Multisensory Control of Homing Behavior of Whip Spiders (Arachnida: Amblypygi)
MULTISENSORY CONTROL OF HOMING BEHAVIOR IN WHIP SPIDERS (ARACHNIDA: AMBLYPYGI)
Patrick E. Casto
A Thesis
Submitted to the Graduate College of Bowling Green State University in partial fulfillment of the requirements for the degree of
MASTER OF SCIENCE
August 2018
Committee:
Daniel D. Wiegmann, Advisor
Verner P. Bingman
Paul A. Moore
© 2018
Patrick E. Casto
All Rights Reserved iii ABSTRACT
Daniel D. Wiegmann, Advisor
Navigation in arthropods has been well studied in terrestrial model organisms such as ants, fiddler crabs, and spiders and there is an emerging understanding that arthropods are much more complex in their spatial abilities than once thought, based on their relatively small nervous systems. Whip spiders (Order Amblypygi) are of interest because, unlike most well-studied terrestrial arthropods, they are nocturnal and live in spatially complex habitats. In addition to the dense terrestrial substrate that creates navigational challenges, navigation at night adds seemingly more adverse conditions for goal-oriented spatial behavior. The integration of multimodal sensory information is hypothesized to facilitate navigation under these conditions, where odor cues are apparently crucial. The goal of this project was to establish a rich dataset of spatial movements of amblypygids under controlled laboratory conditions and determine the relative salience of sensory cues in a multimodal, navigational context. This study used an automated video tracker to record the return paths of amblypygids displaced from a shelter in an arena with a spatially heterogeneous array of sensory cues, where light and odor sources were experimentally manipulated. We found that removal of the light or odor cue did not affect their ability to home to a shelter, but in the absence of the light cue return paths were more circuitous and contained more pauses. Surprisingly, removal of the odor source from the arena had no effect on trajectory kinematics. In addition, our analyses revealed general species differences in return paths. iv ACKNOWLEDGMENTS
I would like to thank my committee; Dan Wiegmann, Vern Bingman and Paul Moore for their constructive criticism and clear investment in my academic growth. I thank both Jake
Graving, and Martin Heck for their help in learning to code in Python and R. Lacy Arnold,
Timothy Huth and Inés Sotelo have been momentous help for their tireless editing of multiple manuscript drafts experimental assistance and unending encouragement. David Gesicki has been an extraordinary help teaching me statistical analyses. I appreciate Vince Coppola for his friendship and help with multiple aspects of the project. Erica Forstater has also been a terrific friend and help. Connie Santangelo was a critical help to the beginning and preliminary stages of the project. Kaylyn Flanigan, Meghan Moore and Brittany Cordova have also been sources of great encouragement and academic conversation. Finally, I am grateful for my family who always have unending faith in me and push me to be better. v
TABLE OF CONTENTS
Page
INTRODUCTION ...... 1
METHODS ...... 6
Subjects ...... 6
Experimental Arena ...... 6
Procedures…...... 7
Return Paths…...... 9
Variables……...... 10
RESULTS…………… ...... 12
Displacement Trials ...... 12
Path Kinematics ...... 13
Homing Path Departure Points ...... 13
Principal Component Analysis ...... 14
DISCUSSION………...... 16
Homing Ability ...... 16
Sensory Stimuli ...... 16
An Unexpected Result of Visual Control ...... 17
Species Comparisons ...... 19
REFERENCES……… ...... 22
APPENDIX A: FIGURES ...... 29
APPENDIX B: TABLES ...... 35 1
INTRODUCTION
The ability for animals to navigate their environment is crucial in supporting behavior such as foraging and relocating a safe refuge. Navigation has been studied intensively in a select group of terrestrial arthropods like fiddler crabs, dung beetles, spiders and desert ants (Cheng,
2012; Perry et al., 2013; Ortega-Escobar & Ruiz, 2014, 2017). These animals mostly inhabit open, two-dimensional environments and the strategies they employ for navigation share a number of properties. Fiddler crabs, wolf spiders and desert ants, for instance, often utilize path integration to relocate their refuge where a time-compensated sun compass is used for heading orientation and idiothetic, proprioceptive cues provide directional and distance information as an animal moves through the environment (Mittelstaedt & Mittelstaedt, 1982; Layne et al., 2003a, b; Wehner, 2003; Reyes-Alcubilla et al., 2009; Cheng, 2012). With distance and direction updated during an often-tortuous outward journey, an estimation of an outbound-path’s tangent can lead to a good approximation of the direction and distance back to the nest (Mittelstaedt,
1983, 1985; Collett et al., 1999, Wehner, 2003). Desert ant species of Melophorus live in the semi-arid shrub deserts of Australia where there are more local cues to help guide them when near their nest (Kohler and Wehner, 2005). In these cluttered habitats, alternative navigational strategies appear to have evolved. They follow learned routes, often using local visual landmarks as guides (Bregy et al., 2008; Bühlmann et al., 2011; Collett, 2010; Fleischmann et al., 2016; Merkle & Wehner, 2008; Schwarz & Cheng, 2010; Steck et al., 2011; Ziegler &
Wehner, 1997; Wehner, 2003), and fail to successfully navigate when such visual cues are disrupted along the route (Cheng et al., 2009).
Nocturnal arthropods are challenged by low light levels which may constrain visually guided navigation. Animals that use polarized light, the skyline panorama, or visual landmarks 2
for navigation are disadvantaged as light decreases (Kelber et al., 2006; Somanathan et al., 2008;
Narendra et al., 2013). However, many of these nocturnal arthropods have optical adaptations to
compensate for a dark environment (Warrant & Dacke, 2010, 2016; el Jundi et al., 2015;
Narendra & Ramirez-Esquivel, 2017). The cursorial wolf spider Lycosa tarantula, for example,
has enlarged median eyes that help collect light at night (Reyes-Alcubilla et al., 2009). In the
Namib desert, the huntsman spider Leucorchestris arenicola likely recovers enough light from the brightest stars and moonlight by temporal summation, characterized by phases of movement and stillness (Nørgaard et al., 2008).
