Do Bats use Olfactory Cues to Find Roosts?

Thesis

Presented in Partial Fulfillment of the Requirements for the Degree Master of Science in

the Graduate School of The Ohio State University

By

Bridget Kay Gladden Brown

Graduate Program in Evolution, Ecology, and Organismal Biology

The Ohio State University

2020

Thesis Committee

Gerald G. Carter, Advisor

Rachelle M. M. Adams

Ian M. Hamilton

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Copyrighted by

Bridget Kay Gladden Brown

2020

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Abstract

Understanding how bats select roosts is crucial to their management and conservation. One way that bats can locate new roosts is by using cues from conspecifics.

Research on this use of social information has mostly focused on conspecific calls. In many species, bats will move toward conspecific echolocation and social calls, which can help bats find new roosts faster. However, many roosts also have olfactory cues from guano and urine. Bat conservationists have long debated whether guano and urine can be used to attract bats to new roosts. If chemical cues can act as an effective bat lure, this would aid in efforts to exclude bats from buildings and attract them to artificial roosts in protected areas like state parks or wildlife preserves. In my thesis, I ask if bats use olfactory cues from guano and urine to locate potential roosts. In Chapter 1, I summarized research to date on the topic of the use of olfaction by roost-searching bats and I discuss reasons that bats might, or might not, use scent to locate roosts. From this research, results are consistent with the hypothesis that guano and urine are not strong enough lures to attract bats to new roosts. In Chapter 2, I describe experiments with three bat species given choices between a roosting area treated with guano and urine and an untreated control roosting area in captivity and in the field. This chapter describes experiments done by me (field experiments 1 and 2, captive experiments 4 and 6) and by other researchers (captive experiments 1, 2, 3, and 5). In field experiment 1 and 2, I used

ii acoustic-based bat detectors to measure bat visits and nearby bat activity at paired treated and untreated artificial roosts for one week deployments at 16 sites in Panama (13 weeks) targeting velvety free-tailed bats (Molossus molossus) and at 7 sites in Ohio (17 weeks) targeting big brown bats (Eptesicus fuscus). Only one visit was detected, and the activity did not show an effect of the treatment. In the captive experiments, I tested vampire bats

(Desmodus rotundus) using paired roost tubes (n=5 bats), using surfaces with different improved test procedures (n=20, n =22, n =33), and using a y-maze that compared their scent response to that of sound cues or the combination of sound and scent cues. I also tested captive velvety free-tailed bats instead (n=18). The overall effect size of scent cues on roost selection by captive bats was near zero. Ultimately, this indicates that guano and urine are not a strong enough lure to consistently draw bats into a roosting area.

Key Words: bats; multimodal; replication; roosting ecology; sensory ecology

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Dedication

I would like to dedicate my thesis to my husband, Tyler Brown, who provided

unconditional support and countless edits to my work during my thesis.

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Acknowledgments

First and foremost, I would like to express my sincere gratitude to my adviser, Dr.

Gerald Carter, for his guidance and support during my master’s thesis. His knowledge and enthusiasm were a crucial resource for my master’s research. I would also like to thank the rest of my thesis committee, Dr. Ian Hamilton and Dr. Rachelle Adams, for their useful advice and constructive criticism.

Besides my committee, I would also like to thank my field assistants, Jessica

Nystrom, Jennifer Stancourt, and Morgan Little, for carrying out the field experiment in

Ohio while I was in Panama for the summer. The preliminary experiments conducted that aided in the creation of my experiments were also done by other research interns. I would like to thank Lauren Leffer, Yesenia Valverde, Nia Toshkova for their contributions.

Part of my project required the procurement of bat guano and knowledge of roosting sites, so I would like to thank the following people for their assistants with these aspects: The Ohio Wildlife Center, Columbus and Franklin County Metro Parks Staff

(particularly Carrie Morrow), Lisa Cooper at NEOMED, and Jenna Kohles in Panama.

While in Panama, I received assistance with mist-netting, resource acquisition and transportation from the Rachel Page Lab at the Smithsonian Tropical Research Institute. I would like to extend my appreciation to the following people for being an unparalleled

v resource while I was there: Dr. Rachel Page, Gregg Cohen, May Dixon, Alexis Heckley,

Camila Santiago, and Sebastian Stockmaier.

I also thank my fellow labmates at Ohio State, Imran Razik, Simon Ripperger,

David Girbino, and Emma Kline, for their assistance in modifying ideas and plans, editing proposals and papers, and extra hands in the field.

Finally, I would like to thank the organizations that provided financial support for me during my thesis: Sigma Xi Grants-in Aid of Research and Ohio State’s Critical

Difference for Women Professional Development Grant.

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Vita

Personal Information

May 2012……………………………………………………..…Beavercreek High School

December 2015…………………..…B.S. Organismal Biology, The Ohio State University

2016-2018………………………………………………..…...…..Bat Survey Coordinator,

Ohio Division of Wildlife

2019-present……………………………….Graduate Teaching Associate, Department of

Evolution, Ecology, and Organismal Biology,

The Ohio State University

Fields of Study

Major Field: Evolution, Ecology, and Organismal Biology

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Table of Contents

Abstract ...... ii Dedication ...... iv Acknowledgments...... v Vita ...... vii List of Figures ...... x Chapter 1. Do Bats Use Scent to Find or Select Roosts? A Literature Review ...... 1 INTRODUCTION ...... 1 FUTURE RESEARCH AND FURTHER QUESTIONS ...... 14 Chapter 2. The Role of Scent Cues in Roost-Finding by Bats...... 17 ABSTRACT ...... 17 INTRODUCTION ...... 18 METHODS ...... 22 Ethics Statement...... 22 Panama Field Experiment ...... 22 Ohio Field Experiment ...... 25 Field experiment statistical analysis ...... 26 Captive Experiment One: Preliminary trials of bats choosing refuges (n=5 bats) ... 27 Captive Experiment Two: Zoo vampire bats (n=20) choosing sides ...... 28 Captive Experiment Three: Wild-born and captive-born vampire bats (n=22) choosing sides ...... 29 Captive Experiment Four: Wild-born vampire bats (n=33) choosing sides ...... 30 Captive Experiment Five: A y-maze with acoustic cues, scent cues, and multimodal cues (n=45) ...... 32 Captive Experiment Six: Velvety free-tailed bats (n=18) choosing sides ...... 34 Captive experiment statistical analysis ...... 35 Comparing scent, sound and bimodal treatments in Captive Experiment Five ...... 35 RESULTS AND DISCUSSION ...... 36 Field Experiments ...... 36

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Captive Experiments ...... 38 CONCLUSION ...... 43 Bibliography ...... 46 Appendix A: Maps of Roost Locations for Field Experiments ...... 55

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List of Figures

Figure 2.1. Paired test boxes used in the field experiments...... 25 Figure 2.2. Test chambers for Captive Experiment One and Captive Experiment Two and Three...... 29 Figure 2.3. Test chamber for Captive Experiment Four...... 31 Figure 2.4. Test chamber for Captive Experiment Five...... 34 Figure 2.5. No increase in bat activity on playback nights...... 38 Figure 2.6. Captive experiments did not detect a consistent or strong effect of scent on roost selection...... 39 Figure 2.7. Isolated vampire bats tested in a y-maze showed a clear attraction towards the roost sound cue but not to the roost scent cue...... 42 Figure A.1. Ohio field experiment locations…………………………………………….55 Figure A.2. Panama field experiment locations………………………………………….56

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Chapter 1. Do Bats Use Scent to Find or Select Roosts? A Literature Review

INTRODUCTION

Habitat selection is a critical process in the lives of all animals as the choice in habitat can lead to greater fitness through more access to resources or mates. If animals choose habitat that facilitates social relations, this can lead to increased protection from predators or more information about food resources (Rubenstein 1978). Certain social cues can aid in this decision-making process. Studying these cues can aid in understanding the maintenance of social groups, which in turn leads to more knowledge about how to potentially conserve these species through reducing interference with said cues. For example, numerous species use communal latrines to mark territories (Delahay et al. 2001; Irwin et al. 2004; Qamar et al. 2008), for information exchange like signaling reproductive availability (Ikeda 1984; Irwin et al. 2004; Dröscher and Kappeler 2014;

Rodgers et al. 2015), or for resource defense (Irwin et al. 2004; Dröscher and Kappeler

2014). Avoiding the destruction of these locations could help to conserve these organisms. For bats, roosts provide a place for females to raise young that is protected from weather and predators (Kunz and Fenton 2003). These roosts are fairly limited as each species requires certain characteristics, such as intact interior forest, certain temperatures, and proximity to water, for an ideal roost (Kerth 2008; Jung and Threlfall

2015). When selecting a roost, group-living bats often consider social cues such as the

1 calls or scents of conspecifics that can indicate the viability of a roost (Kerth 2008).

