VERTICAL NAVIGATION IN THE WHIP SPIDER AND INSIGHTS INTO ITS SENSORY CONTROL

Meghan E. Moore

A Thesis

Submitted to the Graduate College of Bowling Green State University in partial fulfillment of the requirements for the degree of

MASTER OF ARTS

December 2019

Committee:

Verner P. Bingman, Advisor

Daniel D. Wiegmann

Jari G. Willing © 2019

Meghan E. Moore

All Rights Reserved iii ABSTRACT

Verner P. Bingman, Advisor

The sensory mechanisms underlying homing, the act of returning to a shelter after traveling to resource-rich areas, has been rigorously studied. Additionally, investigations into spatial encoding suggest the encoding of spatial information is dependent on how an organism moves through space. However, these studies have focused on organisms that are surface-bound and unfamiliar with navigating in the vertical plane. Amblypygids (whip spiders) successfully return to their home shelter after a night’s horizontal journey on a forest floor and vertically on a home tree. As such, they are ideal animals to study navigation mechanisms used to home in both the horizontal and vertical dimensions. The purpose of this study was two-fold. The first was to examine homing fidelity on a vertical surface under laboratory conditions. The second was to investigate navigation in surface-bound organisms that are familiar with the vertical plane.

Phrynus pseudoparvulus were placed individually in one of nine possible shelters positioned on a vertical surface and tracked for four nights to determine the extent to which P. pseudoparvulus successfully relocate their home shelter on the vertical plane. Subsequently, for four nights the home shelter was swapped with the location of an alternative shelter to determine whether cues originating from the home shelter, presumably chemical in nature, were more important than the actual location of the shelter, and the sensory cues indicating that location, in guiding homing to the shelter. We found that when presented with conflicting homing cues individuals preferred the original shelter over the original location. Additionally, animals made equal number of errors in the vertical and horizontal directions. Our results indicate that amblypygid’s use homing cues originating from the home shelter, rather than homing cues directly associated with the location iv of the shelter. Furthermore, these results imply that the familiarity to the space rather than the locomotive style controls the way sensory information is encoded. v

This is for the love of science, my lab, my friends, my family, and all that provided their endless

support throughout this journey. vi

TABLE OF CONTENTS

Page

INTRODUCTION ...... 1

MATERIALS AND METHODS ...... 9

Subjects and Housing ...... 9

Test Arena ...... 9

Experimental Procedure ...... 10

Phase I: Orientation Trials ...... 10

Phase II: Homing Trials ...... 11

Phase III: Cue Conflict...... 12

Data Analysis ...... 13

Shelter Choice Analyses and Statistics ...... 13

Phase II...... 13

Phase III ...... 14

Phase II and Phase III Comparisons ...... 14

Occupancy and Shelter Entrances ...... 15

Homing Path Analyses ...... 15

RESULTS ...... 17

Shelter Choice ...... 17

Phase I ...... 17

Phase II...... 17

Phase III ...... 18

Phase II and Phase III Comparisons ...... 18 vii

Percent Correct Choices ...... 19

Quadrant Occupancy ...... 19

Shelter Entrances ...... 20

Homing Path Analyses ...... 20

DISCUSSION ...... 22

Demonstration of Homing on a Vertical Surface ...... 22

Sensory Integration ...... 24

Homing on Vertical and Horizontal Planes ...... 25

REFERENCES ...... 27

APPENDIX A. FIGURES ...... 35 1

INTRODUCTION

The need for animals to successfully move within their environment is essential for their survival (Åkesson and Henderstrom 2007; Fagan et al. 2013). As such, there are selective pressures for animals to develop spatial representational strategies that help them efficiently move through their environments and to extract relevant sensory information to control those movements (Åkesson and Hedenstrom 2007). Animal movement occurs across a broad spectrum of temporal and spatial scales but can effectively be divided into two general categories: orientation and navigation (Hansson and Åkesson 2014). Orientation is the directional movement of the body or limbs often guided by external stimuli and navigation is movement of an animal in a goal-directed fashion (Able 2000). An important example of navigation is homing, the return of an organism to a “home” location, most often a nest or shelter (Able 2000).

Homing, as a form of navigation, can be further subdivided based on the source of information that guides movement: idiothetic and allocentric. The use of idiothetic navigation relies on internal information, or information derived from internal cues such as proprioceptive signals and motor movements, to successfully home (Mittelstaedt and Mittelstaedt 1973; Jander

1975). In path integration, a common form of idiothetic homing, an organism might utilize computations gathered from motor movements like step counting to determine distance traveled.

This distance measure would be combined with positional or orientation information relative to the home goal to calculate a home vector (Muller and Wehner 1988; Papi 1992). Desert ants, for example, monitor their leg movements as a measurement of distance (Wittlinger et al. 2007).

Manipulating stride length can cause animals to over-estimate or under-estimate distance traveled due to errors in calculation of distance traveled (Wittlinger et al. 2006). Similarly, when fiddler crabs are displaced or presented with obstacles, they return to the location of their shelter 2 as if they had not been displaced. While homing using path integration they compensate for the distance traveled by continually orientating their body towards the goal-shelter rather than counting steps (Layne et al. 2003a).

In contrast to idiothetic navigation, allocentric navigation uses information provided by stimuli external to an animal. Allocentric-guided movements rely on sensory cues that are spatially related in some fashion to the desired return location. As such, allocentric sources of spatial information are independent of changes in an animal’s position or movement in space.