While vision in dark conditions is still possible, many nocturnal animals use other sensory modes like odors emitted from the environment to serve as landmarks in place of a visual landscape (Jacobs, 2012). Indeed, many animals have evolved the ability to track odors at very precise scales (Svensson et al., 2014). Even the desert ant Cataglyphis fortis, which uses path integration as its primary navigation strategy, has been found to use odors emitted from its nest to pinpoint the entrance on a return journey (Steck et al., 2009, 2011). Additionally, recent evidence suggests that C. fortis might use the distribution of odor gradients within near vicinity of its nest in a map-like manner (Steck et al., 2010).
For amblypygids—commonly known as whip spiders—olfactory cues appear to guide their spatial behavior at remarkably far distances from a spatial goal. Amblypygi are nocturnal arachnids that are typically found in tropical and subtropical rainforests. They can be observed at the base of the same tree for weeks or months, but intermittently wander distances of 30 m or more from their usual residence before they return several nights later (Beck and Görke, 1974 as cited in Weygoldt, 2000; Weygoldt, 1977a; Hebets, 2002; Hebets et al., 2014a, b). The anterior legs of amblypygids, which are not used for locomotion, are thin and elongated, highly 3
articulated and covered with various sensilla (Weygoldt, 2000; Foelix & Hebets, 2001). Beck
and Görke (1974 as cited in Weygoldt, 2000) proposed that orientation after displacement may
be guided by olfactory cues after noticing that displaced Heterophrynus batesii used these
antenniform legs to probe the environment when they were released. Indeed, the distal 20
articles of these structures are covered with multiporous, olfactory sensilla (Foelix et al., 1975;
Beck et al., 1977; Foelix & Hebets, 2001; Santer & Hebets, 2011a). Hence, olfaction is
hypothesized to mediate important aspects of their behavior, different than most cursorial spiders
(Hebets & Chapman, 2000; Chapin & Hebets, 2016).
Field experiments have created much of the foundation we have for the study of
amblypygid behavior. Amblypygids exhibit fidelity to specific areas and refugia in their habitat
(Beck & Görke, 1974 as cited in Weygoldt, 2000; Weygoldt, 1977a; Hebets, 2002; Chapin,
2011, 2014, 2015; Hebets et al., 2014a, b; Chapin & Hebets, 2016; Bingman et al., 2017), which
provide the fixed, stable locations required as navigational goals. Sensory manipulations and
displacement experiments in the field provide evidence that the olfactory receptors on the distal
articles of amblypygid antenniform legs may be necessary for homing (Beck & Gorke, 1974 as
cited in Weygoldt, 2000; Hebets et al., 2014b) while the role of vision may not be as important
for navigation (Bingman et al., 2017). The sensory ablations in these experiments impaired visual input in one group and impaired sensory input from the distal articles of the antenniform legs in another followed by displacements from their home tree. While the distal articles of the
antenniform legs bear unique olfactory sensilla, they are also covered with mechanosensory and
contact chemosensory sensilla, as well as sensilla that may detect humidity (Foelix 1975; Beck et
al., 1977). Because these other sensory sensilla are present elsewhere on the intact segments of
the antenniform legs, the loss of olfactory function was hypothesized to be responsible for 4
decreased navigational ability of olfactory deprived subjects (Hebets et al., 2014a, b; Wiegmann et al., 2016; Bingman et al., 2017). These previous findings are fascinating as they diverge from
many of the previously mentioned arthropods—ants, bees, spiders—that all use visual cues for
navigation. However, the concomitant ablation of inputs from other sensory modalities leaves
open the possibility that olfaction was not involved.
While field experiments are crucial in demonstrating the use of various mechanisms,
namely, the sensory mechanisms of amblypygid navigation, they naturally suffer from a lack of
control (Bingman et al., 2017). Controlled laboratory experiments have demonstrated that
amblypygids exhibit site-fidelity in a laboratory (Graving, 2016; Graving et al., 2017) similar to
what is observed in the field (Hebets, 2002). The subtropical whip spider species Phrynus
marginemaculatus do not appear to rely on trail-following behavior for homing and can
successfully recognize a shelter that is near an artificial odor (Graving, 2016; Graving et al.,
2017). While the current evidence implicates that olfactory cues are crucial in guiding whip
spider navigation, Santangelo (2017) showed that information other than olfaction appears to
influence their navigational behavior. Indeed, P. marginemaculatus can learn to discriminate
between shelters that have unique tactile surfaces (Santer & Hebets, 2009b).
These previous studies provide a necessary knowledge base of the sensory properties and
individual use of the sensory modes that guide amblypygid behavior, emphasizing olfaction.
However, Wiegmann et al. (2016) provides an important argument for studying complex
behavior—in this case navigation—as an integrated multimodal system. Further experiments
need to move toward elucidating how the different sensory modalities of whip spiders are
potentially used together. The aim of this study was to combine visual and olfactory cues in a
sensory heterogeneous arena and systematically manipulate olfactory and visual cues to reveal 5 their relative impact on the homing paths of amblypygids. This study provides a foundation of information on the homing behavior of amblypygids in an arena of heterogeneous sensory stimuli, upon which we can build hypotheses associated with the relative salience of different sensory stimuli in a multimodal context.