Research on how bats use social cues in roost selection has predominately focused on the acoustic cues of echolocation calls or social calls (Chaverri et al. 2018). For example,

Spix’s disc-winged bats (Thyroptera tricolor) roost as small groups in large tubular, unfurled leaves and the group changes roosts frequently, sometimes daily (Chaverri et al.

2010). To coordinate the selection of a new leaf roost, bats in search of a roost produce a distinct inquiry call, and a bat inside a roost makes a response call (Chaverri et al. 2010,

2012). Several other bat species produce stereotypical social calls when entering roost crevices, and conspecifics frequently respond to them (Wilkinson 1992; Schöner et al.

2010; Arnold and Wilkinson 2011; Furmankiewicz et al. 2011). Many bat species (e.g. common noctules bat, Nyctalus noctule; brown long-eared bat, Plecotus auritus; and

Daubenton’s bat, Myotis daubentonii) use echolocation calls from conspecifics to locate roosts (Ruczyński and Kalko 2007; Ruczyński et al. 2009). Far less is known about whether bats use social olfactory cues to locate new roosts.

I conducted a literature review of this topic, reading 81 papers on olfaction, sensory ecology, and roosting ecology in bats, and finding only six studies that tested the use of olfaction in roost selection by bats (Selvanayagam and Marimuthu 1984; Mueller

1966; Barclay et al. 1988; Ruczyński and Kalko 2007; Ruczyński et al. 2009, Brown et al. unpublished). Here, I discuss what is known or not known about the use of olfaction during roost-finding and roost selection in bats.

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Olfaction in bats

To understand the capacity of bats to use scent in roost selection, it is important to review the structures they rely upon for olfaction. In mammals, the key olfactory structures are the main olfactory system and the vomeronasal system, which can include the vomeronasal organ (VNO) and the accessory olfactory bulb (AOB). Greater complexity in the epithelial structures in the main olfactory system can increase the amount of surface area contact a chemical cue has, which in turn allows for greater absorption and information to be derived from the chemical cue (Hecker et al. 2019). The vomeronasal system appears to play a large part in the assessment of chemical cues that are often involved in social behaviors, such as mate attraction (Kelliher 2007).

Nocturnal mammals tend to have larger olfactory structures as they often rely more on scent and less on sight (Barton et al. 1995). However, compared to other mammals, most bats have reduced olfactory systems. Many bats have a reduced VNO,

AOB, or the loss of one or both of these two structures entirely (Hecker et al. 2019).

Despite the reduction in the VNO and AOB, bats have not had a loss of protein-coding genes that would indicate a loss of olfactory function, such as Trpc2 and V1r (Yohe et al.

2017, 2019; Hecker et al. 2019). It is therefore possible that the main olfactory system has developed to incorporate the functions of these accessory systems (Hecker et al.

2019), or that there is a misunderstanding of the purpose of those protein-coding genes

(Yohe et al. 2019). Diet is one factor that is thought to play an important role on the evolution of mammalian olfactory systems (Barton et al. 1995; Eiting et al. 2014). For example, in bats, the phyllostomids (leaf-nosed bats) have the most diverse diet among

3 the bat lineages (with species consuming insects, fruit, nectar, blood, fish, and small vertebrates), and this family has maintained a complex olfactory system, more similar to other mammals, while many other bat families have lost parts of these structures (Cooper and Bhatnagar 1976; Frahm and Bhatnagar 1980; Schmidt 1985; Yohe and Dávalos

2018). In contrast, the vespertillionid bats are primarily composed of insectivores that may not rely as much on scent while hunting, which could explain the loss of the VNO and AOB in at least six of these species (Cooper and Bhatnagar 1976; Bhatnagar and

Kallen 1974). Due to the strong apparent link between the vomeronasal system and the processing of social cues, those bats with more developed VNOs and AOBs might rely on olfaction more during social interactions (Hecker et al. 2019). However, social and ecological factors are difficult to disentangle for many species. For example, the common vampire bats (Desmodus rotundus) has one of the largest relative AOBs recorded in bats and a well-developed vomeronasal organ, and this species is exceptional in both social complexity (Carter and Leffer 2015) and the diversity of prey (hosts) that it can parasitize

(Carter et al. in press).

Many bat species frequently switch roosts, such that individual bats must locate new roosts multiple times throughout the roosting season. Any cue that allows a bat to reliably and efficiently assess roost quality could play a beneficial role in roost selection.

There are several lines of evidence suggesting that bats might use scent to find roosts.

Compared to acoustic cues or signals, odor cues persist much longer and can travel farther distances, especially in comparison to the limited range of high-frequency sounds in a cluttered forest environment (Martens 1980). Large bat roosts accumulate chemical

4 cues of bat urine, guano, and scent marks over years. There can even be other scents at the entrance of roosts, like the smell of corn tortillas near Mexican free-tailed bat roosts, which is noticeable to humans (French 2003). Therefore, bats that roost in interior forests, or other high clutter areas, might depend upon olfactory cues to travel to the general area of a roost and then use acoustic cues as a beacon to find the roost entrance once they are closer. While acoustic cues show the presence of bats in a roost at that moment, olfactory cues, like guano at the entrance of a roost, might be more important when there are no other social cues present. Examples of this include young bats that have dispersed into a new, unoccupied area and temperate bats making seasonal movements to summer sites that are currently vacant at the end of winter.

Scent at the entrance of roosts could also aid in social recognition if bats can discern conspecifics, groups, or individuals by scent. Most bats roost in groups, which can benefit each individual through social thermoregulation, predator avoidance (Lima and O’Keefe 2013), and information about foraging sites (Wilkinson 1992; Kerth and

Reckardt 2003). Many species, even those with frequent roost switching, preferentially roost with certain individuals (De Fanis and Jones 1995; Bouchard 2001; Bloss et al.

2002; Mann et al. 2011; Wilkinson et al. 2016), and maintaining these relationships can be particularly important in some cooperative species (Wilkinson 1984; Boughman and

Wilkinson 1998). At least some bat species can distinguish between familiar and unfamiliar individuals by scent alone (De Fanis and Jones 1995; Voigt and von Helversen

1999; Bloss et al. 2002; Safi and Kerth 2003). Chemicals collected from glands via swabs contain information about individual identity in the Angolan free-tailed bat (Mops

5 condylurus), the little free-tailed bat (Chaerephon pumilus), the common pipistrelle

(Pipistrellus pipistrellus), and the soprano pipistrelle (Pipistrellus pygmaeus) (Bouchard

2001; Bartonicka et al. 2010). Males of these species could also distinguish between the sex of the bat that was swabbed, and females showed a preference for the scent of familiar conspecifics, showing a function beyond mating. In several studies with big brown bats (Eptesicus fuscus), common pipistrelles (Pipistrellus pipistrellus), and

Mexican free-tailed bats (Tadarida brasiliensis), the scent of a bat was collected by either rubbing a cotton swab over the bat multiple times or placing a cloth in a container with the bat for a prolonged period of time; subject bats preferred the scents of familiar roost mates as opposed to unfamiliar conspecifics (Gustin and McCracken 1987; Loughry and

McCracken 1991; De Fanis and Jones 1995; Bloss et al. 2002; Englert and Greene 2009).

In Bechstein’s bat (Myotis bechstenii) and the greater bulldog bat (Noctilio leporinus), the chemical profiles among bats from one colony were found to be more similar than between different colonies (Brooke and Decker 1996; Safi and Kerth 2003). In the

Bechstein’s bat, the chemical profiles were distinct enough to correctly assign 97.8 ±

4.5% of individuals (n=53) to the correct colony (n=4) (Safi and Kerth 2003). These similarities were not driven by genetic similarity as the mean within-colony genetic relatedness in these species is only 0.02. In fact, the scent profiles between mothers and daughters were not consistently more similar than among other groupmates (Safi and

Kerth 2003).