Allocentric cues range from landmarks used by digger wasps (Philanthus Triangulum; Tinbergen and Kruyt 1938), route following used by green turtles (Chelonia mydas; Luschi et al. 2001) and bees (Melipona scutellaris; Lindauer and Kerr 1958), star compass used by redstarts(Phoenicurus phoenicurus; Rabøl 1998) and indigo buntings (Passerina cyanea; Emlen

1967), and magnetic cues used by sea turtles (Lohmann and Lohmann 1996) and spiny lobsters

(Boles and Lohmann 2003).

The vast majority of research into homing mechanisms has been done on animals that principally move with respect to the horizontal or surface plane. Even the navigation of birds, who are free to move vertically (altitude), is generally investigated as a movement across the surface of the earth. Yet vertical movements and vertical navigation are important for the life history of many animals. Despite being an important piece of life for many animals, there has been little exploration of vertical navigation and of organisms that naturally move along the vertical plane. Vertical movement research has largely explored vertical migration. For example, medusae, as well as the larvae of fish, squids, and crabs, vertically orient according to light and gravity when experiencing changes in hydrostatic pressure (Rice 1961; Rice 1964; Sulkin 1973;

Sulkin 1975). Planktonic organisms will similarly display vertical migration patterns in response 3 to the diel light and dark cycle (Cushing 1951; Hardy 1958; Longhurst 1976). While the above is interesting in the context of vertical movements, those movements are rarely directed to a specific location, and therefore, reveal little about navigation in the vertical plane.

The few studies that have investigated how animals navigate in the vertical plane focus largely on animals that are able to move freely in three dimensions. Current theories derived from the rat work suggest that surface-bound animals, like rats, encode three-dimensional space as a patchwork of two-dimensional maps, meaning that horizontal and vertical spatial information is encoded in sperate two-dimensional planes and stitched together to form a three- dimensional understanding of an environment (Jeffery et al. 2015).

In contrast, bats, which naturally move within three-dimensional space, will make horizontal and vertical errors with equal frequency suggesting similarly informative vertical and horizontal spatial representation (Yartsev and Ulanovsky 2013). Fish have previously demonstrated the ability to successfully discriminate between positions in the vertical plane through hydrostatic changes in pressure with the swim bladder (Burt de Perera et al 2005), suggesting that they successfully encode vertical and horizontal orientation cues. The combination of hydrostatic vertical cues from the swim bladder, in addition to the horizontal orientation cues provided by the lateral line system (Montgomery et al. 1997), allows for the formation of a complete three-dimensional map of their environment (Burt de Perera et al 2005).

Similarly, when hummingbirds are offered food in the vertical and horizontal directions hummingbirds will accurately locate the food in both directions. However, they demonstrate a preference for the food located in the vertical direction (Flores-Abreu et al 2014). This would appear to indicate that despite successfully navigating in both directions, there is more emphasis or a preference for the horizontally encoded information. Furthermore, research has been done in 4 rats with the intent of investigating relative success in finding goals in the vertical and horizontal planes. The data suggest that when rats navigate a vertical plane their grid cell firing patterns, as well as the scale of the grid cell map on vertical surfaces, are determined by an interaction of external and internal sensory information, indicating that the integration of allocentric and idiothetic cues are key for effective navigation the vertical plane (Casali et al. 2018).

Thus, evidence suggests that the accuracy of horizontal and vertical spatial information encoding is dependent on how an animal moves through their environment: three-dimensional movement or surface-bound movement (Holbrook and Burt de Perera 2009; Burt de Perera et al

2013; Flores- Abreu et al 2014; Burt de Perera et al 2016). Furthermore, other factors such as the quality of sensory information and energy expenditure are affected by locomotive style (Davis et al. 2018), further impacting the way vertical and horizontal navigation cues are integrated. The differences between vertical and horizontal spatial encoding between surface-bound and three- dimensional moving animals also suggests that how vertical and horizontal space is encoded is dependent on the locomotive style of the animal. As seen in bats, fish, and hummingbirds

(Yartsev and Ulanovsky 2013; Burt de Perera et al 2005; Flores-Abreu et al 2014), if vertical and horizontal space are encoded together, as they appear to be with three-dimensional moving animals, one would expect similar spatial performance in both planes. Indeed, research has indicated that animals routinely moving through three-dimensional space can successfully encode and navigate in both directions (Holbrook & Burt de Perea 2009). By contrast, horizontal-surface-bound animals such as dogs (Drandt & Dieterich 2013), some ants (Grah et al.

2007; Wohlgemuth et al., 2001), and humans (Renier et al., 2006; Brandt et al. 2015) have a more accurate representation of horizontal space compared to vertical space. For these animals, it appears vertical and horizontal space are encoded separately, with more emphasis on horizontal 5 space. An important piece of information missing, however, is how animals that are surface- bound, but additionally naturally traverse vertical space code spatial information. This would provide further insight into vertical navigation and if vertical and horizontal navigation is truly dependent on how an animal moves through space or if familiarity with the plane plays a role in spatial encoding.

From desert ants (Wittlinger et al. 2006) to fiddler crabs (Layne et al. 2003a; 2003b), arthropods show a robust ability to successfully home and are used as model organisms for understanding navigation by path integration. More interesting, however, are examples of arthropod navigation not dependent on path integration (Görner, 1988; McIntyre & Caveney,

1998; Byrne et al., 2003; Reyes-Alcubilla et al., 2009; Nørgaard et al., 2012). Amblypygids, or whip spiders, are arachnids found in refuges like the bark of trees and banks of rivers in tropical and subtropical regions. On a typical night, whip spiders that rest in tree crevices or holes in river banks during the day traverse the vertical surface of their home refuge and the horizontal surface of the ground while hunting (Chapin & Hebets, 2016; Chapin et al. 2018). Amblypygids’ ability to successfully home back to their refuge after a night hunting (Beck and Görke 1974; Hebets et al. 2014a; Bingman et al. 2016), coupled with the robust sensory toolbox of these animals

(Igelmund, 1987; Santer & Hebets, 2011; Weygoldt, 2000), provide an interesting model for the study of navigation in arthropods and in particular, how they navigate along a vertical plane.