6
METHODS
This study involved nocturnal displacements of amblypygids from a shelter in a circular laboratory arena that included visual, olfactory and tactile cues. The visual and olfactory cues
were systematically manipulated to determine their influence on the ability of subjects to relocate
the shelter and on spatial and kinematic attributes of their return paths.
Subjects
The experiment included five Phrynus pseudoparvulus Armas and Viquez, 2002 and five
Paraphrynus laevifrons (Pocock, 1894). Phrynus pseudoparvulus were collected from the bases
of trees in secondary forest at La Suerte Biological Field Station, Limon, Costa Rica in July
2015. Paraphrynus laevifrons were collected at Las Cruces Biological Station, Puntarenas,
Costa Rica in February 2017 from secondary forest along cliff banks of various streams from the
Java River. Animals were collected under permit 144-17-ACLA-P.
In the laboratory, subjects were housed separately in a medium (23 cm x 15 cm x 15 cm)
or large (30 cm x 19 cm x 20 cm) Kritter Keeper® boxes that contained a coconut fiber substrate
and a piece of tree bark for shelter. They were fed crickets two to three times per week and were
provided with continual access to water. The room in which subjects were housed was lit by
overhead broad-spectrum fluorescent lights (400–750 nm) set on a 12:12 h light: dark cycle
(19:00-07:00 dark phase). The room humidity ranged between 25-55% and the temperature
ranged between 23-28 ºC.
Experimental Arena
Two identical experimental arenas were used. They were located in separate rooms lit in
the day cycle with 100 W incandescent bulbs. During the night the rooms were lit with four 7
infrared LED array lights, which are undetected by amblypygids (Graving et al., 2017). The
rooms had a humidity that ranged from 23-65% and a temperature that ranged between 22-27 ºC.
The arenas were circular, constructed with a white acrylic sheet as a base and a thin piece
of white-coated aluminum wrapped end to end to create a wall (Fig. 1). The arenas were divided
into imaginary quadrants. The manipulated visual and olfactory cues were placed in two of the
quadrants. In particular, an LED light with three diodes that emitted red, blue and green light
was positioned on the arena wall of one of the quadrants and a circular acrylic disk (4 cm x 0.5
cm, Diam. x H) with a well in the center that was filled and replenished daily with 5 µl of geraniol (Sigma-Aldrich, Product Number 163333) was placed in another quadrant. In the other two quadrants we placed two additional sensory cues, an acrylic disk (11.5 cm x 0.5 cm, diam. x
H) covered with Velcro® and a cylindrical PVC tower (11.5 cm x 30 cm, diam. x H). Neither of these cues was experimentally manipulated. A shelter made of PVC was positioned in one of five locations in the arena (Fig. 1, 2). The shelter had a single entrance, which could be blocked, and a removable lid so that subjects could be easily extracted for displacements. The quadrants in which stimuli were positioned were identical for all subjects, as depicted, and within subjects the location of the shelter was fixed.
Procedures
Each subject was moved in its housing container to an experiment room two days before it was placed into the experimental arena. The subject was then transferred to the shelter positioned the arena. The transfer took place at least three hours before laboratory lights-out and subjects invariably remained in the shelter. The subsequent nightly activity was video- monitored. If a subject failed to exit the shelter by 1 h after lights-out the lid of the shelter was lifted, and the subject was gently shuttled through the entrance into the arena. The experiment 8
began after a subject was observed to wander around the arena and successfully return to the
shelter by 1 h after lights-on on three nights, referred to as orientation nights. If a subject failed to re-enter the shelter by 1 h after the room lights turned on it was picked up, placed at the shelter entrance and coerced to enter. The shelter entrance was taped closed—with the subject inside— after the third orientation night so that a subject could no longer freely leave the shelter. The entrance was blocked such that light entered the shelter in daylight hours to maintain circadian activity.
Experimental trials occurred each night after the third orientation night and involved displacement of a subject from the shelter under four treatment conditions (control, light- removed, odor-removed and shelter-removed). Treatments included the pre-displacement removal of a sensory cue from the arena or removal of the shelter, plus a control condition in which no cues were manipulated. In particular, the LED light was turned off in light treatment trials; the odor cue—geraniol—was replaced with 5 µl of reverse osmosis water in odor treatment trials; and in shelter removal trials the shelter was temporarily removed.
Displacements occurred 1 h before the room lights turned on and a subject was allowed to
wander in the arena for 2 h before a trial was terminated. Four displacement locations were
used, each of which was 12 cm from the arena wall (Fig. 1). Under light, odor and control
conditions the shelter entrance was open so that a subject could return to the shelter at any time
within the 2-h period. In shelter-removal trials the shelter—entrance open—was replaced 5 minutes after lights came on and a subject was allowed 1 h to relocate the shelter. The goal of this latter condition was to determine how a subject searched the arena when the shelter itself could not serve as a navigational cue. If a subject failed to return to the shelter within the 2-h 9
period—1-h period for shelter removal trials—it was picked up, placed at the shelter entrance
and gently coerced to enter.
The experiment consisted of four repetitions of each condition (control, light removal,
odor removal, shelter removal) within subjects, distributed over four blocks of four nights (Table
1). Treatment sequences and displacement positions were randomly chosen within blocks such
that each condition and displacement position occurred once in every block and every condition-
displacement location combination occurred once over the four blocks. In addition, the order of
conditions was constrained so that no particular condition occurred on two consecutive nights.
The shelters of two subjects, one P. pseudoparvulus and one P. laevifrons, were assigned to each
of the five possible shelter positions (Fig. 1). Subjects were not fed in the experimental period.
Return Paths
An Avemia Vari-focal CCTV infrared camera (model CMBB100) was mounted above
the center of each arena to record movements of displaced subjects as they wandered around the
arena. The cameras were wired to analog converter boxes connected to an iMac that ran POSE,
a tracking program written in Python (Graving, 2016; Graving et al., 2017). Here, we set POSE
parameters so that a time stamp and the x and y coordinates of a subject were recorded every 2 s.