At least some bats use scent-marking with glands at roost entrances (Nelson 1965;

Moulton 1967; Höller and Schmidt 1996; Grant and Bannack 1999; Caspers et al. 2009;

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Englert and Greene 2009). Often male bats have glands to advertise to potential mates or rival males (Haffner 1994; Bouchard 2001; Bartonicka et al. 2010). For example, males of the Pacific flying foxes (Pteropus tonganus), the black flying foxes (P. gouldii), the gray-headed flying foxes (P. poliocephalus), the greater sac-winged bats (Saccopteryx bilineata), the pale spear-nosed bats (Phyllostomus discolor) and the lesser sac-winged bats (S. leptura) mark their territory by rubbing glands on various surfaces (Nelson 1965;

Moulton 1967; Höller and Schmidt 1996; Grant and Bannack 1999; Caspers et al. 2009).

While scent-marking serves to increase mating opportunities, these same cues are also available as a general cue of an occupied roost or territory. In sum, social scent marks at the entrance of a roost, could convey if that roost was occupied by familiar groupmates or unfamiliar conspecifics, which, in turn could influence roost selection.

Even without scent-marking, many entrances to bat roosts have inadvertent chemical cues from accumulated deposits of guano and urine. Predators and dogs can both locate bat roosts using the scent of guano (Threlfall et al. 2013; Chambers et al.

2015). As a result, many conservation organizations have recommended putting guano on artificial roost boxes in an attempt to attract bats to them (Murphy 1993, Ober 2008). The belief that odors can attract bats has led to the sale of chemical lures based on distinct odors present in some bat roosts. However, there have not been any scientific studies showing if this is effective. Finally, even if bats do not possess specific traits for using olfaction in roost selection, individuals could learn to associate the scent of a roost with its rewards through mere operant conditioning.

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There are also several reasons why we might not expect bats to use scent to find roosts. First, the use of social cues in roost selection is expected for group-living bats, but not all bat species roost in groups. Second, while many bats in certain contexts, such as mating and foraging, use olfaction, it may not be a particularly useful cue in roost selection if the primary benefit of a roost is the presence of other bats at that moment.

Scent cues at roosts, unlike acoustic social cues, can be maintained at the roost far past the point of occupancy, so they do not convey the presence of other bats. Moreover, an empty but scented roost could indicate that the roost has since degraded in quality. Third, many bat species have strong site fidelity (Kerth 2008), returning to the same area year after year and using the same set of roosts. As a result, these bats can therefore rely on spatial memory rather than sensory cues. Fourth, scent cues would be most useful for bats that need to find new roosts, but the species that most frequently switch to new roosts will not occupy them long enough to leave large concentrations of olfactory cues, like guano and urine. Finally, as mentioned above, bats in general show a reduction in the mammalian olfactory systems, which implies less reliance on olfaction.

Only six studies have investigated how bats might use olfaction to locate and select new roosts. To test the importance of olfaction in the homing ability of bats,

Mueller (1966) covered the nares of 35 Myotis lucifugus with a glue-like substance and released 10 of these treated bats at a distance of 10 km and another 25 treated bats at a distance of 32 km. As a control group, he also released 25 untreated bats at each distance.

Bats were marked with acetate paint to distinguish them by treatment. At the distance of

10 km, eight of 10 treated bats returned, but only three still had their nares covered and 13

8 of 25 control bats returned. At the distance of 32 km, no bats returned with covered nares.

Six of 25 of the treated bats returned, but none of them still had their nares covered.

Sixteen out of the 25 control bats released from 32 km returned (Chi-square with Yates correction=11.636, p=0.0006467). This number was calculated by removing the treated bats that returned to the hibernacula and had removed their nare covers.

He repeated this study with another bat species, Myotis sodalis by releasing 100 bats with their nares covered and 100 control bats 32.2 km away from the hibernacula.

For the treated bats, 22 out of 100 returned to the hibernacula, but only four still had their nares covered, whereas 18 of the bats from the control group returned. The author did not analyze the data but argued that the study showed that bats could return to a hibernaculum without the use of scent. However, a simple contingency analysis suggests that bats with covered nares were less likely to return (Chi-square with Yates correction=6.1, p=0.01).

It is possible that bats are less likely to rely on olfaction when locating hibernacula than summer roosts because these bats travel long distances (up to 500 km) to return to the same hibernacula year after year (Norquay et al. 2013; Klüg-Baerwald et al.

2017), so they are likely to rely heavily on spatial memory. Homing bats might also use nonsocial scent cues. According to the well-supported olfactory navigation hypothesis

(Wallraff 2004), birds such as homing pigeons exploit natural odors across the landscape to aid their navigation (Papi et al. 1973; Wallraff 2004; Gagliardo et al. 2011). Compared to birds, little is known about the use of scent cues in homing in bats.

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Another study looked at the impact of removing scent cues on the roosting behavior of Schneider’s leaf-nosed bats (Hipposideris speoris) (Selvanayagam and

Marimuthu 1983). The authors observed that when 40 banded bats returned to their roost, the males often sniffed the substrate while relocating their position. To determine if bats used scent to locate their roosting positions, the researchers washed the roosting location and noted the behavior upon the bats’ return. Where the roosting location was not washed, it took bats an average of 7±3 min to locate it (12 observations from eight bats), but when the area was washed, it took bats between two and three hours to relocate roosts

(five observations from five bats). Further experiments with bats in captivity showed that both males and females were able to relocate roosts within 2 cm, seemingly through sniffing behaviors. Males used urine marks to advertise their territory once dominance ranks had been established. When urine from other males was placed in the roost site of a focal male, it would respond antagonistically upon its return and cover the mark with its own urine. Females did not mark roosting sites with their urine, but they sniffed at the roof of the cage, seemingly using scent to relocate their roosting position. In this species, scent seems to aid in maintaining territories. Given no mention of bats marking more at the edge of their territories, a strong indicator of territoriality in other mammals, it is likely that these scents serve more as a way to label one’s own area for reorientation

(Johnson 1973). To date, this is the only study to test whether bats can detect guano and urine as a social cue for roost selection. The Schneider’s leaf-nosed bats were able to detect their own urine and the urine of other individuals. Further studies measuring this

10 ability in other species could address this question through associative learning, habituation, or brain activity.

In another study, researchers tested individual silver-haired bats, Lasionycteris noctivagans (Barclay et al. 1988), in a flight cage with two roosts that were identical, except that one of them had a different conspecific bat placed in it for at least 24 hours before the trial. In 21 trials with 11 bats, bats selected the scented roost 12 times and the unscented roost 9 times. The authors stated that this suggests olfaction is not “vital” to roost location in this species and that they probably use information from vision or echolocation to find roosts. An attraction to roosts with unfamiliar scent cues would not be expected anyhow because silver-haired bats do not normally roost with conspecifics.

Two studies looked at the sensory basis for selecting new roosts in three group- roosting species, the common noctule bat, brown long-eared bat, and Daubenton’s bat

(Ruczyński and Kalko 2007; Ruczyński et al. 2009). These studies tested the effect of various sensory modalities on the success of locating new roosts. Each bat was trained to find one of eight cavities that were drilled into a log. This training involved placing the bats into a room with a log that had cavities drilled into it and rewarding them with mealworms when they found the roost. Once a bat consistently found a roost within six minutes, it was included in the study. To create the scent cue, researchers placed a cloth into each bat’s cage for 24-48 hours and then added some of the bat’s feces. As a control, cloths without any scent were placed in the seven other cavities. Eight noctule bats, six long-eared bats, and nine Daubenton’s bats were each tested in eight trials. Bats did not locate roosts more often or faster with the use of olfactory cues, either when the bats

11 circled the log in flight to locate the roost or when they landed on the log and crawled around until they found a suitable roost. While the olfactory cues did not decrease search time for cavities, conspecific echolocation calls clearly did. In this study, the olfactory cue was from the focal individual. The scent of a conspecific would be a more accurate representation of a bat locating a new roost.

My master’s thesis aimed to investigate the use of conspecific cues in the location of new roosts in three social bat species through analysis of data from a series of eight experiments. Each experiment improved or built upon the experimental design of previous ones, and the data taken together represent a range of contexts in which bats might use scent to locate roosting sites. A preliminary experiment was done with five common vampire bats (Desmodus rotundus) placed in a cage with two roosting tubes that the bats could enter, one scented with guano and urine. This preliminary study suggested that bats chose scented roosts more often because bats selected the scented tube in nine of

10 trials. However, further experiments could not replicate this result. In three experiments (n=20, 22, 33 bats), we did not detect that vampire bat preferred to roost on a surface scented with guano and urine versus an unscented surface, and there was no evidence for attraction to guano and urine scent from 45 individual bats tested in a y- maze, although they were attracted to playbacks of roost sounds. We also failed to detect any preference for roosting at sites marked with guano and urine in 18 velvety free-tailed bats (Molossus molossus). Finally, we failed to detect an effect of guano and urine in two field experiments, one with velvety free-tailed bats in Panama and one with big brown bats (Eptesicus fuscus) in Ohio. In these studies, we used two pairs of identical

12 experimental roosts, each fitted with ultrasonic microphones to record any visits to the scented or unscented box. In Panama, the two pairs of roosts were moved between 16 sites from the middle of May to the middle of August, and in Ohio, the two pairs were moved between seven different sites from the middle of April to the middle of August.

