between diameters of approximately 3.9 µm and 8.3 µm. There appears to be a structural difference between the first and second leg neuromeres similar to what is observed in the Hogna sample (Fig. 3B and Fig. 3C, Fig. 4B). Close to the ventral surface, the first leg neuromere shows the presence of three different regions, with the single most anterior section being of an indiscernible quality and the posterior most sections partially holding glomeruli. At the same depth, the second leg instead shows two regions, with the anterior section being more faded and the posterior section being darker and holding glomeruli (Fig. 4B). Due to difficulties sectioning the brain, only the left half of the brain could be observed. Additionally, the left pedipalp was unfortunately obscured by artifacts as a result from the slide mounting procedure and could not be accurately counted. Sections containing more dorsal views of the neuromeres were also unable to be used in this study.

Figure 4: Tigrosa: A) an overview of the SOG at a magnification of 2.5x. The leg and pedipalp neuromeres are labeled as in the previous figures. B) A detailed close-up of the first and second left leg neuromeres close to the ventral surface and a magnification of 10x. The orange circle contains what may be considered chemosensory glomeruli. Note the varying number of subdivisions of neuropil, indicated by asterisks. C) An overview of the total chemosensory glomeruli that were counted in each individual leg neuromeres. D) An overview of how the chemosensory glomeruli were found throughout sections starting from their earliest appearance.

Nephila clavipes

The glomeruli identified in a sample of Nephila clavipes was found to be most abundant in the left 1st leg neuromere, with a glomerulus count of 84 (Fig. 5D). The greatest number of glomeruli was found more ventrally than dorsally (Fig. 5E). The size of glomeruli varied between diameters of approximately 6.51 µm and 9.33 µm. The structure of the leg neuromere differed greatly when compared to others in this study, appearing much more muddled and decreased in size in the posterior leg neuromeres (Fig. 5B and Fig. 5C). No distinct differences within neuropil organization could be identified in the 3rd and 4th leg neuromeres (Fig. 5A, 5B, and 5C).

The sample that was used in this study was broken during processing, with what appears to be the 1st left leg separated from the central ganglion. Due to this destruction, the pedipalps could not be identified.

Figure 5: Nephila clavipes: A) an overview of the SOG at at a magnification of 2.5x. The leg and pedipalp regions are labeled identically to the previous figures. B) A detailed close-up of the right half of the SOG regions at close to the ventral surface and a magnification of 10x. The orange circle contains what may be considered chemosensory glomeruli. C) A detailed close-up of the same region now 60 µm farther dorsally than B). D) An overview of the total chemosensory glomeruli that were counted in each individual leg neuromeres. E) An overview of how the chemosensory glomeruli were found throughout sections starting from their earliest appearance.

Olios giganteus

The glomeruli identified in a sample of Olios giganteus were found to be most abundant in the left and right pedipalp neuromeres, with glomeruli number estimates of 164 and 141, respectively (Fig. 6D, E). The glomeruli diameters varied between approximately 7.5 µm and

13.7 µm. The organization of neuropil differed somewhat from previously detailed species. As the depth of the images shift from ventral to dorsal, it appears that the darker neuropil surrounds the edges of the neuromere and the lighter neuropil is located more in the center

(Fig. 6B and 6C).

Figure 6: Olios giganticus: A) an overview of the SOG at a magnification of 2.5x. The leg and pedipalp neuromeres are labeled as in the previous figures. B) A detailed close-up of the right half of the SOG regions close to the ventral surface at a magnification of 10x. The orange circle contains what may be considered chemosensory glomeruli. C) A detailed close-up of the same region now 100 µm more dorsally than B).The white asterisk denotes the region of darkened neuropil and the light blue asterisk denotes the region of lightened neuropil. D) An overview of the total chemosensory glomeruli that were counted in each individual leg neuromeres. E) An overview of how the chemosensory glomeruli were found throughout sections starting from their earliest appearance.

Aphonopelma chalcode

The Aphonopelma, more commonly known as a tarantula, proved to be troublesome.

The glomeruli were not clearly defined in a large majority of the sections, and so there is no data for the total number of glomeruli in a leg neuromere or for the varying number of glomeruli within a leg neuromere. However, pictures of the ganglia and the neuromeres were taken (Fig. 7A-C). From these pictures, larger structures that do resemble glomeruli can be observed (Fig. 7B). However, it remains unclear if they are indeed glomeruli.