The homeward path of a subject—if it homed—was isolated based on a backward trace
of recorded coordinates from the point at which the subject entered the shelter for the night to the
point at which the subject was 12 cm from the arena wall, the distance of release sites to the
arena wall. The return path used in analyses—a subset of the homeward path (Fig. 3)—was defined to end when a subject was 12 cm from the shelter, rather than when the shelter was
entered, because at this distance the shelter could be contacted by either of the antenniform legs
and subjects regularly circle a shelter before it is entered (Graving et al., 2017). 10
Path data were plotted and erroneous coordinates, which occasionally arise due to an unevenness of infrared illumination of the arena, were removed. Pixel units were then rescaled into real distance (cm) units and return paths were spatially rediscretized (Bovet and Benhamou,
1988). In particular, the paths were divided into a series of movement vectors, or steps, with a step length R of R = 3.0 cm, where R was chosen so that the angular deviation σ of the distribution of relative step angles, or changes in direction, was between 0.1 ≤ σ ≤ 1.2 radians (Bovet & Benhamou, 1988; Graving et al., 2017). This procedure removed movement artifacts produced by the tracker algorithm and ensured that distributions of kinematic variables derived from the return paths were unbiased (Bovet and Benhamou, 1988). Rediscretization of return paths was performed in the R package trajr (McLean, 2018).
Variables
For each displacement trial we recorded whether or not a subject successfully homed and, if it homed, the time between when a subject was released from a displacement site and the time it reentered the shelter. In addition, we computed a number of variables that characterize the spatial, temporal or kinematic properties of return paths (Table 2). A repeated measures analysis of variance (repeated measures ANOVA) was used to compare variable means between the two species and to determine how behavior depended on treatment conditions.
We plotted the departure points of homing paths and calculated the angle clockwise referenced by an arbitrary north, namely, displacement position (1) (Fig. 1). We used directional statistics to assess distribution patterns based on experimental factors around the arena using the circular package in R (Batschelet, 1981; Agostinelli and Lund, 2017). Departure points were then separated by treatment, shelter location and displacement location and assessed for clustering assuming a null hypothesis of a uniform distribution about the arena. 11
In order to check if any other experimental factors along with treatment were important
predictors of response variability, a scaled principal component analysis (PCA) derived from all
response variables listed in Table 2 was computed with the prcomp function from R. Principal
component (PC) scores for each trajectory were linear transformations of return path
observations after being multiplied by the PC weights for each response variable. The PC scores
for the principal components that contributed sufficient explanations of variability were then
entered into linear mixed models (LMM) and Akaike Information Criterion (AICc) values were used to determine the most efficient LMM. Each model included treatment as a predictor and subject as the random factor, where four other predictors (i.e., block, species, shelter position and displacement location) were added singly, in pairs, in triplets or all together for a comparison of
16 models. The shelter removal treatment was excluded from these models, as shelter availability in this treatment was constrained. Kinematic properties of paths were computed in the R package trajr (McLean, 2018; Table 2). All statistical analyses were conducted in R 3.3.0
(R Development Core Team 2016).
12
RESULTS
Five of the ten subjects successfully completed orientation trials over the first three
nights. The average number of orientation trials was 3.7 nights. Specifically, three subjects required four nights and two subjects required five nights to successfully complete orientation trials.
Displacement Trials
Overall, subjects successfully homed in 143 of the 160 (89%) displacement trials that involved 40 trials under each of the four (i.e., control, light, odor, shelter) conditions. Subjects successfully homed in 37 (93%) trials for both control and light conditions, in 34 (85%) trials under the odor condition and in 35 (88%) trials in which the shelter was temporarily removed.
Under shelter removal conditions, subjects that successfully homed necessarily returned to the shelter after lights were turned on, when the shelter was replaced. Of the 120 control, light, and odor treatments, 108 (90%) successfully homed, from which path kinematics were computed
(Table 3). 80 of 108 (74%) successful homing trials occurred during the night period before laboratory dawn, while the remaining successful homing trials occurred after laboratory lights turned on. A repeated measures ANOVA was used to assess the proportions of each subject’s homing success whether they homed during the night or during the day with treatment as the fixed effect. The mean proportion of nighttime homing for control (C) treatments (0.825 ± 0.102) and odor (O) treatments (0.725 ± 0.102) were similar (t18 = 1.105, p = 0.284) and both higher
than mean proportion of nighttime homing for light (L) treatments (0.5 ± 0.102, F2,18 = 6.763, p
= 0.006; C-L: t18 = 3.590, p = 0.002; O-L: t18 = 2.486, p = 0.023).
Path Kinematics 13
Table 3 shows the path kinematics for the 108 trials in which subjects homed successfully
under the control, light and odor conditions. Results of the linear-mixed model (LMM), with
subject as a random effect and treatment (i.e., control, light or odor) as a fixed effect, are shown in Table 4. Subjects took longer to home during the light treatment than under control or odor conditions. Mean latency to home for control and odor treatments was similar (t95.64 = -0.78, p =
0.438) and shorter (t95.36 = -5.64, p < 0.0001; t95.64 = -4.73, p < 0.0001, respectively) than in light
treatments (Table 3). The mean expected maximum displacement (Emax) in control treatments
was similar to both odor (t97.06 = -1.49, p = 0.140) and light (t96.42 = 1.50, p = 0.138) treatments;
however, odor treatment homing paths showed higher Emax scores than light treatments (t97.06 =
2.95, p = 0.004). These results indicate that return paths were straighter in odor treatments,
when the light cue was available, compared to light treatments, when the odor cue (and all cues
that were unmanipulated) could be used.