Although many bats were recorded flying close to the roost, only one visit was recorded

(at an unscented box in Ohio). There was no observed effect of treatment on the amount of activity near the roost. These results are consistent with the hypothesis that these three species of bat do not rely on social olfactory cues in guano and urine to locate new roosts.

These studies also bring about the question of how and when bats select new roosts. In our field experiments, only one bat visited the new roosts to investigate it. The roosts were almost never inspected over a period of four months, and during a time when juveniles would be volant. Since the experimental roosts were placed near existing roosts, it is possible that the cues from the main roost were too potent and the incremental additional scent from the artificial roosts was not detectable. This might indicate that scent brings bats to the general area, but other cues guide the bat to the specific roost.

Also, bats could possibly search for roosts more often near foraging grounds than near currently existing roosts.

Taken together, what do these studies tell us? First, although bats clearly use scent in many contexts, the evidence suggests that the scent of guano and urine does not as an olfactory beacon or lure in the same way as echolocation and social calls. Compared to acoustic social cues, scent social cues may not be a sufficiently reliable cue of the immediate presence of conspecifics or the current viability of a roost. Second, there is

13 still a lot of uncertainty. Although many of the studies have found no evidence of the use of olfaction in roost selection, most of these studies were statistically underpowered and could also be considered inconclusive (i.e. absence of evidence is not evidence for no effect). Compared to sound, the production and perception of olfaction is more difficult to study.

FUTURE RESEARCH AND FURTHER QUESTIONS

Further study on use of olfaction in roost finding by bats is warranted and many questions remain. What social information is present in bat guano or urine? Rodents

(Bocskei et al. 1992; Hurst et al. 2001) and some carnivores (Hradeck 1985) convey social information, such as individual identity or genetic relatedness in their urine through major urinary proteins (MUP) (Beynon and Hurst 2004; Zhou and Rui 2010). For how long do these chemical cues persist? Do bats that are unintended receivers eavesdrop on scent marks to find, select, or avoid roosts? How does use of social scents differ among bat species and can this be predicted by their socioecology? Are lineages that use olfaction for foraging more likely to use social scents? If bats do use olfactory cues to locate roosts, how do these cues interact with other sensory cues at varying distances?

For example, bats might use sound to home in on the exact tree, but then they might use scent to find the roost entrance, similar to how bat mothers find their own pup in large colonies with ‘crèches’ of many hundreds of tightly packed pups (Gustin and McCracken

1987). If there is an olfactory cue and an auditory cue emanating from a roost, would it elicit a stronger response than either cue by itself, i.e. multimodal enhancement

(Rhebergen et al. 2015)?

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Studying scent cues could elucidate more of a bat’s life history than just scent location. Further information about the differences between chemical compounds produced by males and females could shed light on the use of olfactory cues in sexual selection. Far more is known about how insects use chemical cues and how it affects their behavior than for mammals. Part of the reason for this is likely due to the fact that researchers have been able to isolate the chemical compounds of a large variety of insects and concisely study their behavior (Ali and Morgan 1990). GCMS has been a key component of isolating chemical compounds and should be further used in bat studies.

Also, detailing of behavior is key to determine how animals react to certain chemicals.

Once the volatiles in a compound are identified, exposing the bat to different components and noting their behavior can be a big step towards determining the function of those chemicals. There also have to be trade-offs to producing pheromones. Studying certain characteristics that are a proxy for fitness, like body mass, before, during, and after pheromone production could illuminate these trade-offs (Johansson and Jones 2007).

Making use of these methods, and others used for studying mammalian olfaction, can increase the understanding of the use of olfaction in bats, particularly in a social context.

Beyond sensory ecology, learning more about if and how bats use olfactory cues to locate roosts could also be used in a conservation context. If a chemical lure was possible, it could attract bats to new or protected areas or roosts (like bat houses), and away from others (like buildings). This is particularly critical in light of continuous habitat destruction. Roosts for bats are a fairly limited resource, and they are becoming more limited as development around the globe increases (Kerth 2008). If biologists can

15 learn how and why bats choose roosts, they can also use this information to protect viable habitat. Certain questions need to be addressed to do this. We would need to know when bats are finding new roosts. This could be near the foraging grounds, close to their current roost, or somewhere in between. There are also questions about what time during the year bats are locating these roosts. For temperate species, is this done during the initial arousal from hibernation, while the females are pregnant, or when the young are starting to become volant? Also, what causes a bat to inspect a roost in the first place?

What features of that roost signal to the bat that it is viable? These are also all questions that need to be answered in regard to juvenile dispersal as well. Answering each of these questions will aid in better conserving bats, and the use of social cues is just a small component of this.

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Chapter 2. The Role of Scent Cues in Roost-Finding by Bats

ABSTRACT

Many species use social cues to find refuges (e.g. nests, roosts, dens). Bats are known to find roost sites by following other bats or by eavesdropping on their calls.

Given that the entrances of roosts are often marked by guano and urine, it is possible that bats also use these chemical cues to find and select roosts. We conducted two field experiments to see if these cues would attract free-ranging velvety free-tailed bats

(Molossus molossus) and big brown bats (Eptesicus fuscus) to new artificial roosts. We deployed two pairs of artificial roosts (scented and unscented) for 17 and 13 weeks in

Ohio and Panama, respectively. We also conducted a series of six captive experiments to determine if urine and guano could be an additional cue during roost choice in the common vampire bat (Desmodus rotundus), a group-living species which often lives in particularly odorous roosts and in the velvety free-tailed bat, a group-living species that roosts in crevices with entrances often marked with guano and urine. In field experiments, chemical cues did not lead to visits to artificial roosts and we did not detect differences in flights near the scented vs unscented roosts. In captive experiments, although we initially observed some preliminary evidence for attraction to roosts scented with guano and urine, we could not replicate this effect using more rigorous and controlled captive experiments. Vampire bats were immediately attracted to the playback of roost sounds, but not to roost scents or the combination of sounds and scents. Taken

17 together, these results are consistent with the hypothesis that the scent of guano and urine do not act as a lure during roost selection by bats.

INTRODUCTION

A key benefit for bats living in groups is that individuals can gain social information about resources, such as food or roosts (Kerth 2008). This social information is significant for a few main reasons. For one, there is often assumed to be a limited availability of roosts because most bats cannot make roosts and depend on structures like cavities (Kerth 2008). Information from conspecifics can aid in finding these limited but crucial resources. Also, many bats are social. They roost in large groups to provide thermoregulation and protection from predators as they raise their young (Kunz and

Fenton 2003). These groups can be fairly stable, with many species preferentially roosting with certain individuals. In many bat species, individuals alternate between multiple roosts possibly increasing the benefit of that information (Lewis 1995). For example, many tree-dwelling bats switch among a set of several trees (Wilkinson 1985;

Brigham et al. 1997; Willis and Brigham 2004; Popa-Lisseanu et al. 2008; Kerth et al.

2011). Also, disc-winged bats roost in young furled leaves and must find new roosts daily

(Chaverri 2010). To find unfamiliar roosts and to maintain group cohesion, bats largely rely on the echolocation and social calls produced by conspecifics (Ruczyński and Kalko

2007; Ruczyński et al. 2009; Chaverri 2010; Schöner et al. 2010; Furmankiewicz et al.

2011). However, it remains unclear to what extent, if any, bats find and select roosts using other cues, such as olfactory cues.

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Given that bats use scent cues for a variety of other tasks including the selection of mates (Chaverri et al. 2018), location of food (Theis et al. 2016), and discrimination of familiar individuals (De Fanis and Jones 1995; Voigt and von Helversen 1999; Bloss et al. 2002; Safi and Kerth 2003), olfaction might also play a role in roost selection.

