Gravity Acts As an Environmental Cue for Oriented Movement in the Monarch Butterfly, Danaus Plexippus (Lepidoptera, Nymphalidae)

Gravity Acts as an Environmental Cue for Oriented Movement in the Monarch Butterfly, Danaus plexippus (Lepidoptera, Nymphalidae)

A thesis submitted to the

Graduate School

of the University of Cincinnati

in partial fulfillment of the

requirements for the degree of

Master of Science

in the Department of Biological Sciences

of the College of Arts and Sciences

Mitchell J. Kendzel B. S. Biology, University of Cincinnati, May 2018

Committee Chair: Patrick A. Guerra, Ph.D. Committee Members: Stephen F. Matter, Ph.D., John E. Layne, Ph.D. July 2020 ABSTRACT Gravity is an especially important environmental cue on which to focus animal movement and sensory biology research, both because of its consistency through evolutionary time, and because it is an essential force for which all organisms must compensate for, whether they move on land or in the air. In this thesis, I used the monarch butterfly, Danaus plexippus

(Lepidoptera, Nymphalidae) as a system to study how organisms move and orient their body using gravity as a cue for directionality. To do this, I developed two assays, one designed to study directed locomotion and the other designed to study orientation via righting behavior. By focusing on directed movements and righting behavior, I was able to define how monarchs respond to gravity and identify how other environmental cues (that can provide directional information) interact with gravity when eliciting a behavioral response.

In my locomotion assay, monarchs displayed negative gravitaxis only, manifested by walking opposite the direction of the gravity vector (i.e., up), even in the absence of other cues that could convey directionality, or in the presence of cues that typically elicit their own directional response (e.g., light cues). The upwards movement of butterflies only occurred once the apparatus was 30° above the horizontal. When individuals were forced to have a body orientation opposite the direction of their negative gravitaxis response during trials, butterflies would quickly correct their body position to face upwards and they then resumed negative gravitaxis. Monarchs moved upwards more vigorously when gravity and light cues were in agreement.

Using the righting response assay, I showed that monarchs orient upwards to achieve a head up position and can use gravity when it is the only cue available or the only cue that can be sensed for directionality. In subsequent experiments, I showed that light biased their gravity-

ii based righting orientation. When presented with a lateral light stimulus, the final head up righting orientation of monarchs was deflected toward the light source. When light cues came from below, directly opposite of the preferred head up orientation of the butterflies, their righting response to a head up position was unaffected. The antennae were found to be important sensory organs for a proper righting response. When antennae were removed and light cues were present, monarchs did not perform the righting response, but instead directly oriented towards the light source. When antennae-less monarchs were tested in darkness, monarchs oriented and righted themselves toward their default upward direction, suggesting the presence of a secondary mechanism for sensing gravity. Finally, the magnetic field was also shown to affect the righting response as the head up position of monarchs was deflected equatorward, with this response related to the monarchs using the inclination angle of the presented artificial magnetic field. My study demonstrates that monarch butterflies can use environmental sensory cues from different modalities for orientation behavior. In particular, the orientation response driven by a specific cue can be modulated by the presence of cues from other sensory modalities.

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ACKNOWLEDGEMENTS

I would like to start by thanking my advisor, Patrick Guerra, for his guidance and support during the 4 years of working with him at the University of Cincinnati. Because of that time, I have grown immensely in my ability to conduct research and built a solid skill base that has allowed me to continue my pursuit in academia. I am extremely thankful for your willingness to help me conduct research as an undergraduate and to make it possible to pursue graduate school.

To my research committee John Layne and Stephen Matter, thank you for taking the time to meet with me and discuss my work. Being available to discuss and work with me, was integral in the completion of my thesis on schedule. The suggestions you both made helped center the rational for my work and strengthen my findings.

To my fellow lab members Jered Nathan, Sam Stratton, and Adam Parlin, thank you for helping rear monarchs and discuss experimentations. Jered, we worked together for 4 years and during that time you helped me in innumerable situations to organize, rear, plan coursework, and prepare for deadlines. Thank you for your contributions in and outside the lab. Adam, your R skills are amazing and the reason I was able to prepare this much material in such a short amount of time. Thank you for your willingness to teach me these techniques and to help with my writing. Sam, thank you for helping me graph my circular based data. There are unfortunately not a lot of resources available to express circular data, so your help was vital to making my data presentable.

Finally, I would like to thank my wife, Hannah Kendzel, whose continued support made this thesis possible. Thank you for helping and motivating me to work hard and push into new

v and sometimes uncomfortable situations. I am truly blessed to have a partner who supports me as much as you do in my career.

TABLE OF CONTENTS

Abstract…………………………………………………………………………………………..ii

Acknowledgments………………………………………………………………………………..v

List of Tables and Figures…………………………………………………………………….viii

Chapter 1: Monarch Butterflies, Danaus plexippus, (Lepidoptera, Nymphalidae) Use Gravity as a Directional Cue that can Supersede the Effects of Other Cues, for Oriented Upwards Movement…………………………………………………………………………………….…...1 Abstract……………………………………………………………………………………2

Introduction………………………………………………………………………………..3

Methods……………………………………………………………………………………5

Results……………………………………………………………………………………11

Discussion………………………………………………………………………………..14

References………………………………………………………………………………..21

Chapter 2: Multimodal Sensory Integration for Executing Oriented Movement in the Monarch Butterfly, Danaus plexippus (Lepidoptera, Nymphalidae)………………………………………33 Abstract………………………………………………………………………………..…34

Introduction………………………………………………………………………………35

Methods…………………………………………………………………………………..38

Results……………………………………………………………………………………45

Discussion………………………………………………………………………………..48

References………………………………………………………………………………..55

vii

LIST OF TABLES AND FIGURES

Chapter 1

Figure 1.1 General tube maze assay and corresponding light wavelengths used in each experiment………………………………………………………………………………………..27

Figure 1.2 Monarch butterflies respond to gravity with negative gravitaxis and not positive gravitaxis…………………………………………………………………………………………28

Figure 1.3 Monarchs display negative gravitaxis behavior starting at a 30° incline………….....29

Figure 1.4 Light affects gravitaxis response only when it comes from above the organism………………………………………………………………………………………….30

Table 1.1 Descriptive Statistics and summary of comparisons for Negative Gravitaxis, Positive

Gravitaxis, and Role of Light experiments………………………………………………………31

Table 1.2 Pairwise comparisons for incline trials………………………………………………..32

Chapter 2

Figure 2.1 Rotation assay design to measure orientations on a vertical plane……..……………61

Figure 2.2 Orientation assay used to test the use of gravity and magnetic cues for righting behavior………………………………………………………………………………………..…62

Figure 2.3 Gravity cues on their own are sufficient for monarch butterfly righting responses in the vertical plane…………………………………………………………………………………63

Figure 2.4 Monarchs from Fall and Summer of 2018 display identical righting responses……..64

viii

Figure 2.5 Monarchs antennae are important for proper righting behavior along the vertical plane……………………………………………………………………………………………...65

Figure 2.6 Monarchs can orient without their antennae and without the aid of light cues………66

Figure 2.7 Monarchs use both gravity and magnetic cues together during righting behavior …..67

Table 2.1 Rayleigh’s test results for all trials with a Von Mises distribution……………………68

Table 2.2 V-test results for all trials that did not have a Von Mises distribution………………..69

CHAPTER ONE

Monarch Butterflies, Danaus plexippus, (Lepidoptera, Nymphalidae) Use Gravity as a Directional Cue for Oriented Upwards Movement

Mitchell J. Kendzel and Patrick A. Guerra*

Department of Biological Sciences University of Cincinnati Cincinnati, OH, USA

*Mitchell J. Kendzel principally composed the work presented here, with the assistance of

Patrick A. Guerra. This manuscript is formatted for submission to the Journal of Experimental

Biology.

ABSTRACT: Gravity is an important environmental cue that provides organisms with directional information in the vertical plane. Using the monarch butterfly (Danaus plexippus) as a model system, we studied how animals responded behaviorally to gravity and how this response could be modulated by other cues that also provide directional information. To do this, we tested monarchs in a behavioral assay that examined their walking movement in a tube maze whose position could be adjusted relative to the vector of gravity. This assay design allowed for changing the vertical position of the tube, the manipulation of light cues, and enabled the rotation of the entire apparatus during trials. In controlled trials in which gravity was the only cue that could be used for directionality, monarch butterflies displayed negative gravitaxis. They walked upward and reached the end of the tube maze more frequently when the tube was aligned with gravity as compared to when it was perpendicular (control). These results indicate that monarchs are capable of sensing gravity and that the monarch butterfly responds to this cue with negative gravitaxis. Negative gravitaxis was found to be initiated once butterflies were in a tube that was at a 30° angle relative to the horizontal plane. In trials where the tube was rotated during negative gravitaxis behavior (effectively switching up and down in relation to the apparatus), it was found that monarchs were able to acutely respond to this change by stopping their downward movement, reorienting their body position, and resuming their negative gravitaxis response.

Finally, we found that other sensory cues have the potential to modulate the negative gravitaxis response. Phototactic cues were found to have an additive effect on monarch directionality when it was placed above the apparatus. Time to complete the maze was not significantly different based on the light position, but there was a significant reduction in the total distance (less up and down movement) needed to complete the maze when light was above. When light cues were positioned opposite of the direction of the gravity vector, no effect on time or distance to

2 complete the maze was found, meaning that light cues that normally elicit positive phototaxis were unable to cancel the negative gravitaxis response. This suggests gravitaxis has the potential to be modulated by cues from other sensory modalities that can elicit their own directional response.