To ascertain whether the less directed paths associated with the light treatment were due
do when a subject homed—specifically, whether a subject homed before or after laboratory
lights on—we performed a linear mixed model with night or day homing as a fixed effect and
subject as a random effect on the response variable Emax. There was no difference between
mean Emax (F1,14.89 = 0.020, p = 0.889) for subjects that homed before laboratory dawn (26.94 ±
4.72, n = 18) or after laboratory dawn (25.76 ± 5.02, n = 19). Therefore, light treatments produced a decrement in return path directedness regardless of whether subjects homed in complete dark or after the laboratory lights on.
Homing Path Departure Points
Overall, there was no clustering of homing path departure points (Rayleigh test, n = 108,
r = 0.120, p = 0.200). Treatment did not have any directional effect on the departure points of 14
homing trajectories, while shelter location showed some directionality (Table 5, Fig. 4). When
the shelter was in quadrant B, the departure points of return trajectories were bimodal.
Trajectories when the shelter was in quadrant D showed a nonuniform distribution (75˚ ± 40˚)
toward the west (Fig. 4).
Principal Component Analysis
Table 6 provides results of the ANOVA scaled PCA. The first three principal
components encompassed 61% of return path kinematic variability (PC1 41%, PC2 17%, PC3
13%). The PC scores were the linear transformed data after being weighted by the respective PC
weights. PC1 scores were weighted by positive correlations with homing path duration, standard
deviation of speed, Emax, and pauses. PC2 scores were weighted by positive correlations with
homing path length and mean speed. PC3 scores were weighted by negative correlations with
length of the return path, standard deviation of turning angles and maximum expected
displacement while conversely weighted by negative correlations with sinuosity. The results of
the LMM comparisons on the principal component scores suggest that treatment and species were the most definitive predictors of variability in return path trajectories (Figs. 5, 6; Tables 6,
7). Using the best LMM described by the PC ANOVA where treatment and species were coded as fixed effects and subject as a random effect, mean duration of return paths was longer in P. pseudoparvulus than P. laevifrons (t6.71 = 4.642, p = 0.003). Return path distance for P.
pseudoparvulus was longer than P. laevifrons (t8.32 = 2.566, p = 0.032). However, the mean
speed of steps was slower in P. pseudoparvulus than P. laevifrons (t7.99 = -2.854, p = 0.021). P.
pseudoparvulus showed less variation in walking speed compared to P. laevifrons (t8.52 = -4.057,
p = 0.003). P. pseudoparvulus had less directed paths than P. laevifrons (t7.98 = -3.588, p =
0.007) according to their Emax scores. Mean sinuosity, a function of the standard deviation of 15 turning angles and step length, of smoothed homing paths (weighted by step length) and the mean standard deviation of turning angles between steps of rediscretized homing paths (equal step length) were not significantly different between the two species. P. pseudoparvulus paused more often along their homing paths compared to P. laevifrons (t6.04 = 3.024, p = 0.023).
In summary, subjects showed a robust ability to home regardless of treatment. Light treatments had the largest effect on return path kinematics, namely that latency to home was longer than either control or odor treatments and were less direct compared to odor treatments.
Departure points of homing paths were uniformly distributed regardless of treatment while shelter location showed clustered departure points for subjects with the shelter located in the tactile quadrant (B) and the tower quadrant (D). Displacement location showed a bimodal distribution of homing path departure points, each concentrated in areas nearer the odor quadrant or the tactile quadrant. Finally, the PCA and repeated measures ANOVA of PC scores showed that there were robust species differences for other aspects of homing path kinematic data and that treatment and species were the largest factors in contributing to the variability of the observed trajectory data.
16
DISCUSSION
Homing Ability
The results of this study clearly show that Phrynus pseudoparvulus and Paraphrynus laevifrons reliably home after displacement from a conditioned shelter in a sensory rich arena.
These results are consistent with displacement experiments performed in the field (Beck &
Görke, 1974; Hebets et al., 2014a, b, Bingman et al., 2017) albeit on a smaller scale. The maximum distance subjects were displaced in the arena from the shelter was one meter, except for subjects that had the shelter in the center of the arena (displaced equidistant), while most displacements in the field were within the range of (6-10 m) (Beck and Görke, 1974; Hebets et al., 2014a, b; Bingman et al., 2017). Considering that the movement patterns of P. pseudoparvulus in the field were up to 30 m (Hebets, 2002), the displacement distance in this study might be defined as being within a local range. This is important because animals may use different navigational strategies in familiar areas, as shown in ants (Collett & Collett, 2000;
Collett, 2010). As such, these results may not necessarily correlate with long-distance navigation, but can be informative for navigation within a familiar “home range.”
Sensory Stimuli
The sensory qualities that were experienced in the arena are likely similar to the qualities of sensory stimuli that animals encounter when they navigate in the wild. When travelling on the forest floor between trees or cliff banks, there are many tactile differences, including tree trunks that are similar in diameter as the three-dimensional tower in our experiment (Hebets, 2002).
The tactile cues might be similar to changes in texture on a moss-covered tree or a cliff bank cluttered with foliage. P. marginemaculatus was found to successfully discriminate between refuges with different tactile surfaces in the lab (Santer & Hebets, 2009b). With their 17
antenniform legs covered in numerous mechanosensory sensilla, it would not be surprising to
find that whip spiders may be able to discriminate between various surface textures. While
noting that the tower and tactile cue were not manipulated in this experiment, they were provided
to create stable heterogenous landmarks from which subjects could have used while homing.