Chemical cues can travel farther distances especially in cluttered forests and last for a longer period of time than other social cues such as high-frequency calls (Martens 1980).

A few previous studies have tested the role of scent marking (via glandular secretions) and an individual’s own guano in roost selection (Ruczyński and Kalko 2007; Englert and Greene 2009; Ruczyński et al. 2009), but another overlooked potential cue is the inevitable guano and urine stains from conspecifics that accumulate at occupied roosts.

Guano and urine attract predators to bat roosts (Threlfall et al. 2013) and can be detected by dogs (Chambers et al. 2015; Delpietro et al. 2017). Based on anecdotal evidence, some people report that guano stains are an effective means to attract bats to a bat house, but others argue that it does not have any affect (Murphy 1993, Ober 2014). If bats can be attracted to artificial roosts using a chemical lure, this could have important implications for conservation and management.

A baseline understanding of the use of these sensory cues is also critical to understanding how urbanization and anthropogenic factors might be affecting bats. For example, “sensory pollutants” like increased artificial light or interfering chemical cues from pesticides can mask existing cues or mislead bats by imitating certain cues

(Dominoni et al. 2020). These processes can interfere with habitat selection and social

19 interaction. However, understanding the use of sensory cues is crucial to assess the potential impacts of urbanization.

We ran a series of eight experiments to test whether bats are more likely to visit roosting sites when they are marked with guano and urine. In two of these experiments, we also presented playbacks of acoustic cues. This was done to directly compare the potential attraction of bats to chemical cues versus acoustic cues, and to test if the multimodal combination of cues would be a stronger lure than a unimodal cue (i.e. multimodal enhancement, Partan and Marler 2005).

The eight experiments included a field experiment with velvety free-tailed bats

(Molossus molossus), a field experiment with big brown bats (Eptesicus fuscus), and six lab experiments with captive common vampire bats (Desmodus rotundus). These were ideal species to use because all three species form stable social networks with preferred associations (Wilkinson 1990; Kilgour et al. 2013; Gager et al. 2016) and live within roosts that are often stained with guano and urine. The neotropical velvety-free tailed bat and the temperate big brown bats are both insectivores that roost in artificial structures including buildings making it more likely that they would visit our experimental roosts in the field experiments. The big brown bat regularly switches roosts at some locations

(Willis and Brigham 2004, whereas the velvety free-tailed bats form strong, stable social groups and can occupy the same roost for up to four years (Kohles unpublished data). A similar Molossid bat species (the Brazilian free-tailed bat, Tadarida brasiliensis) uses scent to locate and identify their pups, roostmates, and roosting sites (Gustin and

McCracken 1987, Englert and Greene 2009). Big brown bats also discriminate between

20 familiar and non-familiar conspecifics using scent (Bloss et al. 2002). The neotropical common vampire bat is attracted to the scent of prey (Bahlman and Kelt 2007) and are thought to use scent during social interactions to recognize individuals (Carter and

Wilkinson 2013). Vampire bats often live in roosts that are uniquely odorous because their diet of blood leads to copious urine and tar-like guano with a high ammonia content.

Their pungent roosts can be found at a distance by dogs or humans (Chambers et al.

2015; Delpietro et al. 2017).

Another way to assess the likely dependence on social olfactory cues is to investigate the accessory olfactory system. Most mammals have an accessory olfactory system composed of a vomeronasal organ and an accessory olfactory bulb. These systems work in addition to the main olfactory bulb to detect chemicals, but they primarily aid in the detection of pheromones. Species with these two organs intact and well-developed, meaning they have complex tube structures associated with the organs, are more likely to use scent in social interactions (Hecker et al. 2019; Yohe et al. 2019). Neither the velvety free-tailed bat nor the big brown bat has a vomeronasal epithelial tube or an accessory olfactory bulb. However, they both have a vomeronasal organ suggesting that they do have the capacity to detect social signals in pheromones, even if it is not well-developed

(Wible and Bhatnagar 1996).Bats in the families Vespertilionidae (e.g. big brown bats) and Molossidae (e.g. free-tailed bats) tend to have a reduced accessory olfactory system

(Wible and Bhatnagar 1996). In contrast, bats in Phyllostomidae, the most ecologically diverse family, tend to have well-developed olfactory systems (Hecker et al. 2019).

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Vampire bats possess an intact vomeronasal system and roughly twice as many intact vomeronasal type-1 receptor genes as other bats (Yohe et al. 2019).

To draw an overall conclusion about the role of scent in roost-finding, we conducted a meta-analysis by estimating an effect size and its precision (using bootstrapped 95% confidence intervals) across each experiment.

METHODS

Ethics Statement

This work was approved by the University of Maryland Institutional Animal Care and Use Committee (Protocol R-10-63), the Smithsonian Tropical Research Institute

Animal Care and Use Committee (#2015-0915-2018-A9, #2019-0501-2022) and

Panamanian Ministry of the Environment (#SE/A-76-16, #SE/A-36-19).

Panama Field Experiment

To count visits to possible roosts and measure nearby bat activity, we created four

0.3 x 0.18 x 0.10 m artificial roost boxes (pine wood stained dark brown and sealed with clear silicone), each containing an ultrasonic microphone of a bat detector (Wildlife

Acoustic SMMU2 and SM4+). On each of 13 weeks, we deployed four boxes as two pairs, one scented and one unscented. Each week, from May 10 to August 11, we deployed the paired boxes at two locations in Gamboa, Panama. There were 16 artificial roost locations, but nine were used twice after all sites were used once. Of these 16 locations, 11 were within six m of an existing roost, four were within 30 m of an existing roost, and one was about 110 m away from an existing roost. Roost boxes were placed

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0.75 m apart and approximately four m off of the ground (Fig.2.1). Each week, we swapped the scent treatment between the boxes.

To create scent cues, we mixed one g of guano with 10 g of water and painted this on a cardboard landing pad attached below the opening of the treated roost box. The control box had an unscented landing pad. We collected guano from four nearby roosts of velvety free-tailed bats and used the guano from the existing roost that was closest to the artificial roost location.

To record echolocation calls at the roosts, the settings for the microphone were as follows: a gain (sensitivity) of 0 dB, a 16 kHz high-pass filter, and a 256 kHz sampling rate. For automatic acoustic monitoring, we set a minimum wav file duration to 1.5 ms, a minimum trigger frequency of 20 kHz at 12 dB, a three sec trigger window, and a maximum call length of 15 sec.

To test the effect of acoustic cues, we also conducted playbacks one random night per week, except that we never conducted playbacks on the first or last night of a week, to allow us to compare the playback night to the day before and after. On the playback night, we mounted an Avisoft USG Player BL Pro speaker between the two roosts and broadcast echolocation and social calls for one hour after sunset, when velvety-free tailed bats are active (Holland et al. 2011).

We constructed playbacks from velvety-free tailed bat calls that were recorded during a previous study in Gamboa, Panama (Kohles et al. unpublished). To record and digitize echolocation calls (250 kHz sampling rate with a 16-bit resolution), an Avisoft ultrasonic condenser microphone, connected to a computer with Avisoft UltraSoundGate

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116 and Avisoft Recorder USHG software, was placed 1.5 m off the ground and 5 m from the location of the bat. Soon after sunset, bats were captured and then released from a platform so that they were calling towards the microphone.

To preserve the potentially informative qualities of call recordings, such as inter- pulse interval or variation in relative call amplitudes, we kept partial sequences intact when constructing playbacks. Using Avisoft SASLab Pro, we used a high-pass filter and a time-domain filter to remove sounds below 20 kHz and above 100 kHz. We also removed clipped calls and calls that were about 15% of the maximum possible amplitude by visually reviewing the waveforms in BatSound Pro. We spaced recorded samples with eight sec of silence to simulate natural breaks in calling rates. A total of 10 social calls and 122 echolocation calls were included in the final playback of 427 sec wav file that we played on loop during the one-hour playback period.

We used Avisoft SASLab Pro and R to count echolocation calls of free-tailed bats

(Molossus sp.). These calls were very likely to be M. molossus because this is the most common Molossus species in this area and the trials were near known roosts of this species. We chose a single threshold of -70 dB to detect as many calls as possible. For every possible call, we used Avisoft to measure the onset time, duration, the maximum frequency at the peak of the sound, the peak-to-peak amplitude, and the peak energy.

Once sounds were measured in Avisoft, we used R to filter 30-50 kHz sounds that were

1-8 ms long and recorded during the activity period of velvety free-tailed bats, between

1800 and 2100 h.