While the literature documenting whip spider’s ability to successfully navigate in a complex environment is expansive, an open question is how well this ability translates to their movement in vertical space. As their home shelters are often on vertical surfaces (Chapin &

Hebets, 2016; Chapin et al. 2018), and these animals have shown fidelity to a home shelter (Beck

& Gorke 1975), one would suspect amblypygids possess an ability to locate specific home 6 locations along the vertical surface of a home tree. Indeed, recent research displaced amblypygids on the home trees to test this ability. Animals were placed into two groups (resident and non-resident displace to the same height as the shelter they were located on the opposite side of a tree dependent on the group. Residents were displaced on their own home tree and non- residents were displaced on the tree of another resident animals. They discovered that over half of the displaced residents returned to the marked home shelter and the residents were more successful at locating their home shelter over the non-resident animals. Additionally, the specific tree crevices did not differ in their appearance and thus would have provided similar visual orientation cues. Despite the similarity of the shelters, the lack of visual cues provided by the exterior shelter did not impact the success of the amblypygids’ ability to locate a home shelter

(Hebets et al. 2014b). These data suggest that whip spiders have the ability to successfully home along a vertical surface but does not provide insight into the mechanisms that guide these abilities nor how vertical space may be encoded.

In examining amblypygids’ ability to move along a vertical surface, it is also important to examine the sensory mechanisms that help the animals navigate this space. Amblypygids have a unique sensory adaptation in that the anterior two legs (antenniform legs) are used for sensory perception rather than locomotion. The antenniform legs are covered in sensory sensilla (Foeliz and Hebets 2001) and three types of sensory sensilla (club, porous, and trichobothria; Iglemund

1987) have been described. Club and porous chemosensory sensilla are most abundantly located within the first twenty segments of the distal antenniform legs (Weygoldt 2000) and respond robustly to chemical/odor stimuli (Santer and Hebets 2011). By contrast, the mechanosensory trichobothria sensilla are only found more proximally on the first segments of the antenniform tibia (Weygoldt 2000). Chemosensory perception through these sensilla has been suggested to 7 guide homing in Amblypygids (Hebets et al. 2014b; Bingman et al. 2016). Interestingly, the role the sensilla play in supporting the homing abilities of amblypygids explains in part why route following, and path integration appear to be unnecessary for successful navigation in these animals (Graving et al. 2017; Hebets et al. 2014a), Although found primarily on the walking legs, rather than the antenniform legs, a fourth important sensory receptor is the lyriform organ

(Iglemund 1987; Foelix and Hebets 2001). The lyriform organ is composed of slit-organ sensilla and is analogous to the campaniform sensilla of insects (Weygoldt 2000). The lyriform organ is responsive to mechanical stress, especially when applied at a 90˚ orientation (Pringle 1955), indicating that the lyriform organ is a possible gravity-sensing organ and a source of information important for vertical navigation.

Completing this discussion of sensory abilities, amblypygids have eight simple eyes arranged into a pair of anterior (medial) eyes and two groups of three posterior eyes (lateral).

Although not much is known about the function of these eyes, the medial eyes are hypothesized to be effective at detecting polarized light whereas the lateral eyes most likely do not detect polarized light (Weygoldt 2000). Despite the potential ability to detect polarized light, it is unlikely that visual cues are necessary for homing in amblypygids as there is no disruption in their homing ability when visually deprived (Bingman et al 2016).

The current study seeks to determine how successful whip spiders are in navigating to a shelter on a vertical surface and examine candidate sensory mechanisms that may guide such vertical navigation. Specifically, will whip spiders reliably leave and relocate an established shelter (fidelity) on a vertical surface given other shelter options? Secondly, assuming the animals demonstrate fidelity when presented with two conflicting homing cues, which cue might the animals preferentially use to home? Given the known sensory capabilities of these animals, 8 two predictions emerge. First, when provided with multiple shelter options on a vertical surface, whip spiders will demonstrate fidelity to a familiar home shelter. Secondly, to test which navigational cues are utilized for homing in whip spiders, animals will be presented with conflicting navigational cues such that the original shelter and the original location are uncoupled. Based on the sensory tool-box available and previous homing research, I predict that whip spiders will prefer the navigational cues provided by the shelter itself over the navigational cues provided by the location of the shelter.

9

MATERIALS AND METHODS

Subjects and Housing

The experiment was conducted using Phrynus pseudoparvulus either hatched at Bowling

Green State University from a captive population (n=3) or collected from Las Cruces Biological

Station, Puntarenas, Costa Rica (Permit Number 060-DGVS-2016) (n=8). Prior to being tested, hatched subjects were group-housed in a tank (91 cm x 45.5 cm x 43 cm: l x w x d) and captive subjects were contained in 7.5-liter Kritter Keepers (30 cm x 19 cm x 20 cm: l x w x h). Both containers were filled with coconut fiber substrate with varying sizes of vertically oriented cork bark for shelter. Animals were fed crickets twice weekly and sprayed with DI water daily providing continual access to water. The housing room was climate-controlled; temperature was maintained between 27-29⁰ C and humidity was kept between 65-70%. The room was lit by overhead broad-spectrum fluorescent lights (400-750 nm) set on a 12:12 h light-dark cycle

(19:00 – 7:00 dark phase).