Geraniol, a floral monoterpenoid alcohol was the point source odor for our experiment and has
been shown produce neural responses within the distal articles of the antenniform legs (Hebets &
Chapman, 2000). Geraniol is synthesized by bees (Danke et al., 1990) as well as produced in
small quantities by many plants; thus, it could be a useful stimulus for testing olfactory behavior.
Given the sensory toolbox of amblypygids (summarized in Santer & Hebets, 2011a), the
experimental arena provided an array of sensory cues used could be employed by amblypygids during their natural homing behavior.
An Unexpected Result of Visual Control
The intent of this research project was to identify which of the various sources of sensory
inputs had more control on the successful homing behavior in amblypygids, with a specific
emphasis on the manipulated cues—odor and light. Based on field experiments, olfaction has
more control on whip spider homing behavior (Beck & Gorke, 1974; Hebets et al., 2014a, b;
Bingman et al., 2017). As such, we hypothesized that removal of the olfactory cue would have a
greater effect on homing behavior compared to light removal. However, the results of the
repeated measures ANOVA with the effect of treatment showed, surprisingly, that removal of
the light stimulus had a profound effect on amblypygid homing behavior compared to odor
stimulus. Importantly, this result implies that the principal source of sensory input that guided
subjects’ navigation in our arena was light. Further, subjects showed a clear decrement in their
homing efficiency (higher latency to home and lower Emax scores—a measure of trajectory 18
straightness that accounts for random angular error accumulation) when the light was removed
regardless of whether they homed in complete darkness or after the laboratory lights came on.
Therefore, this supports an argument against a possible motivational effect on the decline of
navigational efficiency.
Why does covering the eyes of the amblypygids in the field not impact navigation, yet
seems to have an impact in our experimental paradigm? We may hypothesize that amblypygids
have a flexible navigational system that can cope with differing magnitudes of different types of
stimuli as needed. If we examine the structure of our controlled paradigm, one possible
hypothesis for our results may be that we decreased the olfactory salience throughout the arena with the single point source of olfactory information compared to the potential array of olfactory information that could be found in the field. In contrast, the LED arena light may have increased the sensory salience of light information by creating a clear gradient in light intensity across the arena.
Another indicator of possible behavioral flexibility in amblypygids may be proposed by looking at the departure points of return paths around the arena. Although treatment appeared to not have an effect on homing path departure points, shelter location within the arena appeared to have a non-uniform effect on the return paths (Fig. 3). Shelters in quadrant B showed a bimodal distribution in a NW-SE diametric direction while shelters in quadrant D showed a more unimodal distribution with an NE direction. In contrast, shelters in any of the other locations (A,
C or X) were uniform. One hypothesis relating these results may be that biased proximity to the
various stimuli in the arena impacted how subjects navigated with respect to the departure points
of return paths. For example, if different proximities to different cues (except for subjects who
experienced shelters in the center of the arena) induced the employment of different navigational 19
strategies, this would be consistent with a general hypothesis of multimodal control of
navigational behavior. In our paradigm, each shelter location had a different spatial relationship
with the various cues imbedded in the arena. The different departure point distributions based on shelter location (A, B, C, D but not X) suggests that where the shelter was located impacted how they used the available spatial information. This hypothesis comes with a critical assumption that the departure points are independent of one another, which is not the case with this data. By following the more appropriate assumption that each should be a viewed as single mean departure point, the low sample size (n = 2) for subjects in each shelter location diminish this argument to speculation. Nonetheless, where the shelter was positioned in proximity to the sensory stimuli within the arena suggests that the relative weights of a particular sensory cue are affected by the proximity to the shelter.
In the absence of the odor cue, subjects displayed no return path differences in the measured kinematic variables compared to control treatments. These results are contrary to the current hypothesis that olfactory information may be important for guiding navigational behavior
(Hebets et al., 2014b; Bingman et al., 2017). It must be stated however, that although geraniol produces an olfactory response from inputs of the antenniform legs of whip spiders, the behavioral effect of this specific compound is largely unknown (Santer & Hebets, 2011a).
Species Comparisons
One expected result from this study was the robust difference in homing between
Paraphrynus laevifrons (Pocock, 1894), subjects collected from Las Cruces Biological Station, and Phrynus pseudoparvulus Armas & Viquez, 2001, from La Suerte Biological Station. The
PCA analysis provided a visual representation of the multiple factor levels correlated with the
array of measured variables. Running a repeated measures ANOVA with the PC scores for the 20 first two principal components and comparison of AICc scores revealed that both species and treatment were the best factors to explain the variation in our trajectory data. Further, pairwise comparisons showed that P. pseudoparvulus moved significantly slower, made longer homing paths that were less direct, and paused more often while homing compared to P. laevifrons.
Taken together, these results suggest that P. pseudoparvulus are less efficient at homing compared to P. laevifrons. A direct comparison of return path kinematics between this study and current field displacement experiments is not possible as the temporal difference in tracking methods are vastly different between field studies (8-12 hrs. between recorded location intervals) and this study (2 s between recorded location intervals). The individuals of each species used in this study exhibit expected differences between of physical differences where most notably, P. laevifrons varied more in size among individuals and were slightly larger than P. pseudoparvulus. Species descriptions indicate however, that both groups are of similar size
(Pocock, 1894; Armas & Viquez, 2001). All subjects used in this experiment were female.
Interestingly, the species differences did not have an impact on homing behavior during light treatments, suggesting that P. laevifrons may have been affected greater by the removal of the arena light. Further speculation might imply that P. laevifrons may be better light adapted, and without such a cue, perform worse than their slower, meandering relatives. Discussion with
V. Bingman revealed that light levels in the understory between the two Costa Rican locations may be dramatically different. Other habitat differences—light intensity, plant composition, species competition, biogeographical population dynamics, habitat use—just to name a few, then may have roles in explaining how populations with similar sized individuals behave differently.