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Figure 2.1. Paired test boxes used in the field experiments. This image is from one of the sites near Columbus, Ohio used for the Ohio Field Experiment. The roost boxes were placed on an unoccupied condo near a house with a bat colony (including big brown bats). The roost box on the left is treated. We installed microphones in the roofs of the bat boxes that point downwards to the entrances to detect inspection visits by bats. Green boxes on the poles below are SM4+ bat detector units that digitize and store the acoustic signals.

Ohio Field Experiment

We repeated this field experiment at seven sites with known bat activity in

Columbus Metro Parks near Columbus, Ohio, but we did not conduct playback trials. As a scent cue, we collected guano from three different big brown bat colonies and alternated the guano source each experimental week (n=17 weeks). We painted a mixture of five g of guano and 10 g of water on to the removable, cardboard landing pads. As in the Panama field experiment, each pair of boxes was deployed for one week at each site. 25

The paired boxes were rotated between the sites from April 15th to August 12th. Four total boxes were used for this project allowing for two sites to be tested each week. Each site was repeated six times.

Using the same procedures described above, we again used Avisoft SASLab Pro and R to count echolocation calls and possible visits. To select big brown bat calls, we selected calls across the whole night that had peak frequencies of 25-40 kHz, and durations of 5-20 ms (Surlykke et al. 2009). Big brown bats are the most numerous species in Ohio, making up over 80% of summer captures (Brown, unpublished data).

Field experiment statistical analysis

To count roost visits for both field experiments, we looked for calls with an amplitude greater than 95% of the maximum possible amplitude indicating that a bat was calling directly into the microphone, which was inside the experimental roost and pointed at the entrance. Calls of lower amplitude were unlikely to be bats landing on the roost or echolocating inside to inspect it. The spectrograms of potential calls that were above that amplitude were inspected manually. We also verified that big brown bats produce calls of this amplitude when visiting roosts. When three captive big brown bats from the Ohio

Wildlife Center were placed onto the landing pad of a roost box, they would produce calls were at 100% of the maximum possible amplitude when echolocating into the box.

To see if the loudest calls were more likely to be recorded at the scented box than the unscented box, we manually inspected the spectrograms of the loudest calls with an amplitude over 5% of the maximum possible amplitude. To ensure calls were independent, we eliminated any calls that were within 10 min of each other. We then

26 conducted a binomial test to see if the loudest calls were more likely to be at the scented box.

For each day within each site, we calculated the amount of activity to both the treated side and the untreated side. Then, we calculated the treatment bias as (treated- control)/ (treated+control), where “treated” is the proportion of a bat calls near the treated box and “control” is the proportion of bat calls near the control box. We estimated the experiment effect size as the mean treatment bias across the sites. To calculate a nonparametric 95% confidence interval (CI) around the mean effect size, we bootstrapped the mean difference using the ‘basic procedure’ in the boot package in R

(5000 iterations). We also looked at the mean amplitude of sounds per site, week, and within treatments. We calculated this by finding the mean peak to peak amplitude for each box. Then, we found the treatment bias in the same way as with the amount of activity. We also calculated the 95% confidence intervals in the same was as described above.

Captive Experiment One: Preliminary trials of bats choosing refuges (n=5 bats)

We conducted several captive experiments with captive vampire bats, each improving on the methods of the previous one. For example, in the first two experiments, we did not film the behavior of the vampire bats, but later experiments were filmed. In a series of preliminary trials in 2014, we repeatedly tested five captive common vampire bats (originally from Chicago Brookfield Zoo) by presenting them with a scented and an unscented dark refuge (tubes constructed from plastic grid mesh covered in paper and black plastic). The scented tube contained a mesh bag at the top of the tube with a paper 27 towel and bedding soiled with guano and urine from the bat’s home enclosure. The control tube was the same, but the paper towel and bedding were not soiled with guano and urine. To induce the bats to choose and roost inside one of the tubes, we used a dim light to make the tubes the darkest roosting location. We tested each bat (n=5) in four different test cages (Fig.2.2), including three small cages (0.3 x 0.3 x 0.4 m) and one flight cage (1.7 x2.1 x 2.3 m). In the flight cage, we also conducted other trials with one scented and 3 unscented control tubes. We opportunistically scored their location after 1-

24 hours until the individual made a choice. A choice was defined as a bat moving into a roost tube. If the bat did not move into a tube, the cage was checked an hour later. When all five bats made a choice, they were swapped to new locations and we swapped the locations of the scented tubes within each cage. Each bat was tested in three to eight trials. We excluded cases where bats did not hide in either tube.

Captive Experiment Two: Zoo vampire bats (n=20) choosing sides

In Captive Experiment One, bats often did not enter the artificial roosts, perhaps due to neophobia, and we only tested five individuals. In 2015, we therefore improved upon the methods by using a familiar test chamber and a larger number of bats to increase the confidence in our effect size estimate. We tested 20 common vampire bats from a long-term captive colony at the Organization for Bat Conservation in Bloomfield Hills,

Michigan (described in Carter and Wilkinson 2013). We placed a subject bat in a 33 x 33 x 19 cm clear plastic tank. We attached a clean paper towel sheet to one side of the tank; on the opposite side, we placed a paper towel sheet stained with urine and guano that was collected daily from the floor below the captive colony. The bat could hang from the

28 treated side, the untreated side, or the ceiling, but it could not hang from the remaining two clear sides that were smooth (Fig.2.2). We left each subject bat in the tank in the dark, and then opportunistically sampled the position of the bat after a period of one to five hours. If the bat was on the floor or ceiling of the cage, we took another sample at least an hour later. Each subject bat was sampled between one and nine times. We swapped the location of the scented towel and cleaned the cage between each tested bat.

Figure 2.2. Test chambers for Captive Experiment One and Captive Experiment Two and Three. In Captive Experiment One (left), a subject bat chose between a scented and unscented tube. In Captive Experiment Two and Three (right) a bat chose between hanging on a scented or unscented surface.

Captive Experiment Three: Wild-born and captive-born vampire bats (n=22) choosing sides

In 2016, we replicated Captive Experiment Two, but we recorded each subject using infrared light and a Sony Nightshot camcorder, and sampled the roosting position of the bat every 10 min for 240 min, instead of sampling their location between one and

29 five hours. We used 22 vampire bats as subjects, including 13 wild-caught adult females and nine captive-born bats (8 females and 7 males) from a captive study colony housed in

Gamboa, Panama. We tested each bat one to three times and calculated the mean number of observations on each side within each bat.

Captive Experiment Four: Wild-born vampire bats (n=33) choosing sides

In 2019, we again replicated this experiment with several improvements to the design. Specifically, we chose a design that forced the bat to more clearly choose a side, we attempted to remove visual cues (in case the guano-stained side appeared darker) and echoacoustic cues (in case the guano-stained side had a different echoacoustic reflection quality). To do this, we placed the soiled paper towels in between two other clean papers towels before taping them onto one side of the glass aquarium. On the control side, we taped together three clean paper towels. Between trials, we also rotated the position of the test chamber inside the room to account for possible biases caused by airflow or lighting.

Between each trial we removed the paper towels, and the entire chamber was cleaned. To create the scented treatment, we collected about 5 g of guano and urine from the entire vampire bat colony by placing paper towels inside their home cage for one to three days.

We alternated the location of the guano treatment between each trial.

We tested 33 vampire bats (28 females and 5 males) captured from three different sites (Tolé, La Chorerra, and Lake Bayano) in Panama that were held in captivity at the

Smithsonian Tropical Research Institute. Bats were placed in a 40 cm x 56 cm x 40 cm glass test chamber and could choose to hang on one of two spaces divided by a barrier

(Fig.2.3). To ensure bats only chose a side while in a state of exploration rather than

30 escape, bats were initially held in a cloth bag, and we placed the bag in the center of the chamber, which allowed for the bat to crawl out of the bag on their own. Each vampire bat was left in the chamber for either one or four hours (n=48 and n=43, respectively).

We recorded the trials using a Sony Nightshot camcorder and an infrared light directed at the middle of the chamber. We tested each bat one to four times and calculated the mean number of observations on each side within each bat.

Figure 2.3. Test chamber for Captive Experiment Four. The glass chamber had mesh on the back and the top where the bat could climb. A plastic covered cardboard divider forced the subject bat to choose between the two sides. The floor was covered with paper towels that were always placed in the same position to ensure consistency between trials.