Test Arena

The experimental room was climate-controlled similar to housing. The test arena was placed in a cube-shaped space with inner dimensions of 100 cm x 100 cm x 20 cm: l x w x d.

The walls of the arena were constructed from clear acrylic plastic (100 cm x 20 cm x 1 cm each: l x w x d) and the floor was white acrylic plastic (101 cm x 101 cm x 1 cm; Figure 1A). Placed in the middle of the arena (Figure 1A) was a white acrylic, vertically oriented board (hence-forth called vertical shelter board) measuring 50 cm x 50 cm x 1 cm with a white acrylic base (20 cm x

50 cm) (Figure 1B). Nine rectangular entrances (4 cm x 1 cm: l x h) arranged in a three by three grid pattern were drilled into the shelter board. Shelters were spaced such that the vertical and 10 horizontal edges of the shelters were 9 cm from the identical edge of other entrances and 10 cm away from the edge of the shelter board (Figure 1A;1B). Nine shelters were constructed from black acrylic (8 cm x 4 cm x 6 cm; Figure 1C) and two strips of hook-side Velcro® were placed on the floor of the shelter. A video camera (Swann Alpha Series) was mounted 10 cm from the edge of the arena opposite the shelter board and 25 cm above the floor of the arena. The camera recorded video for a period of 13 hours starting 30 mins before lights off and ending 30 mins after lights on (19:30 – 7:30). Recordings were stored in a Swann Alpha Series DVR and displayed on a video monitor. The monitor was covered at night with a blackout sheet to prevent the monitor light from disrupting the light-dark cycle of the subjects. Two Univivi Array Infrared

Illuminator lights were placed approximately 55 cm above the arena across from the shelter board to allow for visualization of a test animal on recordings.

Experimental Procedure

Each animal experienced three phases of experimentation, where each phase consisted of multiple trials. Phase I consisted of three trials, Phase II and III each consisted of four trials.

Animals proceeded to the next trial or phase only after completing the current trial/phase. A completed trial was defined as an animal leaving a shelter and returning to any shelter before lights turned on in the morning.

Phase I: Orientation Trials

The intent of orientation trials was to familiarize subjects with the vertical shelter board, establish a starting home shelter, and prepare the subjects to be forced out of the home shelter during Phase III. During orientation trials, a black acrylic box (8 cm x 4 cm x 6 cm), which served as a shelter, was attached to the back of the center entrance. All other shelter entrances 11 were blocked by poster board attached to the back of the entrance with Velcro® to prevent animals entering the empty spaces on the shelter board. A small rectangular sponge

(approximately 2 cm x 3 cm) dampened with reverse osmosis (RO) water was placed in the shelter to provide access to water. The sponge was dampened once a day unless otherwise necessary. If an animal did not return during the night of a trial, it was placed back in the center shelter the following morning and the trial was repeated. During orientation trials one and three, a test animal was required to leave the shelter on its own. During orientation trial two, a subject was forcibly nudged out of the shelter with a paintbrush at 20:30, 90 minutes after lights out, but was otherwise undisturbed during the trial. The active forcing out of the subject during Trial 2 was to prepare the animals to be forced out during Phase III. The criterion to move on to Phase II was three completed trials in Phase I.

Phase II: Homing Trials

Hypothesis: Whip spiders can successfully navigate to a home shelter located on a vertical surface even if they leave the vertical surface during a night’s wandering.

Prediction: With multiple candidate shelters available, whip spiders will faithfully return to their home/start shelter after a night of wandering restricted to the vertical surface as well as when they leave the vertical surface.

After three successful orientation trials, all nine shelters were made available as nine black acrylic shelters were placed on the back of the shelter board in alignment with the shelter entrances (Figure 1B). A small rectangular sponge (approximately 2 cm x 3 cm) dampened with

RO water was placed in all the shelters and dampened daily. A test animal had free access to all shelters on the vertical shelter board and the floor of the arena for the duration of homing trials. 12

Animals remained undisturbed apart from the dampening of sponges at 20:30 (90 mins after lights out). If the animal returned to the shelter it occupied at the beginning of the trial, the choice was considered correct. Otherwise, if the animal chose any of the nine available shelters, the trial was considered successful. If the animal did not choose a shelter, the trial was considered unsuccessful and the animal was restarted in the shelter occupied at the beginning of the failed trial.

Phase III: Cue Conflict

Hypothesis: For successful navigation (Phase II), whip spiders rely on a multimodal spatial representation consisting of local cues originating from the home shelter as well as sensory information from the external environment.

Prediction: If the home shelter is displaced to another location after an animal leaves for the night, thus disrupting interpretation of any multi-modal representation, the capacity of the animals to return to a home shelter will be disrupted. Animals will use cues associated within the established shelter not the location to home.

During Phase III, all nine shelters remained open. During Phase III, the shelter an animal inhabited at the end of the previous night’s trial was defined as the original shelter (to become the moved location) for the next trial. An hour after lights out, a subject was gently forced out of its shelter. A second shelter was then selected from the same row as the original shelter. If the original shelter was in the middle of the row, a coin flip determined whether the shelter to the left or right was switched with the home shelter. If the original shelter was on either the outside of the row the center shelter was switched with the home shelter. The positions of the original shelter and the second shelter were then switched. The shelter occupied at the beginning of the 13 trial, then moved to either side during conflict, was termed “original shelter” (also referred to as moved location). The location (the entrance) that the original shelter occupied at the beginning of the night was termed “original location”. As with the previous phases, if an animal did not return to any shelter, the home shelter was returned to its original location, the animal was placed back in that shelter, and the trial was repeated. If the animal chose a shelter that was not the shelter occupied at the beginning of the trial, the previously occupied shelter (original shelter) was removed, cleaned, and replaced with a clean shelter. The shelter that the animal chose then became the home/start shelter.