In conclusion, this study adds a surprising insight to the current knowledge of sensory control in whip spider homing behavior. As the current hypothesis supports a crucial role of 21 olfaction in guiding whip spiders to a refuge, this research reveals that it is now important to look at how other possible sensory stimuli may influence spatial behavior. This was the first attempt to produce a set of behavioral data that measured movement patterns of whip spiders in a controlled arena where more than only a couple sensory cues were present. A broader understanding of how whip spiders navigate, both locally and distant, will require that we find unique ways to balance complex multisensory interactions with systematic observations to test hypotheses related to sensory integrated behavior.
22
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APPENDIX A: FIGURES
Figure 1. Camera view of an experimental arena. Dashed lines delineate imaginary quadrants
(A, B, C, D) in which sensory cues were positioned. Dashed circles indicate the five possible shelter locations. Numbered points (1, 2, 3, 4) indicate possible displacement locations.
30
Figure 2. Arena shelter. The shelter was lined with Velcro and contained sponge laden with reverse osmosis water for hydration and humidity.
31
Figure 3. Homing path isolation. Dashed lines are the cut-off areas of the wall and shelter (S). 32
Figure 4. Homing path departure points by shelter location. Arrows indicate mean direction and mean vector length. Dashed lines are von Mises bootstrapped 95% CI.
33
Figure 5. Principal component biplot grouped by species. Ellipses indicate 95% confidence of distribution. Vectors were scaled to a unit variance and centered on the centroid of the PC correlation matrix. Each point is a trajectory which preserves the relationship by distance to other points while the angle between any point and vector represents the associated variability explained by the specified vector. Angles between vectors are arbitrary. The PC scores for each trajectory were the linear transformations of the response variables after being multiplied by the
PC weights for each response variable.
34
Figure 6. Principal component biplot grouped by treatment. Ellipses indicate 95% confidence of distribution.
35
APPENDIX B: TABLES
Table 1. Example treatment and displacement location sequence over the four blocks of four nights on which subjects were displaced. The cell entries indicate the condition (control, light, odor, shelter removal) and location of displacement (1, 2, 3, 4) on a particular night (condition, location). Figure 1 shows the displacement locations within the arena.
Night Block Night I Night II Night III Night IV I Shelter, 1 Light, 4 Odor, 3 Control, 2 II Light, 3 Odor, 4 Control, 1 Shelter, 2 III Odor, 2 Control, 3 Shelter, 4 Light, 1 IV Control, 4 Shelter, 3 Light, 2 Odor, 1
36
Table 2. Variables created to characterize return paths with the R package trajr.
Variable Description trajr function(s) Latency to home Time from displacement until subject TrajDuration (min) homes Duration (s) Duration of homing path TrajDuration
Length (cm) Length of rediscretized path TrajRediscretize, TrajLength
Mean speed (cm s-1) Mean linear speed (distance time-1) TrajSmoothSG of smoothed path Standard deviation Standard deviation of linear speed of TrajSmoothSG of speed (cm s-1) smoothed path Standard deviation The standard deviation of turn angles TrajRediscretize, of turn angles between each fixed step length of a TrajMeanVectorOfTurningAngles (rad √m−1) rediscretized path (See Bovet and Benhamou 1988 for details) Sinuosity . TrajSinuosity2 = 2 + , (rad √m−1) ( )2 2 −0 5 (See Benhamou 2004 for details) 1−𝑐𝑐 −𝑠𝑠 2 2 2 𝑆𝑆where �c𝑝𝑝 is� the1−𝑐𝑐 mean+𝑠𝑠 step𝑏𝑏 �length,� s is the mean sine of turn angles and p and b are the expectation and covariance of the step length. Maximum expected Emax, a dimensionless estimate of TrajRediscretize, TrajEmax displacement the maximum expected displacement (See Cheung et al. 2007 for of a trajectory from its staring point. details) Low Emax scores are more sinuous while higher Emax scores are straighter. Number of Pauses The number of times a subject moved TrajSmoothSG, less than 0.4 cm in 2 s TrajSpeedIntervals
37
Table 3. Treatment sums of squares means. Bold indicates significance and groups separated by superscript letters were significantly different.
Response Variable Control Odor Light Overall Latency to home (min) 28.06 (5.95)A 32.28 (6.07)A 57.89 (5.95)B 38.14 (2.82) Homing path duration (s) 109.35 (26.85) 117.46 (27.65) 125.42 (26.85) 116.73 (12.87) Homing path length (cm) 86.60 (9.99) 93.30 (10.40) 102.69 (9.99) 94.31 (5.58) Mean speed of homing path 1.27 (0.24) 1.65 (0.25) 1.33 (0.24) 1.41 (0.12) (cm s-1) Standard deviation of 0.70 (0.13) 0.68 (0.13) 0.94 (0.13) 0.78 (0.06) homing path speed (cm s-1) Sinuosity of homing path 0.48 (0.05) 0.35 (0.05) 0.46 (0.05) 0.43 (0.03) (rad √m-1) Standard deviation of 0.20 (0.02) 0.21 (0.02) 0.24 (0.02) 0.22 (0.01) turning angles (rad √m-1) Maximum expected 45.75 (9.43)AB 65.33 (9.82)A 26.49 (9.43)B 45.24 (5.54) displacement of homing path (Emax) Pauses in homing path 2.66 (0.70) 3.46 (0.72) 3.46 (0.70) 3.16 (0.37)
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Table 4. LMM for fixed effect test of treatment. Subject was a random effect. Bold indicates significance.