Figure 7: Aphonopelma chalcode: A) an overview of the SOG at a magnification of 2.5x. The leg and pedipalp neuromeres are labeled as in the previous figures. B) A detailed close-up of the right half of the SOG regions close to the ventral surface and a magnification of 10x. The orange circle contains what may be considered chemosensory glomeruli. C) A detailed close-up of the same region now approximately 250 µm farther dorsally than B).

Comparison of the fused Postoral Ganglion Across Different Species

Due to difficulties and uncertainties when counting glomeruli, the term “neuropil size”, referring to the general area of neuropil that is thought to contain chemosensory glomeruli (for reference, see Fig. 2A), will be used to generalize the findings of this study. All of these species demonstrate very similar structure and organization of neuropil (Fig. 3-7). They all show the same basic organization of neuropil within the leg neuromeres and the distinction and qualities of the neuropil are all relatively similar. A notable conservation of structure is that glomeruli are found within the neuromeres in a more posterior-position when compared to the mechanosensory neurons. Additionally, the neuropil size across species appears to be moderately conserved. For example, the Hogna and Olios species had, according to the count of glomeruli, relatively equal neuropil size in their left 2nd leg neuromeres (Fig. 3F and Fig. 6D). In the same species, the posterior legs tended to possess the smallest neuropil size, consistently having the lowest glomeruli-like structure count (Fig. 3F and Fig. 6D). However, there also exist notable differences between species. The spiders that rely on hunting to acquire their prey, such as the Hogna and the Olios, have higher numbers of glomeruli when compared to a web- weaving spider, being the Nephila. Therefore, the presence of glomeruli and the overall size of the glomerular neuropil tentatively appear to correlate with the behavior of the spider.

Glomeruli are Not Evenly Distributed Between or Throughout a Leg Neuromere

The data recovered from this study demonstrated that certain leg neuromeres hold a higher glomerular neuropil size when compared to others. Most obviously, the anterior-most legs (including the pedipalps, the first legs, and the second legs) consistently showed higher numbers of countable glomeruli when compared to the posterior-most legs (the third and fourth legs) (Fig. 3F, 5D, 6D).

The findings of this study also showed that certain depths of the leg neuromeres hold a higher number of glomeruli. The overall size, and in particular the dorso-ventral distribution of the glomerular neuropil, differed across species. In all species studied, there were clear examples of a peak in neuropil size count at a certain, species dependent distance from the ventral surface. The extent of the glomerular neuropil also differed across the leg neuromeres of an individual spider. The dorso-ventral depth at which the neuropil was largest often varied from leg neuromere to leg neuromere, likely due to the slightly slanted sections in regards to its y-axis so that one side is lower than the other. This is clearly seen in the Olios brain (Fig. 4), where one full side of the ganglia comes into view prior to the other.

Discussion

Presence of glomeruli-like structures in spider SOG

As stated previously, glomeruli are often used for olfactory information organization

(Pinching & Powell, 1971; Tolbert & Hildebrand, 1982, all as cited in Ache & Young, 2005;

Hildebrand & Shepherd, 1997;). Furthermore, this system of organization appears to be constant across a wide variety of animals, from humans to insects (Ache & Young, 2005). From the results, it appears that these structures have now been identified in the SOG of spiders as well. However, this finding proposes a contradiction to what is currently known about spiders.

As mentioned earlier, pore hairs, which are the distinctive olfactory receptors found in relatives of spiders such as whip spiders (Foelix, 1985) have only rarely been observed in true spiders.

Olfactory input is currently thought to be processed via the tarsal organ (Foelix, 2011;

Blumenthal, 1935; Ehm & Tichy, 1994), but this is not a definitive conclusion. The presence of what appears to be chemosensory glomeruli in the leg neuromeres instead suggests that other chemosensory receptors exist on the legs in addition to the small tarsal organ.