Data Analysis

Shelter Choice Analyses and Statistics

Phase II

A one-sample t-test was used to compare total correct choice, correct choices made while staying on the board, and correct choices made when leaving board, where correct choice was defined as returning to the shelter in which the animals began the night. All measures for correct choice were compared against random chance. Vertical component errors were defined when the animal returned to a shelter (different than the original shelter) where the chosen shelter was either above or below the original shelter. Similarly, horizontal component errors were defined when the animal return to a shelter (different than the original shelter) where the chosen shelter was either to the left or right of the original shelter. Diagonal errors scored as both horizontal or vertical component errors as diagonal includes both a horizontal and vertical component. For

Phase II trials chance was set at 1/9 (11.11%). To assess differences between error types a paired 14 t-test was implemented during Phase II. A chi-squared test was implemented to compare whether leaving the board impacted whether a correct choice was made.

Phase III

To determine the success of animals in locating a home shelter during cue conflict, a one- sample t-test was conducted on the total percent of correct choices made. A paired t-test was conducted to compare the number of correct choices made against chance. For the purposes of this analysis, both the original location and the original shelter (moved location) were considered correct choices. For Phase III trials chance was set a 2/9 (22.22%) as we would expect the animal to return to one of two shelters (the location or the original shelter). A paired t-test was used to compare choice of original shelter against original location. Original shelter was defined as the shelter occupied at the beginning of the night before conflicting cues were presented. Original location was defined as the starting location (shelter entrance) of the original shelter before conflicting cues were presented.

Phase II and Phase III Comparisons

For comparisons between phases, both the original location and original shelter (moved location) were considered correct choices for Phase III. Additionally, to account for differences in chance when comparing phases, chance (11.11% in Phase II and 22.22% in Phase III) was subtracted from that phases data. Tests were then conducted on the transformed data. To examine differences in the ability of animals to successfully locate a home shelter between phases, a paired t-test was conducted on the total percent of correct choices between Phase II and Phase

III. In addition, a paired t-test was conducted to examine differences in the percent of correct choices made during the last trial (trial 4) of both phases to examine learning effects. Tests were 15 then similarly conducted comparing occupancy scores and the number of shelter switches in both phases across trials (total) and the last trial. Finally, paired t-tests were conducted to examine differences in speed, standard deviation of speed, Emax, sinuosity, and duration of the homing track between phases II and III.

Occupancy and Shelter Entrances

Across Phase II and III trials, animals were tracked visually. Videos were randomly recoded such that the researcher could not determine animal or phase. The videos and a start time for videos that included the start of the trial were given to the researcher. To maintain consistent borders and sizes for each quadrant (zone) throughout scoring a 3x3 grid was drawn on a transparency for each trial such that the quadrant around each shelter was equal. The area around the edge of the shelter board not included in any of the other nine quadrants was deemed open space. This additionally included any space that was not on the shelter board. Animals were recorded each time they switched quadrants or moved into open space, as well as entrances into shelters. Data were gathered for time spent in each quadrant and shelter as well as the number of times a shelter was entered (shelter switches). A shelter switch is defined as the animal moving between two shelters and the entirety of the body entering the new shelter.

Homing Path Analyses

Across all phase II and phase III trials described above, a single position on an animal was marked and tracked electronically through Xcitex motion tracking software. The tracks were digitized into X and Y coordinates at 30 points a second using the center of the animal as a reference point. A homing path was determined to start 15 cm from the edge of the last chosen shelter (home shelter) and end once the animal’s body and ligaments had completely entered the 16 home shelter. Digitized homing pathways were analyzed for speed, standard deviation of speed,

Emax, sinuosity, and duration using the TrajR package for R Studio (McLean & Skowron

Volponi 2018). Tracks were analyzed for both Emax and sinuosity for the purposes of determining the straightness of homing paths. Emax is the estimate of the expected displacement of a path, with larger values of Emax indicating straighter paths (Cheung, Zhang, Stricker, &

Srinivasan, 2007). Sinuosity is an indication of the twistedness of a path based on a consistent step measurement and as such smaller numbers are indicative of a straight path (Benhamou

2004). Animals did not consistently leave the board nor made a correct choice (defined as home shelter for Phase II and both original shelter and original location for Phase III) when leaving the board. As such, N’s may differ from the number of animals available for video analysis in each phase. During Phase II, video analysis was only available for six of eleven animals. During

Phase III, N’s might differ as video analysis was available for six of eight animals. 17

RESULTS

Shelter Choice

Phase I

On average, animals (N = 11) took an average of 3.90 ± 0.37 trials to meet criteria of three successful orientation trials.

Phase II

All eleven animals successfully completed phase I and continued to phase II. A correct choice for phase II was defined as choosing the shelter occupied at the beginning of the night.

The mean of each subject’s percentage of correct choice was 54.55% ± 13.59%, which was well above chance (chance = 11.11%, t(10) = 3.75, p = 0.004; Figure 2). To determine the effect of leaving the vertical board on shelter choice, data were subdivided between trials when animals left the shelter board and trials when they stayed on the board. The mean percent correct choices on trials when the animal left or stayed on the board were 61.86% ± 17.26% (N= 7) and 52.00%

± 15.26% (N=8), respectively. A chi-squared test revealed there was no statistical difference between the choice (correct or incorrect) based on movement (left or stayed on the board)(χ²(1) =

0.0971, p = 0.755). Animals chose the previously occupied shelter over chance when both staying on the board (one-sample t; t(7) = 2.68, p < 0.05) and when leaving the board (one-sample t; t(6) =2.75, p < 0.05; Figure 2).