INTRODUCTION: Environmental conditions can elicit specific responses from organisms as they move within the three-dimensional (3D) space that they inhabit. Gravity is an important cue for movement and a force that organisms must compensate for during terrestrial and aerial movement (MacNaughton, et al., 2002; Schaefer, et al. 2010; Sadovnichii et al., 2008). Gravity remains constant regardless of season or time of day for a given location, and it is very consistent in magnitude and direction across the surface of the earth, varying less than half a percentage from its maximum to minimum (Hirt et al., 2013). This consistency makes gravity an ideal environmental cue to provide animals with information for moving in the vertical plane, especially up and down, against and with gravity, respectively. The ecological importance of gravity has caused diverse taxa to evolve a response to its consistent pull and to utilize it as a cue for execution of non-random movements (Hudspeth, 2000; Sadovnichii, et al., 2008;

Hengstenberg, 1993). By focusing on gravity, we can isolate the behavioral response to a strong cue that defines the vertical plane for many organisms (Halstead, 1994).

The monarch butterfly is an ideal system to study how animals use environmental cues for directional movement due to its use of sensory-based compass mechanisms during migration.

Eastern North American monarchs have a complex multi-generational migration that allows them to escape seasonal deterioration in their habitat (reviewed in Reppert and de Roode, 2018).

During the summer, monarchs do not exhibit the oriented flight behavior necessary for migration

(Zhu et al., 2009). These monarchs instead exhibit dispersal patterns in their North American breeding range (reviewed in Reppert and de Roode, 2018). Butterflies in the fall, comparatively, avoid harsh winter conditions by migrating southwards to overwintering sites in Central Mexico

(reviewed in Reppert and de Roode, 2018). Fall monarchs utilize a time-compensated sun compass to facilitate proper oriented flight during migration, using the sun as a visual cue to orient southwards for directed flight (Perez et al., 1997; Mouritsen and Frost, 2002; Froy et al.,

2003). Monarchs also possess a backup inclination-based magnetic compass when sun cues are unavailable due to cloud cover (Guerra et al., 2014). These two distinct generations, fall and summer, provide different behavioral phenotypes that allow for comparative analyses of oriented movement behavior.

Multiple different species from diverse taxa have been shown to orient using magnetic cues (magnetotactic bacteria, reviewed in Blakemore, 1982; fruit flies, Bae et al., 2016; logger head sea turtles, Lohmann and Lohmann, 1994; birds, reviewed in Wiltschko and Wiltschko

2005). Magnetic compasses can utilize different aspects of the magnetic field as a reference for orientation, such as magnetic polarity used by bats (Wang et al., 2007) and the inclination angle utilized by birds (Wiltschko and Wiltschko, 1972). The inclination angle is defined as the angle made by the horizontal plane and the magnetic field vector. Like birds, monarchs have been shown to orient using the inclination angle for their migratory flight (Guerra et al., 2014). It is unknown what the monarchs use as a reference point to identify the horizontal plane and in turn, measure the inclination angle. Birds have been shown to use gravity to maintain their horizontal orientation while flying (Sadovnichii et al., 2008), and it has been suggested that birds reference the vector of gravity to measure the inclination angle (Wiltschko and Wiltschko, 1972). Birds therefore could use the gravity vector to determine the horizontal plane, align with it, and then

4 measure the inclination angle based on this information. The monarch may utilize a similar method, but it remains unknown whether monarchs sense gravity and use this sensory cue in a similar way when using their magnetic compass.

In this study, we examined the movement of monarchs in a tube maze similar to those used previously with other insects (e.g., Drosophila melanogaster, Vang et al., 2012) that allowed for testing gravitaxis. First, we defined how monarchs respond to gravity (either positive or negative gravitaxis). We predicted that monarchs, regardless of season, would respond to gravity with negative gravitaxis behavior because of the consistent need to orient to gravity for mechanical locomotion (Hengstenber, 1993). Then, we determined the angle at which the gravitaxis response was initiated. Next, we investigated if the gravity response was dynamic and if monarchs can adjust their behavior if the gravity vector suddenly changed directions relative to the animal. We predicted monarchs would adjust to the change in orientation and continue negative gravitaxis. Finally, we compared how environmental cues affected the gravitaxis response with a focus on light. We focused on light for this experiment due to the monarch being diurnal and possessing a strong light cue from the sun for moving up and down throughout the day. We predicted that the phototaxis response of monarchs would modulate their gravitaxis response. In D. melanogaster, vertical oriented positive phototaxis required gravity for the phenotype to activate (Kwon et al., 2016). This result suggests that these two cues have the potential to modulate each other’s behavioral responses.

METHODS: Animals

Adult monarch butterflies were collected at the University of Cincinnati Center for Field

Studies (Harrison, Ohio: 39.285293° N, -84.741566° W) and its surrounding fields in the fall of

2017, the summer and fall of 2018, and the summer and fall of 2019. Monarchs were placed in individual glassine envelopes and stored in an incubator (Percival Model I-36LL). Fall monarchs were housed under fall like conditions with a 12:12 light: dark (L:D) cycle (lights on: 06:00; light off: 18:00) with cycling temperatures (lights on: 21° C; lights off: 12° C), and constant 70% relative humidity (RH). Summer monarchs were housed under summer like conditions with a

14.5:9.5 L:D (lights on: 06:00; lights off: 20:30) cycle with cycling temperatures (lights on:

29°C; lights off: 18°C), and constant relative humidity (70% RH). Both populations were fed a

25% honey solution ad lib.

Experimental Apparatus

The trial assay was modified from that used to measure the gravitactic response in D. melanogaster (Vang et al., 2012). It consisted of a 90 cm long wire mesh tube, including a 13.8 cm starting section and a 76.2 cm long test section (Fig. 1.1a). The end opposite the starting section was open. The tube was illuminated equally by a 108 cm long LED light (1-light 30-Watt

White Utility LED Shop light, Commercial Electric) held at a distance of 61 cm. The mesh tube had a diameter of 10 cm, which is approximately 1.5 times the body length of the butterfly, to permit walking and allow enough room to turn in any direction. A wooden dowel ran along the inside of the mesh tube, serving as an attachment point for the mesh and as more substrate for the organism. No movement bias was observed due to the dowel and the dowel provided no reference point in the vertical plane, as it ran equally from the top to the bottom of the tube.

Animals were removed from their individual glassine envelopes and placed in a mesh cage for 10 minutes outside the testing room to allow free movement before the trial. After the

10 minutes, monarchs were then placed in the tube’s starting area. This starting area was separated from the rest of the tube by a wall, allowing monarchs to freely move, without

6 allowing them access to the trial area. The wall was removed after one minute, which granted monarchs free access to the entire tube for a maximum of 10 minutes. Measurements included total distance walked (in any direction) to serve as a proxy for motivation and maximum distance reached (toward the end of the tube) to serve as a proxy for directionality. Trials were recorded using a video recording system (I DVR-PRO; CCTV Camera Pros) and the variables were measured using the software IMAGEJ (Schneider et al., 2012) from these videos. Measurements only included distances within the trial area itself and not the starting area. Trials were always conducted in a pair-wise fashion, with an individual completing each trial within an experiment.

Monarchs were only used in one experiment each.

Trial Lighting conditions

Trials were done in a dark room with the light source used for the trial as the only source of illumination. Trial light conditions were measured using a spectrometer (Ocean Optics Inc.,

Dunedin, FL, USA) and an optic fiber (QP230-1-XSR, 235 microns; Ocean Optics Inc.) with cosine corrector (CC-3-UV-S; Ocean Optics Inc.). Spectrographs were generated using the

‘pavo’ R-package to convert the values from radiance to photon flux. The light chosen had negligible amounts of ultraviolet-A (UV-A) and ultraviolet-B (UV-B) wavelengths (light source measurements at the location of the butterfly during a trial: peak at 580 nm; range: 300-700 nm; total irradiance: 8.74 x 1012 photons s-1 cm-2 nm-1; Fig. 1.1 b). Exposure to UV-A and UV-B wavelengths have been shown to be necessary for magnetoreception as part of the

Cryptochrome-based (CRY) light-sensitive mechanism of magnetoreception hypothesized for insects such as monarch butterflies (Guerra et al. 2014) and fruit flies (Gegear et al., 2008). For

CRY function to occur, irradiance levels are required to be at least in the range of 1011 photons s-

1 cm-2 nm-1 between 380 nm and 420 nm (VanVickle-Chavez and Van Gelder, 2007; Helfrich-

Forster et al., 2002). The light we used had irradiance values between 380-420 nm that were below the range necessary for CRY-dependent biological responses; our light had a total irradiance of 1.27 x 1010 photons s-1 cm-2 nm-1 between wavelengths 380 nm and 420 nm (Fig.

1.1 c). These irradiance values are significantly lower than the required irradiance threshold for

CRY function. Butterflies in our trials could therefore not use magnetic field cues for orientation, as their magnetic sense would not have been activated.

The gravitaxis experiments were separated into five tests, with each test using the same apparatus and methodology for running the trials. These tests were as follows: (i) negative gravitaxis, (ii) positive gravitaxis response, (iii) eliciting gravitaxis (incline response), (iv) dynamic gravitaxis response, and (iv) phototaxis and gravitaxis.