Response Variable Fixed Effect Ratio Prob > F
Latency to home (min) F2,95.54 = 18.483 < 0.0001 Homing path duration (s) F2,96.03 = 0.154 0.858 Homing path length (cm) F2,96.52 = 0.736 0.482 -1 Mean speed of homing path (cm s ) F2,96.31 = 1.109 0.334 Standard deviation of homing path F2,96.24 = 2.529 0.085 speed (cm s-1) Standard deviation of turning angles F2,96.73 = 1.1494 0.321 (rad √m-1) Sinuosity of homing path F2,98.22 = 1.879 0.158 Maximum expected displacement F2,96.84 = 4.355 0.015 of homing path (Emax) Pauses in homing path F2,95.11 = 0.576 0.564
39
Table 5. Distributions of homing path departure points by different effects. Bold indicates a significant mean direction (after transformation if indicated).
α Prob > C.I. Effects Level Distribution Transformation n r (deg) r (deg) Control uniform - 37 65 0.08 0.799 [165,-158] Treatment Light uniform - 37 136 0.20 0.236 [227,61] Odor uniform - 34 128 0.13 0.551 [259,16] A uniform - 24 170 0.04 0.955 [369,40] B bimodal double-angle 19 149 0.65 < 0.001 [172,120] Shelter C uniform - 22 203 0.15 0.621 [322,61] location D von Mises - 24 75 0.41 0.015 [116,36] Center uniform - 19 122 0.28 0.239 [220,56] 1 uniform - 25 59 0.16 0.524 [166,-76] Displaceme 2 uniform - 26 111 0.28 0.135 [181,42] nt location 3 bimodal double-angle 28 125 0.42 0.006 [160,87] 4 uniform - 29 210 0.14 0.549 [302,48]
40
Table 6. Repeated measures ANOVA comparisons of PC scores. Subject was included as a random effect in all models. Bold indicates the model with the best set of AICc values.
Abbreviations are fixed effects in each model: T, treatment; Sp, species; SL, shelter location; B, block; DL, displacement location.
Model PC1 AICc PC2 AICc PC3 AICc T 454.808 360.005 332.024 T + Sp 448.454 357.146 333.666 T + SL 459.551 369.221 338.429 T + B 461.961 370.965 340.978 T + DL 461.962 370.867 335.184 T + Sp + SL 447.851 367.940 340.047 T + Sp + B 455.615 368.336 342.913 T + Sp + DL 455.633 368.047 337.293 T + SL + B 467.403 380.725 347.562 T + SL + DL 467.230 380.643 342.017 T + B + DL 469.651 382.231 344.916 T + Sp + SL + B 456.189 379.611 349.511 T + Sp + SL + DL 455.617 379.499 344.176 T + SL + B + DL 475.700 392.610 352.019 T + Sp + SL + B + DL 464.572 391.649 354.490
41
Table 7. Species sums of squares means. Bold indicates significantly different means. The final three columns are weights
(eigenvectors) for the first three principal components of response variables. Principal component weights equal to or greater than an absolute value of 0.35 are underlined as the largest influencers of each principal component.
Phrynus Paraphrynus PC1 PC2 PC3 Response Variable Overall pseudoparvulus laevifrons Weights Weights Weights Latency to home (min) 38.47 (4.18) 37.82 (3.83) 38.14 (2.82) 0.22 0.19 -0.31 Homing path duration (s) 170.59 (16.31) 64.88 (15.90) 116.73 (12.87) 0.45 -0.20 -0.20 Homing path length (cm) 107.88 (7.41) 81.32 (7.23) 94.31 (5.58) -0.30 0.35 -0.37 Mean speed of homing 1.00 (0.20) 1.82 (0.20) 1.41 (0.12) -0.02 0.73 -0.28 path (cm s-1) Standard deviation of 0.52 (0.09) 1.03 (0.09) 0.78 (0.06) 0.37 0.19 -0.13 homing path speed (cm s- 1) Sinuosity of homing path 0.45 (0.04) 0.42 (0.04) 0.43 (0.03) 0.29 0.33 0.48 (rad √m-1) Standard deviation of 0.25 (0.02) 0.19 (0.02) 0.22 (0.01) -0.30 -0.32 -0.50 turning angles (rad √m-1) Maximum expected 31.10 (5.57) 59.00 (5.42) 45.24 (5.54) 0.38 -0.13 -0.36 displacement of homing path (Emax) Pauses along homing 4.25 (0.50) 2.12 (0.49) 3.16 (0.37) 0.45 -0.11 -0.14 path
42
Table 8. LMM for fixed effect tests of treatment and species. Subject was included as a random effect. Bold indicates significance (p ≤ 0.05).
Response Variable Factor Fixed Effect Ratio Prob > F
Latency to home (min) Species F1,7.45 = 0.0236 0.882 Treatment F2,95.67 = 18.459 < 0.0001 Homing path duration (s) Species F1,6.71 = 21.544 0.003 Treatment F2,96.14 = 0.117 0.890 Homing path length (cm) Species F1,8.33 = 6.585 0.032 Treatment F2,97.68 = 0.702 0.498 Mean speed of homing path Species F1,8.00 = 8.143 0.021 (cm s-1) Treatment F2,96.82 = 1.049 0.354 Standard deviation of homing Species F1,8.52 = 16.457 0.003 path speed (cm s-1) Treatment F2,97.39 = 2.591 0.080 Standard deviation of turning Species F1,8.17 = 2.873 0.128 angles (rad √m-1) Treatment F2,96.69 = 1.110 0.334 Sinuosity of homing path Species F1,8.65 = 0.295 0.601 Treatment F2,98.01 = 1.844 0.164 Maximum expected Species F1,7.98 = 12.874 0.007 displacement of homing path (Emax) Treatment F2,98.1 = 4.126 0.019 Pauses along homing path Species F1,6.04 = 9.144 0.023 Treatment F2,95.14 = 0.518 0.598