The experiment was designed to determine if, when making errors, the whip spiders would be more likely to make vertical or horizontal errors with respect to the home shelter.

Therefore, to further examine how amblypygids encode vertical and horizontal spatial information, error data was divided into horizontal and vertical (diagonal errors were scored as 18 both horizontal or vertical errors). The mean percent error for vertical space was 58.33% ±

22.05% and for horizontal space was 88.1% ± 33.3%. There was no significant difference between vertical and horizontal errors (paired t; t(6) = -1.88, p = 0.109; Figure 3).

Phase III

Eight animals that successfully completed phase II continued to phase III. During phase

III, chance was determined to be 2/9 or 22.22% as there were 9 potential shelters and two possible correct shelters (original location or original shelter). The mean percentage choices made to one of the two correct shelters during phase III was 50 ± 8%, which exceeded chance

(one-sample t; t(7) = 3.39, p < 0.05; Figure 4). Choices to the moved shelter location (original shelter) was statistically different than chance (one-sample t; t(7) = 4.64, p < 0.005). By contrast, choices to the original location were not statistically different than chance(one-sample t; t(7) =

1.01, p =.345; Figure 4). Notably, the number of choices to the location of the moved location

(12 ± 0.33) was greater than to the original location (4 ± 0.27 ; paired t; t(7) = -2.00, p = 0.043).

The percent of total correct choices to the location of the moved location (75 % ± 14%) was not statistically different than the original location (25.00% ± 14.00%; paired t; t(7) = -1.82, p <

0.056; Figure 4) The former result suggests that cues emanating from the shelter itself have more control over the final shelter choice than spatial information originating from the surrounding environment.

Phase II and Phase III Comparisons

The results from the Phase III analysis described above suggest that home-shelter-derived cues had more control over the final shelter choice of the subjects than other external cues.

However, that final choice was preceded by a search phase where perhaps external spatial 19 information may have been relevant. If in fact both sources of spatial information were being used, and therefore in conflict, then other aspects of the homing behavior of the whip spiders should have reflected a greater degree of “confusion” during Phase III compared to Phase II. To test for possible confusion during Phase III, the paths of the 6 animals’ video-tracked during

Phase II and III were examined with respect to three dependent measures and other measures of movement path characteristics (Homing Path Analyses).

Percent Correct Choices

To draw comparisons between phase II and phase III a linear transformation was done on the data as described in the methods. The mean proportion of correct choices made during phase

II after the transformation was 54.51% ± 19.27% for phase II and 27.78% ± 9.8% for phase III.

The mean percent correct choice for trial four of phase II and phase III was 62.39% ± 22.06% and 62.39% ± 22.06%. With the hypothesis that animals would be more successful in phase II than phase III as phase III creates confusion with conflicting cues, there was a significant difference between the percent of correct choices made throughout all trials in phase II and phase

III(paired t; t(7) = 1.89, p = 0.05; Figure 5). Additionally, there were no differences in the percent of correct choices during trial 4 between phase II and phase III (paired t; t(7) = 0.00, p = 1.00;

Figure 6).

Quadrant Occupancy

The average time spent in the home shelter quadrant in phase II, defined as the quadrant of the shelter chosen at the end of the night, was 2063.04 ± 421.12 (s). The average time spent in all other quadrants in phase II was 6461.33 ± 1318.91 (s). In phase III, the average time spent in the moved location and original location was 1771.83 ± 361.67 (s) and 1567.42 ± 319.95 (s) 20 respectively. The average time spent in a quadrant other than the moved location or original location was 3016.25 ± 615.69 (s). There was not a statistical difference between the total correct zone occupancy between phase II and phase III (paired t; t(5) = -.634, p = 0.554; Figure 6). There were no statistical differences between occupancy proportions for trial 4 (paired t; t(7) = 0.748, p

= 0.479; Figure 6).

Shelter Entrances

If the conflict design of Phase III resulted in more uncertainty on the part of the subjects in recognizing the home shelter, then one would predict more shelter entrances and exits throughout the night during Phase III trials. For Phase II, animals on average made 5.17 ± 0.86 entrances into the shelters and 5.66 ± 0.94 entrances during trial 4. During phase III animals made 3.78 ± 0.63 entrances into shelters and 2.66 ± 1.09 entrances during trial 4. There were no differences between the total number of shelter switches made in phase II and phase III (paired t; t(5) = 0.512, p = 0.631; Figure 7). Additionally, there were no differences between the number of shelter switches (entrances) between trial four of phase II and phase III (paired t; t(5) = 1.603, p =

0.170; Figure 7), indicating that as training progressed the lack of difference between the two groups did not change.

Homing Path Analyses

The average speed of the homing path in phase II was 4.40 ± 0.90 (cm/s) and 3.53 ± 0.72

(cm/s) in phase III. The average sinuosity of the homing path for phase II and III was 10.20 ±

0.21 and 11.62 ± 2.37 respectively. The average Emax score of the homing paths for phase II and phase III were 2508.37 ± 512.02 and 72.54 ± 14.81 respectively. A paired t-test was conducted to test for differences between the speed, sinuosity, and Emax scores of the homing paths of 21 phase II and phase III. The speed of the homing paths were not significantly different between phase II and phase III (paired t; t(5) = .935, p = .393). Additionally, Emax did not differ between phase II and III (t(5) = 1.01, p = 0.360). Finally, there were no difference in sinuosity between phases (paired t; t(5) = -0.912, p = 0.404). A repeated measures ANVOA was run to test for differences in the homing path between the original shelter and the moved shelter. There were no differences in the speed and sinuosity of the homing paths between shelter choice (F(1,2) = 4.06, p