(i) Negative Gravitaxis Assay

We determined if monarchs had a negative gravitaxis response by testing if they moved up against gravity, i.e., in the opposite direction of the gravity vector. In these trials gravity was the only cue available for orientation, as other directional cues were either controlled (light) or made unavailable (magnetic field). Two sets of trials were conducted: (1) the apparatus was perpendicular to the ground (vertical trial) and (2) the apparatus was horizontal with the ground

(horizontal trial). Individual monarch butterflies from both fall and summer were tested in both trials to compare the results of a migratory population against a non-migratory population. The experiment was also repeated in both fall of 2017 and fall of 2018 to test for repeatability from year to year.

(ii) Positive Gravitaxis Assay

To test for a positive gravitaxis response, a design similar to the negative gravitaxis assay was used. The first trial was the same as the above, with individuals tested in the same vertical orientation, with individuals starting at the bottom of the apparatus. The second trial rotated the vertical tube upside-down, which effectively rotated the gravitational cue in relation to the apparatus and had the animals start at the top. Animals tested were from a different subset of migratory monarchs from fall 2018 in this experiment.

(iii) Eliciting Gravitaxis Assay

To determine the tilt angle at which the gravitaxis response is observed in monarchs, the same assay was run at different tilt inclinations of the tube. The initial trial was exactly like the horizontal trials from the negative gravitaxis assay and will be referred to as the 0° trial. After the initial trial, the apparatus was angled upward at 15-degree increments. The trials continued until the gravitaxis response was observed for all individuals. From these trials, we calculated the median angle at which all monarchs began oriented walking towards the end of the tube maze, i.e., oriented walking behavior towards the end of the tube maze as observed in our negative gravitaxis assay. We also calculated the 95% confidence interval that contained all the observed angles at which monarchs exhibited oriented walking.

(iv) Dynamic Gravitaxis Response

To test if the gravitaxis response was able to switch directions with a changing substrate

(i.e., test if monarchs were able to adjust their response if the vector of gravity was changed during the trial), we rotated the apparatus 180° while the butterflies exhibited their negative gravitaxis behavior. A modified tube, with no starting area or opening on either side, was used so that both ends were identical. The light was the same used in the other gravitaxis trials and was

9 positioned 61 cm and parallel to the tube, to ensure equal illumination of the trial area. Once the butterflies reached the halfway point in the tube, the apparatus was manually rotated clockwise at the midpoint of the apparatus. The movement of the monarchs (i.e., either up or down the tube after rotation) was recorded until they reached one of the tube’s ends. Once the butterflies reached one of the ends, the tube was rotated to reset the monarch’s position back to the bottom of the apparatus. This process was repeated an additional two times once the monarch reached the midpoint, resulting in three total rotations while monarchs exhibited their negative gravitaxis response. The tube was 60 cm long and flipped three times to provide the monarch approximately the same total available distance to move as with the longer negative gravitaxis apparatus. The tube was rotated at a pace slow enough so as not to startle the butterflies during trials, yet fast enough to complete the rotation before monarchs moved a significant distance

(about a body length) in either direction (15° s-1). Monarchs from both fall and summer of 2019 were tested to compare migratory and non-migratory populations.

(iv) Phototaxis and Gravitaxis

To test if the butterflies can use information from both gravity and light when exhibiting oriented upward walking in our study as seen in our negative gravitaxis trials, the position of the light source was manipulated to be perpendicular to the apparatus: either above or below it. Fall migratory monarch butterflies were initially tested in the vertical tube with light on the side. This trial would serve as the baseline negative gravitaxis response to later be compared to the trials that involved light manipulation. After the baseline trial, monarch butterflies were split into two groups randomly: (1) light cue below the apparatus (phototaxis against gravitaxis) and (2) light cue above the apparatus (phototaxis with gravitaxis).

Data Analysis

For each of the 4 experiments, ‘maximum distance reached’, ‘total distance walked’, and

‘time to complete trial’, did not meet assumptions of normality and some lacked variance. We therefore analyzed all variables using non-parametric statistical tests. For single comparisons of maximum distance reached, total distance walked, and maze completion time, a paired two-sided

Wilcoxon signed-rank test was used (α = 0.05). For multiple pairwise comparisons, a Friedman-

Conover test was used with an adjusted p-value based on the methods outlined for a False

Discovery Rate (FDR) with an alpha-value set at 0.05 (Benjamini & Hochberg, 1993). Data were analyzed using the ‘stats’ and ‘PMCMR’ packages on R.

RESULTS

Negative Gravitaxis

Consistent with negative gravitaxis, all three populations of monarchs walked significantly closer to the end of the maze in the vertical trial than in the horizontal trial

(Wilcoxon signed-rank; fall 2017: n = 20, V = 190, p < 0.001; fall 2018: n = 21, V =190, p <

0.001; summer 2018: n = 21, V = 231, p < 0.001; Fig. 1.2a). Monarchs from fall 2017 and summer 2018 walked the same total distance between their vertical and horizontal trial

(Wilcoxon signed-rank; fall 2017: n=20, V = 157, p = 0.053; summer 2018: n=21, V = 157, p =

0.154). Butterflies from fall 2018 walked significantly more in the horizontal trial than in the vertical trial (n = 21, V = 43, p = 0.0123). Median distances and comparisons for all generations are summarized in Table 1.1.

Positive Gravitaxis

Monarchs did not express positive gravitaxis, as fall of 2018 monarchs walked significantly farther toward the end of the maze during the vertical trial than during the upside-

11 down trial (Wilcoxon signed-rank; n=21, V = 231, p < 0.001; Fig. 1.2b left panel). Animals walked more within the apparatus when they started the trial at the bottom (Wilcoxon signed- rank; n = 21, V = 231, p < 0.001; Fig. 2b right panel). Median distances for both trials are summarized in Table 1.1.

In these trials, starting at the top of the apparatus, the butterflies remained active, but did not directionally move downward. In fact, many individuals in the positive gravitaxis trial failed to leave the starting area. These specific individuals were still counted because they remained active (e.g., continuously walking horizontally in the starting area) during the entire trial.

Animals that remained active but that did not advance from the starting area further down the tube maze in these trials received a value of 0 cm for both the total distance walked and the maximum distance reached in the tube maze.

Initiation of Negative Gravitaxis

Monarchs required a threshold angle for their negative gravitaxis to activate. The maximum distance reached, toward the end of the maze, was significantly different between the inclines ranging from 0°- 60° (Friedman-Conover; X2 = 18.64, df = 4, p < 0.001; Fig. 1.3a). 0° had the lowest median distance reached, with 15°- 45° having intermediate distances, and 60° having the largest median. The total distance walked within the apparatus was significantly different based on the incline (Friedman-Conover; X2 = 15.89, df = 4, p = 0.003; Fig. 1.3b).

Median total distance walked within the apparatus significantly decreased after reaching the 30° incline and remained the same even at 60°. The multiple pairwise comparisons for both the maximum distance reached and the total distances walked are summarized in Table 1.2 with corresponding “FDR” adjusted p-values.

Dynamic Gravitaxis Response

Monarchs’ negative gravitaxis was found to adjust with a shifting substrate. After rotations, monarchs walked significantly farther upward (against the pull of gravity) for both fall and summer populations (Wilcoxon signed-rank; fall 2019: V = 55, p = 0.002; summer 2019: V

= 55, p = 0.002). Monarchs from fall 2019 had a median positive distance of 81.48 cm and a median negative distance of 8.67 cm. Fall 2019 butterflies had an average positive distance of

77.42 cm +/- 4.73 cm and an average negative distance of 7.52 cm +/- 2.15 cm. Monarchs from summer 2019 had a median positive distance of 113.43 cm and a median negative distance of

7.63 cm. Summer 2019 butterflies had an average positive distance of 106.73 cm +/- 7.72 cm and an average negative distance of 6.72 cm +/- 1.64 cm. Once the monarchs’ head was pointed upward, after the tube had completed the rotation, the negative gravitaxis behavior was observed.

Phototaxis and Gravitaxis

Light modulated the negative gravitaxis response of monarchs. Monarchs reached the same maximum distance (the end) when the light was on the side and when the light was above the apparatus (Wilcoxon signed-rank; V = 10, p = 0.1003; Fig. 1.4a left panel). Monarchs walked a greater total distance, regardless of direction, when the light was on the side, compared to when the light was above the apparatus (Wilcoxon signed-rank; V = 21 p = 0.036; Fig. 1.4a right panel). Monarchs completed the maze in the same amount of time when light was above and to the side (Wilcoxon signed-rank; V = 10, p = 0.1). Monarchs walked the same distance toward the end of the maze when the light was to the side or below the apparatus (Wilcoxon signed-rank; V

= 3, p = 1; Fig. 1.4b left panel). Monarchs walked the same total distance, regardless of direction, when the light was on the side and underneath (Wilcoxon signed-rank; V = 12, p = 0.28; Fig.

1.4b right panel). Monarchs completed the maze in the same time when light was below and to the side (Wilcoxon signed-rank; V = 11, p = 0.36).