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Captive Experiment Five: A y-maze with acoustic cues, scent cues, and multimodal cues (n=45)

To test for multimodal enhancement, we placed 45 vampire bats from the colony used for Captive Experiment Three. Here, the test chamber was a y-maze with interchangeable arms (Fig.2.4)., and we tested the immediate responses of bats to either sound cues, scent cues, or both cues that would typically emanate from a roost.

To record the sounds of a vampire bat roost, we placed an Avisoft CM16 ultrasound condenser microphone (frequency range 10-200 kHz, Avisoft Bioacoustics,

Berlin, Germany) in the entrance to a single, large hollow tree in Tolé, Panama that contained more than 200 vampire bats. Seventeen of the 45 tested vampire bats were originally captured from this roost. We digitized the roost sounds (echolocation and social calls) continuously with constant gain and 16-bit resolution at a sampling rate of

250 kHz through an Avisoft UltraSoundGate 116 to a laptop running the program Avisoft

Recorder. To create acoustic playbacks, we compiled five 60-sec samples of recordings where no sounds reached maximum amplitude. During each trial, we played back the five acoustic samples in random order. To create scent cues, we again gathered guano and urine each day from below the captive colony on paper towels.

We conducted three trial types. In a scent trial, a bat chose between maze arms with either a balled-up paper towel scented with guano and urine (test arm) or a balled-up clean paper towel (control arm) at the end. In a sound trial, a bat chose between arms leading towards a playback speaker playing the roost sounds (test arm) or a cardboard box the same size as the speaker (control arm). In a bimodal trial, we presented both the acoustic and scent cues at the test arm and both control cues in the control arm. The arms 32 of the maze were swapped between bats. If a bat urinated or defecated in an arm, both arms were cleaned, and the plastic wrap was replaced before testing the next bat. A Sony

Nightshot camera with an infrared light was set-up facing the y-maze to record the movement of the bats. At the start of each trial, we placed the bat inside the y-maze then filmed for 300 s. If the bat did not enter either arm in this time, a new test with the same bat was started immediately. The experimenter and camera were centered to prevent side bias.

Each bat was randomly assigned to a trial type, then the numbers were balanced across trial types. To increase the power of the experiment, we also presented 35 of the

45 bats with the remaining two trial types in random order. We analyzed both “first choices” and ‘all choices’ (39 scent trials, 38 sound trials, and 38 bimodal trials). In each trial, we measured the latency from the start of the trial until the bat entered each arm and the total duration of time the bat spent in each arm. For ease of interpretation, we calculated “decision speed” as trial duration minus the latency.

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Figure 2.4. Test chamber for Captive Experiment Five. The center of the Y-maze consisted of a four-way junction of a PVC pipe. The left and right openings led to plastic- grid-mesh tubular arms wrapped in clear plastic wrap with the ends left open. These allowed the bat to enter the central junction and walk down each arm of the Y-maze towards the stimulus. The central opening was covered with clear plastic to allow the bat’s movements to be recorded by video. Bats were introduced into the back opening.

Captive Experiment Six: Velvety free-tailed bats (n=18) choosing sides

We repeated Captive Experiment Four using velvety free-tailed bats rather than vampire bats. We captured 18 velvety free-tailed bats from three different roosts in

Gamboa, Panama. To create scent cues, we collected 2-3 g of guano from the roost where the subject bat was captured and combined it with a few drops of water so that it could be spread onto the paper towels. The procedure was similar to Captive Experiment Four, expect each bat was tested only once and all trials were four hours. The velvety free- tailed bats were released the same night they were captured after being given water and mealworms.

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Captive experiment statistical analysis

To calculate effect sizes, we had to first develop a standardized statistic that could be applied to the data from all captive experiments. For each subject bat within each experiment, we calculated treatment bias as (observed- expected) / (1- expected), where

“observed” is the proportion of a bat’s choices (or time) on the scented side and the value

“expected” is the proportion expected by chance. For example, if a bat spent 60% of its sampled time on the scented side in a binary choice test, the treatment bias would be (0.6

- 0.5)/(1-0.5)=0.2. The treatment bias therefore ranges from values of -1 to 1. This treatment bias gave us a measure of the degree to which bats were spending more time on the scented side (a value closer to one), the control side (a value closer to negative one), or neither (a value around zero). We estimated the experiment effect size as the mean treatment bias across all bats. To calculate a nonparametric 95% confidence interval (CI) around the mean effect size, we bootstrapped the mean treatment biases using the ‘basic procedure’ in the boot package in R (5000 iterations). We applied the same procedure to all experimental data. Across the captive experiments, we calculated a weighted mean of the standardized effect sizes scaled by the number of bats used in each experiment. We bootstrapped these values to get a nonparametric 95% CI.

Comparing scent, sound and bimodal treatments in Captive Experiment Five

For the choices of the bats in the y-maze trials (Captive Experiment Five), we used the bootstrapping procedure describe above to calculate a treatment bias for the scent treatment, sound treatment, and bimodal treatment. To get p-values, we used permutation tests where we compared the observed mean difference in speed or duration

35 to 5000 expected differences when the response data were randomly swapped between arms within each trial and swapped between trial types within each bat (i.e. we never permuted data between bats).

RESULTS AND DISCUSSION

Field Experiments

We found no strong evidence that guano and urine can act as a chemical lure for artificial bat roosts. Out of the 24 loudest independent calls (passes) detected at the experimental roosts in Panama, only 8 were at a treated roost (binomial test, p=0.15). In

Ohio, only 11 of the 27 loudest independent calls (passes) were at a treated roost

(binomial test, p=0.44). We also did not detect a difference in general activity between the scented and unscented boxes(Ohio: t=0.58, p=0.5; Panama: t=-1.86, p=0.08). In

Panama, the sample mean activity was higher at the unscented box. We detected only one clear case of a “visit” where a bat echolocated directly into a treated roost box in Ohio.

Given that bats do sometimes move into bat houses, they must at least inspect potential roosts, but it is unknown how often they do. Despite deploying our roost boxes in 7 areas of high bat activity in Ohio for 16 weeks and 16 areas of high bat activity in

Panama for 13 weeks, bats almost never visited and inspected the artificial roosts by echolocating into the interior. This result is consistent with the idea that roost inspections by bats are relatively rare or that they primarily occur at certain times of the year. The scent cue we used was either ineffective or not strong enough to lure bats to inspect the interior, but it is also possible that the relatively weak scent cues at our roost boxes were masked by existing scent cues from nearby occupied roosts.

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A similar effect may also explain the unpredicted result that we did not detect more activity during the playbacks in Panama (Fig.2.5). If there were already many bats calling in the area from the other nearby roost, additional calls may not have been noticed by bats flying through. The Molossus at these sites quickly depart and return from their roosts using relatively fast and straight flight paths. During another experiment in

Panama, Molossus activity did not strongly increase in response to playbacks of echolocation or social calls outside roosts (G. Carter, personal observation), but activity did increase in response to playbacks of distress calls (Carter et al. 2015).

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700

e

t a

r 600

l

l

a

c

y ●

l 500

t

h g

i ● n

400

n

a e

m 300

control night playback night playback night

Figure 2.5. No increase in bat activity on playback nights. The mean number of calls per hour did not increase during playback hours. Error bars are bootstrapped 95% confidence intervals.

Captive Experiments

In our captive experiments, we did not detect an overall preference for either vampire bats or velvety free-tailed bats to roost in locations scented with guano and urine

(Fig.2.6). Although initial trials with vampire bats were suggestive of an effect, this effect was not observed in experiments with more rigorous methods (Fig.2.6). Any possible attractive effect of guano and urine was not robust across the different scenarios. To get the effect size across all the experiments, we weighed the mean effect size within each experiment by the number of bats used in that experiment. We estimated the overall effect size to be essentially zero (weighted mean with all experiments=0.025, 95% CI: -

0.003 to 0.055; weighted mean with only vampire bat experiments= 0.036, 95% CI: -

0.00138 to 0.0768). Below we discuss each experimental result and give possible explanations for differences in the observed effect sizes.

38

Figure 2.6. Captive experiments did not detect a consistent or strong effect of scent on roost selection. The distribution of treatment biases (left) and the means and 95% Cis (right) are shown for Captive Experiments One through Six. The size of the circles in the right figure are representative of the number of bats used for that experiment.