= .182; F(1,2) = 2.26, p = .272). However, there was a difference in the Emax scores between shelter choice (F(1,2) = 66.6, p = 0.015). 22

DISCUSSION

Demonstration of Homing on a Vertical Surface

The goal of this study was to examine the ability of whip spiders to navigate vertically and attempt to reveal some of the sensory mechanisms that guide that navigation. Our results demonstrate that amblypygids reliably return to a familiar home shelter given multiple shelter options. Additionally, the ability of these animals to reliably return to a home shelter does not falter when these animals leave a vertical surface. Thus, amblypygids can leave their shelter to explore their arenas on the vertical and horizontal plane and return to their previous shelter with high fidelity. In Phase III of the experiment, a sensory conflict was created by translating the previously visited shelter with an unoccupied shelter. Even under these conflict conditions, animals reliably return to one of two home shelters (original location or moved location). In examining choices made between these two possible home shelters, amblypygids preferred to return to the original shelter (the shelter occupied at the beginning of the night) over the original location of the shelter. This result would seem to indicate that the homing animals are using a sensory cue associated with the shelter as opposed to a sensory cue associated with the original spatial location of the shelter. The most likely candidate for this cue is an olfactory one.

When displaced within the field, amblypygids were able to successfully home when their vision was obscured, however, their ability to home diminished when their antenniform legs were ablated or covered (Hebets et al. 2014; Bingman et al. 2016). As the antenniform legs are covered in tactile and olfactory sensilla (Iglemund 1987; Weygoldt 2000) it is likely that the cues being used to home would be tactile, olfactory, or a combination of the two. However, when homing cues were uncoupled, the overall success of choosing the home shelter occupied the previous night diminished. This indicates that uncoupling the homing cues created confusion 23 over the location of the home shelter. Furthermore, confusion due to the uncoupling of homing cues suggests that the combination of multiple cues associated with the original shelter and the original location are used for homing with a preference for homing cues associated with the shelter itself over the location. This is further supported by the discrepancy in occupancy time between Phase II and Phase III. As animals were confused by the conflicting homing cues, it is possible that a secondary homing technique was utilized to explore the board and locate other cues associated with the home shelter and thus less time was spent in the correct zone.

Environmental stimuli are frequently detected by multiple sensory modalities simultaneously and this multisensory integration results in robust behavioral responses ranging from escape to orientation (Meredith 2001). Particularly, inputs from multisensory integration are well suited for enhancing detecting, localizing, and orientation type behaviors (Stein et al.

1988; Stein et al. 1989) given the spatial-temporal relationship of the stimuli to each other. The integration of input from multiple spatio-temoral stimuli with each other and to their spatial relationship to the shelter would result in a stronger behavioral response to the home shelter. The redundancy of spatial information creates a more reliable set of behavioral choices than those from a single input due to the uncertainty of information from a single stimulus. Interestingly, the mushroom body of amblypygids are suspected to be the site of multimodal integration in these animals (Menda et al. 2014) and is additionally the same area where tactile and olfactory input is detected (Strausfeld and Barth 1993; Strausfeld et al. 1998). Furthermore, the mushroom body is the site of sensory integration in moths and several other insects (Namiki et al. 2013).

Although olfactory cues can be used for long-distance homing, with the dense and complex nature of the forests in Costa Rica, the dispersion of olfactory cues through the environment through the trees would be highly variable. With this variability comes uncertainty as to where a 24 home shelter might be, meaning olfactory cues alone might not be viable for amblypygids traveling long distances. Thus, it would be unsurprising if amblypygids are using a combination of tactile and olfactory cues to successfully home.

Sensory Integration

In examining the potential homing cues utilized by amblypygids, the preference of these animals for the original shelter over the original location is indicative of homing cues associated directly with the home shelter. As the exterior of the shelter board and thus the visual and the mechanosensory stimuli surrounding the shelter entrance does not vary between location, it would be unlikely that the animals are using visual or tactile cues to locate the original shelter.

Thus, the main homing cues associated with the original shelter would be stimuli emanating from inside the home shelter. Given the literature on homing cues, it is most likely that the preference to the original shelter is indicative of olfactory dependent homing using an odor present within the shelter. A variety of olfactory homing cues are used by animals when homing although the two most common olfactory techniques used are route following and landmarks. Olfactory route following uses a series of associated olfactory stimuli which animals can follow to a home shelter (Willson 1962; Bossert & Willson 1963). Route following allows animals to successfully home after long, complicated outbound paths by identifying several cues along an outbound path to follow back to the homing goal (Collett & Zeil 1996). Landmark- based homing uses an external olfactory cue that have a predictable spatial relationship to the goal (Able,2000). Route following, whether using point-source or continuous trails, is unlikely guiding homing within amblypygids as if this were the homing technique used, it would lead the animals directly to the original location, not the original shelter (Collet & Ziel 1996; Kohler &

Whener 2005; Hölldobler & Wilson 1990). Thus, it seems likely that amblypygids are using 25 olfactory landmarks laid within the shelter itself. Furthermore, often when using a beacon, animals employ a secondary search strategy to locate the actual homing goal (Wehner &

Srinivasan, 1981; Schultheiss & Cheng, 2012), which could explain some of the confusion between phases once the cues were uncoupled.