DISCUSSION:

In our study, we found that monarchs have a clear behavioral distinction when they are provided with the opportunity and space to move up or down (Fig. 1.2a). Monarchs in these trials were not able to sense the earth’s magnetic field because of the lack of adequate irradiance levels for UV-A and UV-B wavelengths in our trials. These wavelengths are needed at a sufficient level in order to activate light-based magnetoreception in monarchs (Guerra et al., 2014) and other insects (fruit flies; Gegear et al. 200), and there were no directional light cues toward the end of the apparatus that could elicit positive phototaxis. We conclude that monarchs only respond with negative gravitaxis in response to gravity, at least within our study. It is still possible that monarchs might exhibit positive gravitaxis under other contexts, e.g., use gravity as a directional cue for landing when flying during the day. For example, pea aphids can exhibit positive gravitaxis, a behavior that might help with host finding (Zhang et al., 2016), and the single-celled alga Euglena gracilis has been shown to exhibit both negative and positive gravitaxis based on solar radiation levels (Ozasa et al., 2019). This response changed from the non-oriented walking phenotype (back and forth movement and not reaching the end of the maze) found in the horizontal trial, to directed (moving toward the end of the maze and reaching the end) once the tube was at a 30° incline (Fig. 1.3), which suggested an incline threshold is necessary to activate the behavior. The dynamic gravitaxis response trials showed that monarchs were monitoring their body position in relation to gravity. Once the tube was rotated and the monarchs were heading downward, they righted their body upwards to the vertical position, and resumed their negative gravitaxis response. Finally, we found that monarch positive phototaxis modulates their

14 negative gravitaxis response when both cues can attract the butterfly upward (Fig. 1.4a). When the light was above the butterfly, they completed the maze with less total distance walked as compared to when lights were in other positions during trials.

Gravitaxis Response

Monarchs consistently reached the end of the maze when the tube was vertically upright, but not when the tube was completely horizontal (Fig. 1.2a). This directional movement was found for each of the seasons and years tested, which suggests that the migratory syndrome does not determine this directional behavioral response, unlike oriented flight (reviewed in Reppert and de Roode, 2018). The positive gravitaxis trials did not result in individuals directionally moving downward (Fig. 1.2b). These individuals underwent trials where they could display either positive (upside down tube) or negative (vertical tube) gravitaxis, and all subjects moved directionally upward.

The additional comparisons of the total distance walked in the apparatus showed that the motivation of monarchs when tested did not influence the distance reached toward the end of the maze. Horizontal trials had the same total distance for two of the generations tested and for one of the generations tested had a greater distance when compared to their vertical trial. This is indicative that the motivation to walk in our animals is the same between the two tube orientations and that the maximum distance reached and the probability to reach the end of the maze are not related to motivation, but instead result from negative gravitaxis behavior.

One possible ecological explanation for negative gravitaxis relates to flight take-off. An initial hurdle in flight kinematics is the ability to generate enough acceleration to initiate flight.

Butterflies (Blimberd et al., 2013), other insects (Burrows et al., 2019), and birds (Heppner &

Anderson, 1984) have been shown to overcome this initial hurdle by using a coordinate initial leap followed by wing flapping. It is possible that the monarch is looking to increase their physical elevation prior to this initial leap; in fact, it was observed repeatedly that butterflies initiated flight upon successfully reaching the end of the maze. Both migratory and non- migratory generations could utilize this technique for initiating flight by jumping from a higher location.

Another ecological explanation for negative gravitaxis could be related to thermoregulation. Monarchs have been observed to utilize the sun to warm their flight muscles prior to flying (McCord and Davis, 2010). By walking upward on a substrate, monarchs could reach higher locations with less cover to bask in the sun. This behavior would be especially helpful during colder months when monarchs undertake their migratory journey and during early morning when temperatures are typically lower.

Initiation of Negative Gravitaxis

The total distance walked and the maximum distance reached by the butterflies in the trials both switched from the non-oriented movement observed at 0° and 15° (greater total distance walked and lower maximum distance reached) to directed at 30° (lower total distance walked and higher maximum distance reached). As the angle increased, gravity’s pull would be directed toward the bottom of the apparatus, causing the butterflies to resist more as the pull toward the bottom increased. At 30°, a threshold was reached, which activated the negative gravitaxis behavior and caused the organism to direct their movement up and toward the end of the maze. This threshold hypothesis has been supported in the taxis responses of other animals, such as D. melanogaster. It has been found that the directed movement of D. melanogaster

16 toward a light source (positive phototaxis) was based on light level (Hecht and Wald, 1934).

Lower light levels were unable to elicit responses, while higher levels activated their positive phototaxis. Gravity’s pull would be equal regardless of the orientation of the apparatus, however, the direction of this pull on the monarch (in relation to the apparatus itself) would change. As the apparatus angled upward, the gravity vector’s pull toward the bottom of the apparatus would increase. The stick insect, Carausius morosus, has been shown to measure their body orientation in relation to the gravity vector using thorax-coxa joint hair plates and antennae (Bässler, 1971).

Monarchs could be utilizing their own hair plates and antennae to determine their body orientation with respect to gravity while in the incline apparatus (see below). Once that orientation reached the 30° threshold, the monarch negative gravitaxis response was elicited.

Dynamic Gravitaxis Response

The gravitaxis response was found to adjust to a shifting substrate. As the monarchs walked up, the tube was rotated so that they were suddenly walking downward. In response to the rotation, the monarchs righted themselves to have a head up position and continued walking upward. After each rotation there was some distance moved toward the bottom, resulting in a median distance moved downward of about 8 cm (n=15) for both fall and summer. This downward movement suggests that monarchs might have a lag for the behavioral response after sensing that their body position relative to the direction of the gravity vector has changed. It is unknown if this lag is the result of a delay in expressing the negative gravitaxis response or if it suggests that monarchs have a lag in processing the sensory information.

Phototaxis and Gravitaxis

When the light’s position was changed to be vertically above the apparatus (positive phototaxis drive was up and the negative gravitaxis drive was up) there was a significant reduction in the total distance walked prior to reaching the end of the maze compared to when the light was on the side. Butterflies in both trials took the same amount of time to complete the maze, meaning trials with light cues on the side moved up and down at a higher pace than trials with light cues from above. When light and gravity were aligned, individuals traveled slower, yet more directionally upward than in trials with the light on the side during which butterflies traveled faster over a greater distance (both up and down). Based on this information, phototaxis and gravitaxis have an additive effect on directionality when the cues drive oriented movement in the same direction. This is consistent for what is predicted for a day active animal, that throughout most of the day, the animal would have both gravity and light aligned in this manner.

When the light was opposite the gravity cue (positive phototaxis drive was down and the negative gravitaxis drive was up) the monarchs had no alterations in behavior. They reached the end of the maze and accomplished this with the same total distance traveled and time; thus, the cues can be additive, but not subtractive, and indicates gravity is the primary cue for determining up and down in the butterflies. This particular hierarchy has been found in Euglena gracilis, with their negative gravitaxis response able to be enhanced and even reversed with powerful enough light cues (Ozasa et al., 2019). This response to multiple cues at the same time has broad implications in how organisms react in natural habitats and future work isolating the ecological drives of gravitaxis.

Gravity and Magnetism

Wiltschko and Wiltschko (1972) suggested that the magnetic inclination angle compass birds utilize during migration requires gravity as a reference point. Gravity provides a cue that

18 can help determine up and down and the horizontal plane because it is the plane perpendicular to the vector of gravity. We found that monarchs respond to the earth’s gravitational pull and that this vector could be the reference point used to measure the magnetic inclination angle. With the rotating tube trial, we found that gravity is used to determine body orientation, and our incline trials showed that gravity can be used to determine body tilt in relation to the horizontal. By consistently knowing the direction of gravity when in different positions in 3D space, monarchs could use this information to measure the angle that the magnetic field vector intersects with the vector of gravity.

Mechanism

There are several possible mechanisms for gravity perception in invertebrates and monarchs specifically. The three likely gravity-sensing mechanisms in monarchs are the campaniform sensilla (CS), the Johnston organ (JO), and the hair plates (Bohm’s Bristles) (Dey et al. 1995; Sant and Sane, 2018). The CS is a mechanoreceptor imbedded in the cuticles near the attachment points of the appendages (Dey et al. 1995). These mechanoreceptors are capable of sensing straining forces on the cuticle placed on it by the associated appendages, effectively being able to sense the weight on the individual appendages (Zill et al.,2012). Hair plates at the base of these appendages provide information about joint angle (Tuthill and Wilson, 2016), which may provide key information about the direction of gravity in relation to these appendages. The CS and hair plates have been linked to gravity perception in walking D. melanogaster, allowing them to resist the pull of gravity on a substrate (Mendes et al., 2014). It has also been found that Carausius morosus can use their thorax-coxa hair plates to determine the magnitude of their orientation in relation to gravity (Bässler, 1972). This mechanism was

19 most likely utilized in the inclination trials and dynamic gravitaxis trials given that these assays also focused on substrate-based body positioning.

The antennae are ideal gravity sensors given their ability to be deflected in response to the pull of gravity. The JO is a group of specialized scolopidia cells located in the pedicel of the antenna of insects (Kristensen, 1981). It has been linked to the perception of several environmental cues including sound, vibration, wind deflection, and gravity (Kamikouchi et al.,

2009; Yorozu et al., 2009). D. melanogaster, Formica rufa, and Aedes aegypti have been found to utilize this structure for gravity perception (Armstrong et al., 2006; Vowles, 1954; Boo and

Richards, 1975). In D. melanogaster, the JO has been shown to have isolated neural circuits and specialized channels in their scolopidia cells that activate based on different frequencies allowing the structure to be used for varying sensory stimuli (Armstrong et al., 2006; Toma et al., 2002;

Matsuo and Kamikouchi, 2013). Lepidoptera use the JO for the perception of mechanical deflections from wind currents and use this information for flight stabilization (Sane et al.,

2007). This requires the JO to be activated in response to high frequency, small-amplitude deflections (Sane et al., 2007). It is unknown if this specialization precludes the ability to use the

JO for gravity perception or as a multimodal sensory organ. However, the JO’s ability to respond to a multitude of deflection frequencies based on different sources of mechanical deflection makes it a likely candidate for gravity perception.