In Captive Experiment One, bats frequently did not enter any of the tubes, but when they did, all five bats chose scented tubes over unscented tubes. Initial results were compelling. In nine out of 10 trials in the small cages with only two tubes, the bat chose to roost in the scented tube over the unscented tube. In the larger flight cage trials, the bat chose the scented roost rather than the three unscented roosts in five out of eight cases. If we assume these choices were unbiased and independent, the binomial probability of seeing these outcomes by chance is only 0.02 and 0.03, respectively. However, choices were not independent because bats were tested multiple times. So, a bat may have acquired a familiarity bias for the scented tube over the course of several trials. More importantly, because the experiment was not filmed, there was no way to look for possible experimenter biases in how the bats were released into the cage. In fact, the

39 results for an attempted replication of this experiment conducted in Panama are not presented here because a clear experimenter bias could be seen in the videos (i.e. the experimenter tended to release the bats towards the treated side). Such biases could have also exaggerated the effect size of in Captive Experiment One. In Captive Experiment

Five, bats also chose between a scented and unscented tube in the form of the two arms of the y-maze, but in this experiment, we did not observe an effect of the scent treatment

(Fig.2.7).

In Captive Experiment Two and Three, we gave isolated vampire bats a choice to hang on two walls that were only 33 cm apart. In the first set of trials, observers reported that the bats were more likely to hang on the surface stained with guano and urine than on the unscented surface. However, these observations were haphazard and not filmed.

When we replicated this experiment and filmed the results, we no longer observed the effect. One problem here was the lack of an objective and clear distinction over whether a bat had chosen a side. Captive Experiment Four resolved this issue using a barrier and also removed any possible visual or echoacoustic cues from the guano. In this experiment, we did not detect a clear effect of the treatment. We also did not detect an effect of the treatment when velvety free-tailed bats were tested with this same procedure.

There are two reasonable interpretations of these experiments. The first possible interpretation is that vampire bats prefer scented roost sites, as seen in Captive

Experiments One and Two, under some conditions, but that this preference is not particularly robust. It is likely that other cues may play a larger role in this decision. Also, as previously mentioned, there may be particular moments in which a bat is actively

40 searching for a new roost and would be more likely to use scent cues. The use of aversive light in the Captive Experiment One might be an example of a situation where a bat would be motivated to find a new roost and one of the roosts was more familiar due to the scent. The second interpretation is that there is no preference of bats for roosting in locations scented with guano and urine, or any such preference is small enough to be biologically insignificant. Whatever the case, it is clear from these trials, as previously mentioned, that guano and urine, unlike acoustic cues, are not a strong enough lure to draw bats to the treated side across the variety of conditions offered in these experiments.

The y-maze trials in Captive Experiment Four compared the immediate responses of an isolated bat to the sound of a roost, the smell of a roost, or both. Vampire bats spent longer and moved faster towards the sounds of a roost rather than an empty arm (mean bias=108 sec sooner and 77 sec longer, p<0.0002). They did not show a clear bias towards the scented arm (mean bias= 27 sec sooner and 11 sec longer, p=0.2, Fig.2.7).

They also were attracted to the bimodal arm with both sound and scent cues (mean bias=144 sec sooner, 121 sec longer, p<0.0002), but this bias was not greater than the bias towards the sound cue alone (Fig.2.7).

41

Figure 2.7. Isolated vampire bats tested in a y-maze showed a clear attraction towards the roost sound cue but not to the roost scent cue. Panel a shows the mean and bootstrapped 95% CIs for bias towards the stimulus arm (with either the scent, sound, or both) compared to the empty arm. Panel b shows the observed mean compared to the 95% CI of expected null values from permutations of the data where choices were randomly swapped within each trial (grey error bars).

The attraction to sounds of the roost is not surprising given that vampire bats are attracted to contact calls (Carter and Wilkinson 2016), and many other bat species are attracted to the the acoustic cues at conspecifics at roosts (Barclay 1982; Ruczyński and

Kalko 2007; Ruczyński et al. 2009a; Chaverri 2010; Schöner et al. 2010; Arnold and

Wilkinson 2011; Furmankiewicz et al. 2011).

42

CONCLUSION

Our results overall suggest that the scent from guano and urine may not be strong enough to attract bats into new roosts. If guano could act as a chemical lure, this would be a useful tool for bat conservation and management, but we did not find clear evidence for this. Vampire bats, are highly social, have exceptionally odorous roosts, and have well-developed accessory olfactory systems compared to other bats, but females were not strongly attracted to the guano and urine scent cues of a roost like they were to roost sounds (Fig.2.6 and 2.7).

There are several reasons why we might expect bats to use scent to locate roosts.

Chemical cues from guano and urine might last longer and travel farther in cluttered environments where acoustic calls do not travel as well (Martens 1980), and bats might be able to recognize conspecifics from these scents (De Fanis and Jones 1995; Bouchard

2001; Bloss et al. 2002; Mann et al. 2011; Wilkinson et al. 2016). Males Schneider’s leaf- nose bats (Hipposideros speoris) use urine to relocate roosts (Selvanayagam and

Marimuthu 1984), and urine marks appear to aid in maintaining male territories.

However, other studies found that some bat species do not seem to prefer to roost in areas with guano and urine, such as Daubenton’s bat, the common noctule bat, and the brown long-eared bat (Ruczyński and Kalko 2007; Ruczyński et al. 2009). The current evidence does not entirely exclude the possibility bats might use guano and urine scent in roost location, but it suggests that these particular cues are not as attractive as other cues such as calls (Ruczyński and Kalko 2007; Ruczyński et al. 2009; Chaverri et al. 2010;

43

Furmankiewicz et al. 2011) or possibly scent marking (Nelson 1964; Moulton 1967;

Höller and Schmidt 1996; Grant and Bannack 1999; Caspers et al. 2009).

One limitation with our field experiments was that each of the paired experimental roosts was only left at a site for a week. It is possible that a week is not long enough for the bats to have found the roosts or to overcome any neophobia. Another limitation is that the additional scent from the guano on the experimental roost may not have been sufficiently detectable given the stronger scent of the nearby occupied roost. It is also possible that experimental results varied based on the bat’s motivational state or differences between the subject bat’s relationship with the source of guano and urine.

Another difference between the experiments is the distance between the scented and unscented side. It is likely that scent influences decisions in a way that depends on the spatial scale. For example, if the general area smells relatively similar to a roost, then other cues might become more salient than scent.

Our captive experiments also highlight the importance of replication in experiments. The first three initial experiments showed evidence for an effect of scent, but when repeated with more rigorous methods, these effect sizes did not replicate. Given a mix of some positive and null results, one might, intentionally or unintentionally, cherry-pick results that provide a more straightforward, clear, and simple narrative. For example, we could have excluded data from trials that were not filmed. However, the common practice of selectively publishing results has led to obvious biases in the literature that can be seen in meta-analyses (Møller and Jennions 2001; van Assen et al.

2014; Mlinarić et al. 2017). One partial solution is to move away from the false binary

44 outcomes of null hypothesis-testing towards a focus on effect size estimation (Ho et al.

2019). The key advantage of this approach is that it better communicates the actual uncertainty in the data.

The role of scent as an informative habitat cue in bats is surprisingly understudied. Future research could look at the variation in use of olfactory cues by additional species. Bats that use scent more in location of prey, like frugivores and nectivores, may use also scent cues more in roost location, compared to the insectivores and sanguinivores that we focused on. Another more rigorous approach would be to analyze the chemical components of guano and urine to isolate and test the response to specific compounds. For example, information such as individual identity or genetic relatedness, but not age and gender, can be communicated via urinary proteins in rodents

(Beynon and Hurst 2004). Finally, further work that manipulates existing chemical cues could test whether bats use the scent cues from guano and urine to relocate familiar roosts

(rather than find new roosts).

45

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Appendix A: Maps of Roost Locations for Field Experiments

Figure A.1. Ohio field experiment locations. This is a map of all of the metro parks in Franklin Country, Ohio. All of the locations with squares around them are locations that were used for the Ohio Field Experiment. Each location was visited six times from April 15th to August 12th. This map was modified from the map provided by the metro parks (https://www.metroparks.net/parks-and-trails/park-locations-map/).

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Figure A.2. Panama field experiment locations. This is a map of all of the site where the paired experimental roosts were placed in Panama. Site 150 and 152 both had two locations on either side of the house located there, and site 145 had four locations around that parcel of property. Nine of these locations (site 150A, 152A, 152B, 145A, 145D, 255A, 257A, 251A, and 269A) were each repeated twice. All other locations only had the pair of roosts there once for a whole week. 56