Additionally, as there were no artificial odors present in the current experiment, if odors were the homing cue used by these animals, it is likely that the odor beacon would be a naturally produced by amblypygids. Recent research has indicated that amblypygids are able to discriminate between an odorless shelter and a shelter laid with their scent, preferring the scented shelter (Casto et al. 2019). Green turtles (Chelonia mydas) use chemical landmarks carried by the wind for the last portion of their homing path when returning to their nesting grounds (Luschi et al. 2001; Hays et al. 2003). Similarly, fire ants (Solenopsis saevissima) use an odor gradient given off by an olfactory landmark to home a temporary home shelter (Wilson, 1962; Bossert &

Wilson, 1963). Thus, the use of olfactory cues would be a robust homing cue and successfully traverse the horizontal and vertical planes, allowing animals to successfully locate the home tree

(shelter board) as well as the individual home shelter.

Homing on Vertical and Horizontal Planes

Research has previously suggested that the accuracy of vertical and horizontal coding varies according to how an animal moves in space (Holbrook and Burt de Perera 2009; Burt de

Perera et al 2013; Flores- Abreu et al 2014; Burt de Perera et al 2016). Amblypygids make equal error rates in the vertical and horizontal direction when homing, indicating that vertical and horizontal space is encoded equally. This result is interesting as previous research suggests that surface-bound animals make more errors in vertical encoding (Drandt & Dieterich 2013; Grah et al. 2007; Wohlgemuth et al., 2001; Renier et al., 2006; Brandt et al. 2015). This result could be 26 explained due to the familiarity that amblypygids have with vertical space. Unlike many surface- bound animals previously explored amblypygids are familiar to vertical space, and quite often reside on a vertical surface. One implication here is that it is not the locomotive style, but more likely the familiarity with the plane that controls the way spatial information is encoded.

Additionally, the equality in the rate of error suggests that the homing cues used are robust enough to successfully guide homing in both planes.

We found that amblypygids orienting on either vertical and horizontal planes make the equivalent amount of homing errors. This result would appear to indicate that homing information associated with the vertical and horizontal space is encoded with similar fidelity in these animals and furthermore, that the familiarity of a plane, rather than the locomotive style, affects the way space is encoded. Furthermore, the homing success of these animals, after transitioning between planes, is not different than homing only on the vertical plane. This result implies that the homing cues used by the animals provide robust information regardless of the planes traversed during homing. Given the homing choices of these animals during the cue conflict studies, it appears that olfactory homing cues are the primary cues utilized by these animals with possible other cues used as redundant information. 27

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APPENDIX A. FIGURES

A B C

Figure 1 (A, B, C). Vertical Shelter Board and Surrounding Structure. (A) Enclosure, equipment, and surrounding structure. An enclosure (a) with clear acrylic walls and a white acrylic floor is place in the center of the surrounding wooden structure. The shelter board (b) is placed inside of the arena approximately 25 cm away from the wall of the arena. A camera (c) is attached to the wooden structure opposite of the board. Two infrared lights (d) are attached to the wooden structure 75 cm from the floor of the arena and opposite of the board. A thick black sheet (e) is hung from the top of the wooden structure and falls behind the board, arena, and surrounding structure. (B) A white acrylic board (a) measuring 50 cm x 50 cm x 1 cm nestles within a slot measuring (50 cm x 1 cm x 3 cm: l x w x d) in the white acrylic base (b) (20 cm x 50 cm). (A)

Front View. Nine shelter entrances (c) measuring 1 x 4 cm (w x l) are arranged in a three by three grid pattern. Shelters entrances are positioned 12 cm from the top of the board and the base, and 10 cm from the side of the board. Shelter entrances spaced are 10 cm vertically and 9 cm horizontally from other shelters. (C) Side View. Behind each of the nine shelter entrances are black acrylic shelters (d) attached with hook and loop fasteners. 36

Figure 2. Percent Correct Choice in Phase II. Percent correct choices on all trials, trials when a subject did not leave the vertical board and trials when a subject left the vertical board.. All values were statistically different from chance (11.11%) and no statistical difference was found between on and off trials. * p < .05 37

Figure 3. Vertical and Horizontal Error in Phase II. Percent errors made in the vertical and horizontal planes (note the combined values exceed 100% as diagonal errors were scored as both vertical and horizontal errors). There were no statistical differences between error types. * p <

.05 38

Figure 4. Percent Correct Choice in Phase III. Percent of total correct choices, and of those correct choices, percent choices made to the moved location (original shelter) and the original location. Chance for total was set at 22.22% (blue), and chance for moved location and original location was set to 11.11% (orange). Total proportion correct, and proportion of correct choices made to the moved location were statistically different than chance. Additionally, percent of correct choices made to the moved (original shelter) location statistically exceeded those made to the original shelter location. * p < .05 39

Figure 5. Percent Correct Comparisons between Phase II and Phase III. Percent correct choice between Phase II and Phase III. In order to compare Phase II and Phase III chance for each phase was subtracted from the scores of each animal to correct for the difference in chance (11% in

Phase II; 22% in Phase III). The total percent correct between Phase II and Phase III was significant, although there were no differences between trial 4 of both phases. * p < .05 40

Figure 6. Occupancy Score Comparisons between Phase II and Phase III. Proportion of time spent in the quadrant of the home shelter per animal. Home quadrant was defined as the shelter chosen at the end of the trial. Occupancy score was calculated as time spent in home quadrant (s) divided by the time spent in all quadrants multiplied by chance ( Time Home/ (Time Other *

Chance)). For Phase III, time in the home shelter was calculated by adding time spent in both the moved location and the original location. 41

Figure 7. Shelter Switch Comparisons between Phase II and III. Total number of shelter entrances across animals per night. A shelter switch is defined as the animal moving from one shelter entrance to different shelter entrance. There were no significant differences in shelter choice between phases.