The hair plates at the base of the antennae (Bohm’s bristles) provide general antennal position information in Lepidoptera (Sant and Sane, 2018). They also are important in determining the position of antennae due to mechanical deflections (Krishnan et al., 2012). Like the other hair plates’ role in gravity sensation, Bohm’s bristles could be used to sense the direction of the deflection of the entire antennae due to the pull of gravity, and use this

20 information to determine antennal position. This system could be used in isolating the direction of gravity. In conjunction with the JO, Bohm’s bristles might be a mechanism Lepidoptera additionally rely on for gravity perception. Future studies should focus on isolating both appendage and antennae-based mechanisms to sense gravity and their individual role in gravity- based responses in monarchs.

ACKNOWLEDGEMENTS:

We thank Jered Nathan, Adam Parlin, Sam Stratton, and Michael Soellner for assistance with collecting monarchs for trials. We thank Molly Albright, Michael Paddock, and Hannah

Dawson for assistance with husbandry. A. Parlin helped with R-script and figure construction.

M. Paddock helped in inclination experiment trials and apparatus construction.

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FIGURES:

Figure 1.1: General tube maze assay and corresponding light wavelengths used in each experiment. A) Diagram of the tube maze used in each experimental trial. The maze consisted of a wire mesh frame that is 10 cm in diameter. Trial conditions either oriented the tube or the light differently in relation to gravity. B) Full light spectrum (nm) of the light used in each trial.

C) Focused light spectrum for UVB and UVA wavelengths for trial conditions. The irradiance range for values between 380 and 420 nm in our trials fall below those needed to activate the

27 magnetic sense in monarchs (Guerra et al., 2014) and other insects (fruit flies; Gegear et al.,

2008).

Figure 1.2: Monarch butterflies respond to gravity with negative gravitaxis and not positive gravitaxis. A) Results of negative gravitaxis assay from fall of 2017 and fall and summer of 2018. Median distance reached toward the end of maze graphed as solid black squares with 95% confidence intervals. Wilcoxon signed rank test shows significant difference between both trials for each generation. B) Results of positive gravitaxis assay from fall of 2018.

Left panel shows median distance reached with 95% confidence intervals. Right panel shows median distance walked within trial area.

Figure 1.3: Monarchs display negative gravitaxis behavior starting at a 30° incline. Black boxes represent medians with 95% confidence intervals for each angle. Significant groupings based on post-hoc p-value adjustments using the False Discovery Rate with alpha at 0.05.

Groups with different letters are statistically different from each other.

Figure 1.4: Light affects gravitaxis response only when it comes from above the organism.

Black squares represent the median with 95% C. I. for the respective trials. Grey dashed line on left panels is the distance needed to get to the end of the maze. Left panels are the median of the maximum distance reached and right panels are the median of the total distance walked within the apparatus. A) Results from the light above trials. The total distance needed to walk to the end of the maze is significantly less when the light is above the apparatus. B) Results from the light below trials. Light below apparatus shown to have no effect on either maximum distance reached or total distance walked.

CHAPTER TWO

Multimodal Sensory Integration for Executing Oriented Movement in the Monarch

Butterfly, Danaus plexippus (Lepidoptera, Nymphalidae)

Mitchell J. Kendzel and Patrick A. Guerra*

Department of Biological Sciences

University of Cincinnati

Cincinnati, OH, 45221 USA

*Mitchell J. Kendzel principally composed the work presented here with the assistance of Patrick

A. Guerra. The manuscript version of this chapter is formatted for submission to the Journal of

Experimental Biology

ABSTRACT:

How animals move in response to their environment is crucial for determining which environmental conditions are important for facilitating those movements. Gravity is an important environmental cue since it has been present for the entirety of evolutionary history on this planet and organisms moving across land and air must respond to it accordingly. In this study, we used the monarch butterfly, Danaus plexippus, as a model system to study how an organism uses gravity as a cue for orientation and how that cue is integrated with other environmental cues for directed movement. We used a righting response (from head down to head up) on a vertical substrate to determine how monarchs orient using cues that provide directionality. We found that monarchs can execute this righting behavior using gravity as an orientation cue. In particular, when no other cues that could provide directional information were available, monarchs oriented their bodies upward using gravity as a cue. When presented light cues, we found that monarch positive phototaxis mediated the response to the gravity cues, resulting in the alteration in the righting response with final vertical orientations deflected toward the light sources. This deflection toward the light was only found when light cues were presented perpendicular to gravity along the plane of rotation. When light cues were below the animal (effectively making cues causing positive phototaxis to be against the direction of the righting behavior) monarchs ignored the phototaxis cue and oriented upward as normal. The antennae were found to be important in the righting response, since without their antennae, monarchs did not exhibit the vertical righting response, but instead performed positive phototaxis towards the light source.

Without light or antennae, monarchs were able to rotate as normal, suggesting monarchs possess another gravity sensing mechanism outside the antennae, but this mechanism is unable to override their positive phototaxis response when light cues are present. The addition of the

34 magnetic field also deflected the monarch vertical righting response, resulting in bearings consistent with inclination-based orientation.

INTRODUCTION:

Natural environments are dynamic systems in which a multitude of sensory cues are available simultaneously to individuals. The ability to derive information from multiple sources and integrate this information has been shown to be important in animal migration (reviewed in

Muheim et al., 2006), finding mates (Griffith and Ejima, 2009), and predator avoidance

(Stynoski and Noble, 2012). Previous work has found that some cues, when presented together, can have their information combined, resulting in a new behavioral output that is not observed when either cue is produced in isolation (Krstic et al., 2009). Additionally, it has been observed that cues can drive contradictory behavioral responses, forcing animals to either compromise between cues or choose between them (Lewis et al., 2015). The way organisms integrate this sometime-conflicting information helps illuminate the programming of the behavioral decision- making processes of animals.

The integration of different sensory stimuli has the advantage of taking information from multiple cues to get a better understanding of the environment, compared to when utilizing only one cue in isolation (Wessnitzer and Webb, 2006). This combination of information can cause organisms to re-calibrate their response to a cue by using the information from another. Birds have been found to integrate the information from the polarized light on the horizon to re- calibrate and respond appropriately to the information from their magnetic sense (reviewed by

Muheim et al., 2006). Monarchs also may use information from other sensory sources in order to use their compasses for directionality during oriented movement, e.g., flight during the fall migration (Reppert and de Roode, 2018).

The monarch butterfly is a useful study system to investigate the effect sensory integration has on orientation given their sensory modalities and the amount of previous research on their orientation behavior. Eastern North American monarchs undergo a complex multi- generational migration every year that allows them to escape the seasonal deterioration of their northern habitat ranges (reviewed in Reppert and de Roode, 2018). Fall monarchs orient and direct their flight southwards toward overwintering locations in Mexico (reviewed in Reppert and de Roode, 2018) using a time-compensated sun compass (Perez et al., 1997; Mouritsen and

Frost, 2002; Froy et al., 2003) as their dominant compass sense. When cloud cover obscures the sun cue, fall monarchs have been shown to maintain the proper southwards flight orientation by using a backup light-dependent magnetic inclination angle compass (Guerra et al., 2014).

The inclination angle of the magnetic field is the angle that the magnetic field vector intersects with the horizontal plane. It would thus be important to accurately determine the horizontal plane to orient with this aspect of the magnetic field. It has been suggested birds utilize the vector of gravity to determine the position of the horizontal plane and to measure the inclination angle based on this information (Wiltschko and Wiltschko, 1972). This would suggest information from both gravity and the magnetic field are integrated so that migratory birds can utilize their magnetic compass. How gravity and magnetic field components integrate is important in understanding how this magnetic compass functions. To date, there has not been a study directly testing if gravity and the magnetic field are integrated or modulate behavioral responses when presented together.

Under natural conditions, the primary light cue for a diurnal animal such as the monarch is the sun. The monarch has been shown to orient in the horizontal plane using the sun (Perez et al., 1997; Mouritsen and Frost, 2002; Froy et al., 2003) and its location above the organism

36 during the day can provide up and down information. However, it is unknown which cue (gravity or light) is more important for determining the vertical axis or if information from both is used.

How monarchs orient when provided both light and gravity, especially when information from these cues is contradictory, can help establish how the animal uses the two cues and which is more important to determine the vertical plane.

Animals can self-right their bodies to re-orient from poor positioning to proper positioning for locomotion (Camhi, 1977; Frantsevich and Mokrushov, 1980). Failure to right to the proper position can have fitness costs and can end in death (Delmas et al., 2007). Monarchs will alter their position on a vertical substrate, by orienting their head and body to have an upright position and will adjust their body position when rotated and are placed upside-down.

The switching of head down to head up is essential for successful emergence of adult monarchs from their chrysalises and requires the accurate identification of up for proper rotation. How these animals are determining up for this behavior, in which individuals can use either light, gravity, the vertical component of the magnetic field, or some combination of these cues, is unknown. In this study, we used a righting response assay to isolate the orientation of monarchs on a vertical plane using directional cues. By focusing on how monarchs take directional information from gravity, light, and the magnetic field, we can isolate the importance of individual, combined, and contradicting environment cues and how these are manifested behaviorally.