Chemosensitive hair sensilla, which are thought to function as contact chemoreceptors, have been greatly detailed to be found mostly on the distal ends of the legs and the palps of spiders (Foelix & Chu-Wang, 1973). These hair sensilla are unique in that they possess an opening at the tip of the hair that exposes the ends of the chemosensitive dendrites to the outside (Foelix, 2011; Foelix & Chu-Wang, 1973). These contact chemoreceptors probably

respond to physical contact with certain chemical stimuli (Foelix & Chu-Wang, 1973; Foelix,

1985), and a comparison can be made to a human’s sense of taste. A possibility that the finding of this study reveals is that these putative contact chemoreceptors are also capable of responding to the airborne, volatile chemical compounds that are commonly referred to as odorants. An extension from this possibility is that the glomerular structures found represent generalized chemical stimuli from both volatile and non-volatile compounds. Because chemical stimuli in the environment can come in many forms and levels of volatility, it would make sense for there to exist glomeruli that combines and integrates several manners of chemical input from (not necessarily contact-only) chemoreceptors. Furthermore, the glomeruli-like structures identified in the leg neuromeres could also belong to the tarsal organs. However, the tarsal organ has been shown to only comprise only a limited selection of neurons (in the large wandering spider Cupiennius; (Anton & Tichy, 1994). The tentative number of synaptic connections shown in this study far exceeds that previously described. This implies that, while some of the glomeruli-like structures may indeed receive input from the tarsal organs, it is likely there is another source of olfactory input. Further experiments are needed to determine the exact role that these glomeruli-like structures play. A good starting point would be to determine if these structures are linked to the hair sensilla found on the legs of a spider and if they are capable of detecting airborne, volatile chemical stimuli.

Glomeruli-like structures were found in all studied species

In all species of spider studied, structures that resembled glomeruli were identified. This suggests that the function and purpose of these structures, whether they be airborne or contact chemical glomeruli, is similar across species. However, drastic differences in neuropil were observed between the hunting species, including the Hogna, Tigrosa, and Olios, and the web-weaving species, Nephila (Nyfeller et al., 1994; Nyfeller, 1999; Airamé & Sierwald, 2000).

There was a greater number of these structures in the hunting species, whereas the Nephila appeared to have a smaller number of glomeruli. If the structures identified are indeed olfactory glomeruli, then this suggests that hunting spiders have a greater capacity for olfaction or chemosensation when compared to ’sedentary’ web-building spiders. This difference may reveal an evolutionary adaptation for hunting spiders as they are much more dependent on their ability to locate prey than the web-weaving spiders. This idea is supported by the well- documented phylogeny. Taken with the results of this study, it appears that the hunting spiders, as they lost the tendency to spin webs (Fig. 8, Garrison et al., 2016), instead gained higher ability for chemosensitivity.

Figure 8: Phylogeny of Spiders: (Top Arrow) Demonstrates the location of the species Aphonpelma icivci, which is a close relative of the Aphonpelma chalcode used in this study. (Middle Arrow) Demonstrates the location of the species Nephila clavipes, which is a species used in this study. (Bottom Arrow) Demonstrates the location of the species Schizocosa rovneri, which is a relative of the Hogna and Tigrosa species used in this study. Image taken from Garrison et al. 2016.

Anterior Leg and Pedipalp Regions Hold Higher Significance for Chemosensensitivity

The results of this study also demonstrated, in the Hogna, Nephila, and Olios species, a higher amount of glomeruli-like neuropil in the neuromeres of the anterior limbs (the pedipalp, first leg, and second leg). This would imply that there exists an emphasis on these regions to detect and process chemical stimuli in the environment. However, the results from this study are not conclusive. More experiments that utilize a greater sample size and more consistent quality of the sections utilized and pictures taken are necessary to affirm this claim.

Limitations

While successful in its purpose of identifying chemosensory glomeruli in spiders, this study was beset by a variety of setbacks that inhibited it from collecting all the data that was originally planned. Sections of the brains studied were often obstructed in some way, such as an inconsistent section thickness resulting in a thicker, darker, more heavily stained section often followed by a thinner, lighter, and less stained section. Some sections were destroyed, altered, or obstructed during the mounting process, leading to an inability to use them for general observation or counting. Additionally, the heavily disrupted world state due to the

COVID-19 outbreak prevented access to the laboratory that held the microscope and collection of slides, limiting the data set and level of detail that was able to be utilized in the study.

Acknowledgements

This project was supported by the funding provided from the NSCS Undergraduate Research

Award.

This project could only have been completed with the help of Dr. Wulfila Gronenberg, whose support, patience, and guidance are much appreciated.

Special thanks to Irina Sinakevitch, who was kind enough to lend her time and vast expertise to this project.

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