In this study, we first isolated gravity’s role in the righting response behavior of monarchs by omitting light cues and preventing magnetoreception. We then compared the effect light had on the righting response by either setting the available light cues to be opposite of the direction of the gravity cue or had the light cues along the horizontal plane perpendicular to

37 gravity. Next, we isolated morphological sensory structures that are potentially important for the righting response and how that mechanism could be manipulated by the presence or absence of light. We tested the antennae as potential gravisensors in monarchs, as the antennae were found to be important in gravity perception in D. melanogaster and C. morosus (Armstrong, 2006;

Bässler, 1971). Finally, we tested how the magnetic field influenced the gravity based righting response to see if both cues were important for righting. The magnetic field intensity and vertical component of the magnetic field have been found to be important in vertical based orientation in other systems (fruit flies, Bae et al., 2016; salmon, Putman et al., 2018).

METHODS:

Experimental Organisms

Adult monarch butterflies were collected at the University of Cincinnati Center for Field

Studies (Harrison, Ohio 39.285293, -84.741566) and surrounding fields in the fall and summer of 2018 and 2019. Butterflies were placed in individual glassine envelopes and stored in an incubator (Percival model I-36LL). Fall butterfly populations were stored under fall like conditions and had a 12-hour light: dark cycle (12L:12D); the light cycle temperature was 21°C and night temperature was 12°C with a constant 70% relative humidity (RH). Summer butterfly populations were stored under summer like conditions and had a 14.5-hour light: dark cycle

(14.5L: 9.5D); the light temperature was 29°C and dark temperature was 18°C with a constant

70% RH. Both populations were fed a 25% honey solution every other day. Individuals were acclimated for two weeks under laboratory conditions to standardize photoperiod and behavior

(Mouritsen and Frost, 2002; Reppert et al., 2004; Guerra et al., 2014).

Righting Response Assay

The orientation assay consisted of a black cardboard box (dimensions: 68.6 x 33 x 36.8 cm; Fig. 2.1a). One side of the box was open, either to the left, right, or underneath the organism.

This opening served as the illumination point and allowed for the manipulation of where the light and visual cues were located. A mesh wall (dimensions: 36.8 x 33 cm; Fig. 2.1b), was placed in the center of the black box, perpendicular to the ground. This served as the plane of movement for the butterflies and allowed for full 360° rotations in the vertical plane (Fig. 2.1b). A rubber clamp was threaded through the back wall, behind the monarch butterflies, and was used to manually hold the monarch in position via the wings, prior to the start of a trial. The rubber clamp was able to be opened and closed without the need to reach inside the box. Once the trial commenced, the rubber clamp was removed from behind the wall and the organism could move and rotate freely.

Animals were placed in a mesh cage for 10 minutes outside the testing room to allow free movement before the trial. After the 10 minutes, the monarch butterflies were clamped within the apparatus and held for 1 minute in 1 of 2 possible orientations: (1) head pointed up, or (2) head pointed down. After the clamp was removed, animals were given 5 minutes to select an orientation: either walking a bodies length away from their starting position or holding an orientation for 5 minutes. Orientations were measured using the video recording system (I DVR-

PRO; CCTV Camera Pros) and IMAGEJ program (Schneider et al., 2012). The reference point for measurements put 0° at “up” and 180° at “down”.

Light Conditions for Magnetic Sense

UV-A and UV-B wavelengths have been shown to be necessary to activate magnetoception in insects that possess a magnetic sense (monarch butterflies, Guerra et al.,

2014; fruit flies, Gegear et al., 2008). The irradiance range required to activate light-dependent

39 cryptochrome-based magnetic sensing must reach 1011 photons s-1 cm-2 nm-1 between 380 nm and 420 nm (VanVickle-Chavez and Van Gelder, 2007; Helfrich-Forster et al., 2002). For trials where we wanted to prevent monarchs from using the magnetic field for orientation, we used a light that had negligible amounts of ultraviolet-A (UV-A) and ultraviolet-B (UV-B) wavelengths

(light source measurements at the location of the butterfly during a trial: peak at 580 nm; range:

300-700 nm; total irradiance: 8.74 x 1012 photons s-1 cm-2 nm-1; Fig. 2.1 c). The light we used had irradiance values between 380-420 nm that were below the range necessary for CRY- dependent biological responses; our light had a total irradiance of 1.27 x 1010 photons s-1 cm-2 nm-1 between wavelengths 380 nm and 420 nm (Fig. 2.1 d). For trials where responses to the magnetic field were allowed, we used a light with adequate irradiance levels at the relevant UV-

A and UV-B wavelengths (total irradiance from 380-420 nm was 7.02 x 1013 photons s-1 cm-2 nm-1; Fig 2.2 c & d).

Trial light conditions were measured using a spectrometer (Ocean Optics Inc., Dunedin,

FL, USA) and an optic fiber (QP230-1-XSR, 235 microns; Ocean Optics Inc.) with cosine corrector (CC-3-UV-S; Ocean Optics Inc.) at the trial position within the apparatus.

Spectrographs were generated using the ‘pavo’ R-package to convert the values from radiance to photon flux. Magnetic conditions were measured using an Applied Physics Systems tri-axial fluxgate magnetometer (model 520A) at the head position of the butterflies during trials. Using these values, the inclination angle and total field intensity were calculated for each trial condition.

Orientation experiments were separated into five explicit tests that asked separate questions. First, we focused on determining if monarchs had a righting response when the only directionally available cue was gravity. With the default righting response defined, we assayed

40 the effect on orientation when both gravity and light cues were present. Next, we isolated the location of the mechanism responsible for both the default orientation and the orientation with light and gravity cues. Finally, we assayed the effect on the righting behavior when both gravity and magnetic sense were available to orient with.

Defining Monarch Righting Behavior

To determine how monarchs right themselves using the gravity vector alone and prepare for further trials, an initial, or baseline, experiment was used to measure the righting response without the use of light or magnetic cues. Each butterfly went through three trials: (1) light on - from the left – starting position head down to serve as a control, (2) light off - starting position head up, and (3) light off - starting position head down. Point 1 was done as a control, just in case the gravity response and orientation behavior were light dependent. Points 2 and 3 were utilized to determine if starting position affected final orientation and together provide the baseline orientation of monarchs using only gravity.

Trials were conducted in complete darkness. Observations were made with an infrared camera system (HD-Q28; IR Bullet Camera). Prior to the trial, butterflies were held in an acclimation cage under darkness for 1 hour. This hour ensured enough time for the organisms to acclimate to the dark trial conditions. Fall and summer monarch butterflies from 2019 were tested to compare migratory vs. non-migratory populations.

Orientation with Gravity and Light

To test whether information from gravity and light were used together during righting behavior, two trials with three different light orientations were conducted. In each trial, the light equally illuminated the box, was set along the axis of rotation, and lacked adequate irradiance at

UV-A and UV-B wavelengths. Three light positions relative to the butterfly were used: left, right, or underneath. This effectively gave two trials where the light was perpendicular (left and right) and one opposite (light underneath) to the gravity cue. Butterflies were tested with both starting positions (head up and head down) for each of the three light positions, resulting in six trials per individual. Trial order was randomized using a random number generator to eliminate order effects. Monarch butterflies from both fall and summer of 2018 were used to compare resulting orientations between migratory and non-migratory generations.

After initial testing, an additional light orientation was added to test how animals responded when the light source was no longer in the plane of rotation. This additional trial placed the light behind the organism, effectively making the light cue both perpendicular to the wall and the axis of rotation. A new subset of individuals from fall of 2018 were tested in three trials: (1) light behind with starting position head up, (2) light behind with starting position head down, and (3) light on the right with starting position head down. The light on the right trial served as an internal control for each individual and a comparison trial.

Mechanism for Gravity Orientation

In order to isolate the mechanism that allowed the butterflies to accurately orient in response to the gravity vector, trials were conducted after surgically removing a likely candidate structure (the antennae), given their ability to deflect in response to gravity. For each trial, the light position was controlled and set to the left of the organism with their starting position facing head-down. This provided two trials per organism, an initial baseline response trial and a second post-surgery or sham operation trial. After creating a pool of animals that had completed their initial trial, a random number generator split the pool into two groups: one whose antennae were surgically removed and another group who underwent a sham operation. Surgeries were

42 conducted under a compound microscope. Micro-forceps were used to hold the antennae in place, while micro-scissors were used to remove the antennae in its entirety, where the joint connects with the head. Silicon grease (Danco: Plumbing Repair) was used to seal the wound and prevent the organism from desiccating. The second trial was conducted the following day of surgery or sham, giving animals a minimum of 24 hours to recover from the stress of surgery.

Both fall and summer monarch butterflies from 2019 were tested to compare migratory versus non-migratory populations.

Orientation Without Light Cues or Primary Gravity Cue

Based on results from the previous experiment, we tested how monarchs orient when their primary gravity sensor (antennae) was removed along with light cues. A pool of animals was tested, initially in the dark with a head-down starting position. The pool was then randomly split into two groups, one set who had a sham operation and another group that had their antennae removed. Surgeries were conducted in the same way as described above. After 24 hours from the sham or surgery, individuals were re-tested in the dark with a starting position of head- down. Both fall and summer monarch butterflies from 2019 were tested to compare migratory vs. non-migratory populations.

Orientation with Gravity and Magnetic Cues

To test if monarchs use gravity and magnetic cues together during righting behavior on a vertical plane, a modified orientation box was constructed to allow for the manipulation of the magnetic field surrounding the trial (Fig. 2.2a). The black box apparatus for this set of trials

(dimensions, L x W x H: 41 x 31 x 21 cm) was nested within a Helmoholz coil system. The coil system consisted of two coils arranged orthogonally, with each coil powered by its own power

43 supply, and was set in accordance with previous work (Guerra et al., 2014). The horizontal coil allowed for the magnetic north and south to be aligned with the wall and axis of rotation, so magnetic south (mS) was on the left side and magnetic north (mN) on the right side (Fig. 2.2b).

The vertical coil allowed for manipulation of the vertical component of the magnetic field. An opening above the apparatus allowed the illumination of the trial by a full-spectrum light source that ran through a diffuser. This diffuse light source possessed wavelengths between 380-420 nm at the necessary irradiance that is necessary for the active response to the magnetic field in monarchs and other insects (Fig. 2.2c and 2.2d) (Guerra et al., 2014; Gegear, et al. 2008).

Three different artificially generated magnetic stimuli were tested: the ambient magnetic field at the testing location (University of Cincinnati, Cincinnati, Ohio: 39.133940, -84.517072) based on the World Magnetic Model (WMM), double the magnetic field strength for both vertical and horizontal components, and the ambient field but with vertical component inverted

180°. Monarch butterflies were acclimated to each of the trial magnetic fields in a mesh cage contained within a separate Helmholz coil system, with the same light source, for 1-h in accordance with previous work (Guerra et al., 2014). Fall migratory monarch butterflies from

2019 were used to ensure the organisms possessed the ability to respond to the magnetic field.

An additional experimental test was done to compare orientations under no magnetic inclination angle. Fall migratory monarchs were tested using two magnetic fields: an ambient field (as described above) and a field possessing no vertical component. A field with no vertical component resulted in an inclination angle of 0°, generated by setting the vertical coil to the equal and opposite strength as the natural ambient field.

Data Analysis

Significant mean orientations were calculated using a Rayleigh’s uniformity test. For multiple parametric pair-wise comparisons between trial groups, we used Hotelling's Paired Test

(Zar, 1999) with adjusted p-values calculated using the False Discovery Rate (FDR) method

(Benjamini & Hochberg, 1993). For antennae manipulation experiments, non-parametric analyses were used given the low sample sizes of these experiments and their departure from the

Von Mises distribution. This included the Moore's paired sample second order test and a modified Rayleigh’s test with an a priori mean angle called the V-test given their increased statistical strength with low sample sizes (Landler et al., 2018). Mean orientations were calculated using the R package ‘circular,’ adjusted p-values were calculated using the R package

‘stats,’ and comparisons between trials were calculated using the program Oriana 4 (Kovach

Computing Services).

RESULTS:

Preferred Gravity Orientation in Monarchs

Fall and summer butterfly righting responses had significant mean orientations in the dark (Rayleigh’s test; p<0.0001 for all trials; Table 2.1). The starting position, i.e., the butterfly’s initial head position, resulted in the same orientation in the dark for both seasons (Hotelling’s paired test; fall 2019: n = 15, F = 1.91, p = 0.19; summer 2019: n = 15, F = 0.89, p = 0.43).

For both seasons, organisms’ dark trials had an orientation upward (Fig. 2.3 a & b right panels). The trials in the light had a significant deflection toward the light source resulting in a difference between the two trials (Hotelling’s paired test; fall 2019: n = 15, F = 17.3, p < 0.001; summer 2019: n = 15, F = 6.17, p = 0.013; Fig. 2.3).

Gravity and Light

For fall of 2018, each of the six trials had a significant mean orientation (Rayleigh’s test; p < 0.001 for all trials; Table 2.1). Monarch orientations were not affected by the starting position given that there was no significant difference between the head-up and head-down trials for all three light positions (Hotelling’s Paired Test; left: n = 21, f = 2.817 pfdr = 0.12; right: n =

21, f = 2.591, pfdr = 0.12; underneath: n = 21, f = 2.425, pfdr = 0.12). Light position affected final orientation, causing an orientation shift toward the light source and a significant difference between the three light positions (Hotelling’s paired test; p < 0.001 for all; Fig 2.4a). This pull shifted both light on the left and right trials away from 0.

When light was presented behind the organism (i.e., not on the plane of rotation), monarchs oriented upward as normal regardless of starting position (Hotelling’s paired test; n =

10, f = 0.68, p = 0.53; Rayleigh’s test; p <0.001 for all trials; Table 2.1). The final head-up orientations of butterflies when the light was behind them were not deflected from the vertical as when the light was placed laterally to the butterfly (Hotelling’s paired test; light on right vs. light behind: n = 10, f = 7.6, p = 0.01).

For summer of 2018, each of the six trials had significant mean orientations (Rayleigh’s test; p < 0.0001 for all trials; Table 2.1). For all three light positions, orientations were not affected by starting position resulting in no significant difference in mean angle between the head up and head down trials (Hotelling’s paired test; left: n = 21, f = 1.7 pfdr = 0.167; right: n =

21, f = 3.0, pfdr = 0.111; underneath: n = 21, f = 4.23, pfdr = 0.09;). This season also saw an effect of light on orientation, causing the same deflection as seen in fall, based on the location of light

(Hotelling’s paired test; p<0.0001 between light on the left vs. right). Again, when the light was positioned on the left and right, butterflies oriented away from 0 and slightly toward the

46 phototaxis cue (Fig. 2.4b). However, light underneath was not significantly different than light on the left (Hotelling’s paired test; p=0.12).

Mechanism for Gravity Orientation: Antennae

For both fall and summer, all pre-operation trials had significant mean orientations (V-

Test a priori angle 0°; p<0.05 for all; Table 2.2). Sham trials had no effect on orientation for the fall population (Moore’s paired test; n=5, R’ = 0.62, 0.5 > p > 0.1; Fig. 2.5a left panel). Sham trials were significantly different than baseline trials for the summer population (Moore’s paired test; left panel; n=5, R’ = 1.32, p < 0.001; Fig. 2.5b). However, clustering had overlap and was in line with previous results when the light was on the left.

Antennae manipulations had significant changes in mean orientations for both fall and summer (Moore’s paired test; fall 2019: R’ = 1.33, p < 0.001; summer 2019: R’ = 1.32, p <

0.001). Antennae-less butterflies shifted their orientation toward the light more than butterflies with antennae intact (Fig. 2.5 a & b right panels). Antennae-less butterflies were not significantly clustered with an a priori mean orientation set to 0° (V-test; fall 2019: n = 5, r = 0.25, p = 0.23; summer 2019: n = 5, r = 0.0023, p = 0.5; Table 2.2). Antennae-less butterflies had a significant mean orientation when the a priori angle was set to the light’s location at 270° (V-test; fall 2019: r = 0.94 n = 5, p < 0.05; summer 2019: n = 5, r = 0.91, p < 0.05; Table 2.2).

Orientation Without Light and Antennae - Fall and Summer of 2019

Fall and summer butterflies’ initial baseline trial in the dark had a significant mean orientation for both fall and summer (Rayleigh’s test; fall 2019: n=10, r = 0.94, p < 0.0001; summer 2019: n=10, r = 0.86, p = 0.0001; Fig. 2.6 a & b left panels). Post antennae removal trials had significant mean orientation (V-Test a priori 0°, p < 0.05 for all trials, Table 2.2).

Sham trials had no effect on orientation and were not significantly different than the baseline trials (Moore’s paired test; fall 2019: n = 5, R’ = 0.62, 0.5 > p > 0.1; summer 2019: n = 5, R’ =

0.01, p > 0.999; Fig. 2.6 a & b left panels). Butterflies from both seasons were still able to rotate toward up after removal of their antennae and this trial was not significantly different than their initial baseline trial (Moore’s paired test; fall 2019: n = 5, R’ = 0.62, 0.5 > p > 0.1; summer 2019: n = 5, R’ = 0.20, p > 0.9; Fig. 2.6 a & b right panels).

Gravity and Magnetic Integration

All three magnetic trial conditions resulted in significant mean orientations (Rayleigh’s test; Ambient: n = 10, r = 0.904, p < 0.0001; Double: n = 10, r = 0.73, p = 0.0026; Inverted

Vertical Component: n = 10, r = 0.96, p < 0.0001; Fig. 2.7a). Both the ambient and double magnetic field trials resulted in the same mean orientation (Hotelling’s paired test; f = 1.0, pfdr =

0.41). Butterflies in these trials oriented more toward the left side of the apparatus and were significantly different than the inverted vertical component trial which oriented more toward the right side (Hotelling’s paired test; Ambient vs. Inverted: f = 6.3, pfdr = 0.03; Double vs. Inverted: f = 8.3, pfdr = 0.03). Orientations of butterflies in ambient and double magnetic fields shifted equatorward (left side of the apparatus) and orientations of the inverted vertical component trial shifted toward the right side of the apparatus (Fig. 2.7a).

The no inclination experiment found significant mean orientations for the ambient and zero inclination angle trial (Rayleigh’s test; Ambient: n = 6, r = 0.919, p = 0.002; Zero

Inclination Angle: n = 6, r = 0.975, p <0.0001). These trials were significantly different with ambient trials shifted toward the left and zero inclination trials not shifted to either side (Moore’s paired test; R’ = 1.39, p < 0.001; Fig. 2.7 b).

DISCUSSION:

Monarch butterflies exhibit righting behavior, by which they aim to maintain a head up orientation. We found that this behavior was facilitated via the use of gravity as a directional cue.

This occurred in the absence of other cues that could be utilized for directionality (Fig. 2.3 a & b right panels). Information from light cues were shown to integrate with the gravity cue, modulating monarch righting behavior when the light was placed perpendicular to the butterfly, but no modulation was observed when light was below the apparatus (Fig. 2.4) or when placed behind. Antennae were found to be important in the normal righting response; once antennae were removed, monarch orientation was consistent with positive phototaxis to the light cue present during trials (Fig. 2.5). Monarchs were still able to right themselves when gravity was the only cue available, even without antennae, suggesting there is another mechanism used for sensing gravity to facilitate the righting behavior (Fig. 2.6). Finally, aspects from the magnetic field were shown to modulate the monarch righting response and served as a cue for body positioning. Fall migratory monarchs shifted their orientation equatorward when presented with ambient fields, a directional response consistent with their migratory status and the use of an inclination-based magnetic compass for orienting towards their overwintering sites in Mexico

(Fig. 2.7).

Orientation via Gravity

Without any other directional cues besides gravity, monarchs orient with their head pointed directly upward on the vertical plane. We therefore can determine that this is their preferred orientation using gravity and this orientation can be reached without any other directionally based cues. A likely explanation for this orientation behavior could be the preparation for a negative gravitaxis response, a directed movement upward and against the pull

49 of gravity observed in other insects and monarchs (Armstrong et al., 2006; Kendzel and Guerra, unpublished).

Gravity and Light Integration

The head up monarch orientation on the vertical plane, which appears to be the preferred position of monarchs, can be influenced by various environmental cues, one of the greatest influencers being light. During the gravity and light trials, when the light source was from the left or right, monarchs showed a clear orientation skew in both head down and head up trials, in which monarchs had a final head up position that was then turned towards the light when light could be localized from either the right or left (Fig. 2.4). Monarchs, like many other insects, display positive phototaxis (Jander, 1963). This light-dependent behavior in insects has been shown to be influenced by the spectral composition and intensity of the light source (van

Grunsven et al., 2014; Sun et al., 2014). This photo-driven orientation exhibited an integration with the gravity driven orientation that resulted in a net shift of about 10 - 15° toward the light source (Fig. 2.4). When the light cue contradicted the gravity cue (phototaxis down while gravity up), the monarchs ignored the light cue in favor of the gravity cue. Thus, gravity appears to be the primary orientation driver on the vertical plane with light being secondary, as indicated by the shift (towards light) or lack of effect (light below) in the righting response of monarchs. If both types of sensory cues were weighted equally, monarchs would have been expected to have a shift of 45° relative to the vertical when the light was on either side, or a random orientation when the two cues were in conflict.

Light and gravity cues can both possess directional information, and the directed movements they elicit have been found to modulate each other’s responses in several taxa (fruit flies, Kwon et al., 2016; alga, Daiker et al, 2011). In Euglena gracilis, results have found that

50 their gravity and light driven responses can be equal, light favored, or gravity favored depending on environmental conditions and intensity (Daiker et al., 2011; Ozasa et al., 2019). In D. melanogaster, vertical based light orientations required gravity to activate. Both these cues can possess information about up and down, here we show that gravity forms the basis of monarch vertical orientation with light being able to modulate the response.

Gravity and Magnetic Integration

After providing the necessary light wavelengths to activate the magnetic sense in monarchs and other insects (Guerra et al., 2014; Gegear, et al. 2008) the orientation of monarchs on the vertical plane in our trials was shifted. When tested under artificially generated magnetic fields with either ambient field strength or twice the ambient field, the righting response of monarchs was shifted equatorward from the vertical position. This shift is consistent with the equatorward flight orientation of migrant monarchs in magnetic compass trials conducted in a flight simulator (Guerra et al. 2014). When tested under conditions in which the inclination angle was reversed during trials, monarchs adjusted their righting behavior to have their shift from the vertical toward the right side of the apparatus. This confirmed that monarchs are responding to the magnetic field, as they adjusted their righting response to also orient with the altered magnetic field conditions (Fig. 2.7a). In fact, the monarchs’ righting response was not altered when butterflies had no inclination angle information available (zero inclination angle trial), this confirms that monarchs are responding to the inclination angle of the magnetic field for orientation as observed during migratory flight (Guerra et al., 2014). There was also a significant difference between ambient, zero inclination angle, and inverted inclination angle fields (Fig.

2.7b). These results show that information from gravity and the magnetic field are integrated, resulting in a significant shift in the orientation of monarchs on the vertical plane.

Orientation responses due to inclination angle manipulations supports the radical pair mechanism hypothesis for magnetoreception whereby the magnetic field is sensed through the creation of molecules with varying spin states in their unpaired valence electrons (Schulten et al.,

1978). The inclination angle can affect the proportion of spin states while opposite polarities create equal spin states of equal proportion (reviewed in Zhang et al., 2015; Rodgers et al.,

2009;). This mechanism allows for the detection of changes in inclination and not polarity. No significant difference was found between ambient and double ambient fields, and it is unclear as to why. It is possible that our results suggest that the monarch is either unable to respond to the field strength at these levels, that the information that field strength provides is ignored in this context, or that both field strengths elicit the same response. Magnetic field strength has been shown to be key in vertical plane movement in young salmon (Putman et al., 2018), suggesting a possible difference in the mechanism used in monarchs.

For bird, insect, and sea turtle systems, an aspect of the magnetic field being sensed and oriented to is the inclination angle (Schwarze et al., 2016; Guerra et al., 2014; Lohmann and

Lohmann, 1994). It is likely that animals measure this inclination angle based on a reference point or have an environmental cue that helps determine the horizontal plane consistently. In birds, it has been suggested that they utilize the earth’s gravitational vector as a reference point to measure this inclination angle (Wiltschko and Wiltschko, 1972). Birds have been shown to respond to earth’s gravitational pull to orient their body (Sadovnichii et al., 2008), but there has not been a study showing both stimuli integrating in a single behavior. In this study, we demonstrated that information from both the magnetic field and gravity can be used together to inform orientation and body positioning. This offers valuable information to determine how these animals are measuring the inclination angle. Within the monarch system specifically, our

52 research supports that monarchs orient their body using the earth’s gravitational vector, and this vector could serve as the information these animals need to interpret the earth’s magnetic inclination angle.

Gravity Orientation Mechanism

After removal of the monarch antennae, there was a clear orientation shift away from 0° and toward the light in the monarch righting response (Fig. 2.5 a & b right panels). This suggests that intact antennae are vital in orienting toward the preferred upward direction. The organs in the antennae that are potentially responsible for this default orientation are the Johnston’s organ

(JO) and the Bohm’s bristles (BB). The JO is an organ within the antennae that has been found to be significant in gravity perception in several species from diverse insect taxa (fruit fly,

Armstrong et al., 2006, ants, Vowles, 1954; mosquitos, Boo and Richards, 1975). In D. melanogaster, this structure is used for sensing multiple cues through specialized cells and neural circuits (Armstrong et al., 2006; Sun et al., 2009). In Lepidoptera, the JO is capable of sensing high frequency, small-amplitude deflections for flight stabilization purposes (Sane et al., 2007).

It is unknown if this specialization precludes the use of the JO to sense gravity or as a multimodal sensory structure. The BB are hair plates located at the base of the antennae; these hair plates provide gross localization information about where the antennae are in relation to the head of the butterfly (Sant and Sane, 2018). By knowing the position of and in what direction they are being deflected, monarchs can utilize this system to make measurements of the direction of the gravity vector. This suggests that the antennae are primarily used for determining up and down via gravity. After removing both these structures, the monarchs no longer utilized gravity, but instead used the light cue for orientation. This caused an orientation directly towards the light

53 cue (positive phototaxis) that was perpendicular to the vertical plane and adjacent to the apparatus.

When we removed the antennae along with light cues, monarchs oriented upward again and toward the default orientation at 0° (Fig. 2.6 a & b right panels). This suggests monarchs have a secondary mechanism to sense gravity’s direction. A possible candidate is campaniform sensilla (CS) and hair plates (HP) located at the thorax-coxa joint (Bässler, 1971). CS are specialized mechanoreceptors imbedded in the appendages of insects capable of determining the forces put on said appendages (Dey et al., 1995) and the HP are important for proprioception

(Tuthill and Wilson, 2016). The integration from both sensory organs has been linked to body orientation of a vertical plane in stick insects (Bässler, 1971). By knowing the weight (CS) and location of limbs (HP), monarchs could be utilizing this same information to determine their orientation in respect to gravity.

We provided evidence that antennae are important in responding to gravity in monarchs.

They have also been found to be important in perceiving the earth’s magnetic field (Guerra et al,

2014). The magnetic trials in this paper showed that the antennae are a multimodal sensory organ, in which cues from different sensory modalities are sensed, and then likely sent and integrated downstream, e.g., the central complex (Heinze and Homberg, 2007). How these senses are integrated can reveal how the magnetic compass in monarchs works and what neural circuits are important in perceiving the cues necessary for that compass to be utilized for oriented flight.

ACKNOWLEDGEMENTS

We thank Jered Nathan, Adam Parlin, Sam Stratton, and Michael Soellner for their assistance with the collection and rearing of monarchs. We thank A. Parlin and S. Stratton for

54 assistance with statistical analyses and figure construction. We also thank Hannah Dawson,

Michael Paddock, and Briana Thompson for their assistance with monarch husbandry.

REFERENCES: Armstrong, J. D., Texada, M. J., Munjaal, R., Baker, D. A., and Beckingham, K. M. (2006).